Language selection

Search

Patent 2487858 Summary

Third-party information liability

Some of the information on this Web page has been provided by external sources. The Government of Canada is not responsible for the accuracy, reliability or currency of the information supplied by external sources. Users wishing to rely upon this information should consult directly with the source of the information. Content provided by external sources is not subject to official languages, privacy and accessibility requirements.

Claims and Abstract availability

Any discrepancies in the text and image of the Claims and Abstract are due to differing posting times. Text of the Claims and Abstract are posted:

  • At the time the application is open to public inspection;
  • At the time of issue of the patent (grant).
(12) Patent Application: (11) CA 2487858
(54) English Title: METHODS, COMPOSITIONS, AND GROWTH AND DIFFERENTIATION FACTORS FOR INSULIN-PRODUCING CELLS
(54) French Title: PROCEDES, COMPOSITIONS ET FACTEURS DE CROISSANCE ET DE DIFFERENCIATION POUR CELLULES PRODUCTRICES D'INSULINE
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 5/02 (2006.01)
  • C12N 5/071 (2010.01)
  • C12N 5/00 (2006.01)
  • A61K 35/12 (2006.01)
(72) Inventors :
  • SCHARP, DAVID (United States of America)
  • PRESNELL, SHARON C. (United States of America)
  • HEIDARAN, MOHAMMAD (United States of America)
  • HAALAND, PERRY (United States of America)
  • LATTA, PAUL P. (United States of America)
  • COUTTS, MARGARET (United States of America)
  • MCINTYRE, CATHERINE (United States of America)
(73) Owners :
  • NOVOCELL, INC. (United States of America)
  • BECTON, DICKINSON AND COMPANY (United States of America)
(71) Applicants :
  • NOVOCELL, INC. (United States of America)
  • BECTON, DICKINSON AND COMPANY (United States of America)
(74) Agent: GOWLING LAFLEUR HENDERSON LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2003-05-28
(87) Open to Public Inspection: 2003-12-04
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2003/016734
(87) International Publication Number: WO2003/100026
(85) National Entry: 2004-11-29

(30) Application Priority Data:
Application No. Country/Territory Date
60/384,000 United States of America 2002-05-28
10/443,733 United States of America 2003-05-22

Abstracts

English Abstract




A method of converting differentiated non-hormone producing pancreatic cells
into differentiated hormone producing cells is disclosed. The method comprises
two steps: first, culturing cells under conditions which convert
differentiated non-hormone producing cells into stem cells; and second,
culturing stem cells under conditions which provide for differentiating stem
cells into hormone-producing cells. The invention defines growth and
differentiation factors that are presented to the stem cells to result in
their differentiation into hormone-producing cells, especially insulin-
producing cells. The invention provides a new source of large quantities of
hormone producing cells such as insulin-producing cells that are not currently
available for therapeutic uses such as the treatment of diabetes.


French Abstract

L'invention concerne un procédé de transformation de cellules pancréatiques différenciées non productrices d'hormone en cellules différenciées productrices d'hormone. Ce procédé consiste d'abord à mettre en culture des cellules dans des conditions qui permettent de transformer des cellules différenciées non productrices d'hormone en cellules souches; puis à mettre en culture ces cellules souches dans des conditions qui permettent de les différencier en cellules productrices d'hormone. L'invention définit les facteurs de croissance et de différenciation qui sont mis en contact avec les cellules souches pour induire leur différenciation en cellules productrices d'hormone, particulièrement, en cellules productrices d'insuline. Par ailleurs, l'invention concerne une nouvelle source d'obtention de cellules productrices d'hormone en grande quantité, notamment les cellules productrices d'insuline, qui ne sont pas encore disponibles pour un usage thérapeutique, tel que le traitement du diabète.

Claims

Note: Claims are shown in the official language in which they were submitted.





WHAT IS CLAIMED IS:


1. A method of converting differentiated non-hormone producing pancreatic
cells into
differentiated hormone-producing cells, comprising:
a) culturing said differentiated non-hormone producing pancreatic cells in
a first cell culture system with a first cell culture medium comprising a
basal medium, with or without serum, and with or without growth factors;
under conditions which provide for converting said differentiated non-hormone
producing pancreatic cells into stem cells; and
b) culturing said stem cells in a second cell culture system with a second
cell
culture medium
comprising at least one compound selected from Group A, wherein the
compounds of Group A are selected from the group consisting of:
Betacellulin, Activin A, BMP-2, TGF-.beta. SRII, DMSO, Sonic Hedgehog,
Laminin, Met-Enkephalin, DMF, and Cholera Toxin A;
and at least one compound selected from Group B, wherein the compounds of
Group B are selected from the group consisting of:
Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone Morphogenic
Protein (BMP-2), Bone Morphogenic Protein (BMP-4), C natriuretic
peptide (CNP), Caerulein, Calcitonin Gene Related Peptide (CGRP-.alpha.),
Cholecystokinin (CCK8-amide), Cholecystokinin octapeptide (CCK8-
sulfated), Cholera Toxin B Subunit, Corticosterone (Reichstein's substance
H), Dexamethasone, DIF-1, Differanisole A, Dimethylsulfoxide (DMSO),
EGF, Endothelin 1, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I,
Gastrin Releasing Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1),
Glucose, Growth Hormone, Hepatocyte Growth Factor (HGF), IGF-1,
IGF-2, Insulin, KGF, Lactogen, Laminin, Leu-Enkephalin, Leukemia
Inhibitory Factor (LIF), Met-Enkephalin, n Butyric Acid, Nerve Growth



72




Factor (.beta.-NGF), Nicotinamide, n-n-dimethylformamide (DMF),
Parathyroid Hormone Related Peptide (Pth II RP), PDGF AA + PDGF BB
MIX, PIGF (Placental GF), Progesterone, Prolactin, Putrescine
Dihydrochloride Gamma-Irradiated Cell Culture, REG1, Retinoic Acid,
Selenium, Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor,
Substance P, Superoxide Dismutase (SOD), TGF-.alpha.., TGF-.beta. sRII, TGF-
.beta.1,
transferrin, Triiodothyronine (T3), Trolox, Vasoactive Intestinal Peptide
(VIP), VEGF, Vitamin A, and Vitamin E;
under conditions which provide for differentiating said stem cells into
hormone-
producing cells.

2. The method of Claim 1, wherein the second cell culture medium comprises at
least
two compounds selected from Group A and at least two compounds selected from
Group
B.

3. The method of Claim 1, wherein the second cell culture medium comprises at
least
three compounds selected from Group A and at least three compounds selected
from
Group B.

4. The method of Claim 1, wherein the second cell culture medium comprises at
least
four compounds selected from Group A and at least four compounds selected from
Group
B.

5. The method of Claim 1, wherein the second cell culture medium comprises at
least
five compounds selected from Group A and at least five compounds selected from
Group
B.

6. The method of Claim 1, wherein the second cell culture medium comprises at
least six
compounds selected from Group A and at least six compounds selected from Group
B.

7. A method of culturing stem cells into differentiated hormone-producing
cells,
comprising culturing the stem cells in a cell culture system with a cell
culture medium



73




whereby said stem cells are differentiated into hormone-producing cells
wherein said
culture medium comprises basal medium without serum and at least one compound
selected from Group A wherein the compounds of Group A are selected from the
group
consisting of: Betacellulin, Activin A, BMP-2, TGF-.beta. SRII, DMSO, Sonic
Hedgehog,
Laminin, Met-Enkephalin, DMF, and Cholera Toxin A;
and at least one compound selected from Group B, wherein the compounds of
Group B
are selected from the group consisting of: Activin A, Atrial Natriuretic
Peptide,
Betacellulin, Bone Morphogenic Protein (BMP-2), Bone Morphogenic Protein (BMP-
4),
C natriuretic peptide (CNP), Caerulein, Calcitonin Gene Related Peptide (CGRP-
.alpha.),
Cholecystokinin (CCK8-amide), Cholecystokinin octapeptide (CCK8-sulfated),
Cholera
Toxin B Subunit, Corticosterone (Reichstein's substance H), Dexamethasone, DIF-
1,
Differanisole A, Dimethylsulfoxide (DMSO), EGF, Endothelin 1, Exendin 4, FGF
acidic,
FGF2, FGF7, FGFb, Gastrin I, Gastrin Releasing Peptide (GRP), Glucagon-Like
Peptide
1 (GLP-1), Glucose, Growth Hormone, Hepatocyte Growth Factor (HGF), IGF-1, IGF-
2,
Insulin, KGF, Lactogen, Laminin, Leu-Enkephalin, Leukemia Inhibitory Factor
(LIF),
Met-Enkephalin, n Butyric Acid, Nerve Growth Factor (.beta.-NGF),
Nicotinamide, n-n-
dimethylformamide (DMF), Parathyroid Hormone Related Peptide (Pth II RP), PDGF
AA + PDGF BB MIX, PIGF (Placental GF), Progesterone, Prolactin, Putrescine
Dihydrochloride Gamma-Irradiated Cell Culture, REG1, Retinoic Acid, Selenium,
Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor, Substance P,
Superoxide
Dismutase (SOD), TGF-.alpha., TGF-.beta. sRII, TGF-.beta.1, transferrin,
Triiodothyronine (T3),
Trolox, Vasoactive Intestinal Peptide (VIP), VEGF, Vitamin A, and Vitamin E.

8. The method of Claim 7, wherein the cell culture medium comprises at least
two
compounds selected from Group A and at least two compounds selected from Group
B.

9. The method of Claim 7, wherein the cell culture medium comprises at least
three
compounds selected from Group A and at least three compounds selected from
Group B.

10. The method of Claim 7, wherein the cell culture medium comprises at least
four
compounds selected from Group A and at least four compounds selected from
Group B.



74




11. The method of Claim 7, wherein the cell culture medium comprises at least
five
compounds selected from Group A and at least five compounds selected from
Group B.

12. The method of Claim 7, wherein the cell culture medium comprises at least
six
compounds selected from Group A and at least six compounds selected from Group
B.



75

Description

Note: Descriptions are shown in the official language in which they were submitted.




CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
METHODS, COMPOSITIONS, AND GROWTH AND DIFFERENTIATION
FACTORS FOR INSULIN-PRODUCING CELLS
Background of the Invention
Related Applications
[0001] This application claims priority to U.S. Provisional Application No.
60/384000, filed May 28, 2002 which is incorporated herein by reference in its
entirety.
Field of the Invention
[0002] This invention relates to the culture media, mode, conditions, and
methods
for converting non-insulin producing pancreas cells into stem cells that can
be proliferated
and differentiated into pancreatic hormone producing cells.
Description of the Related Art
[0003] The ability to selectively control the in vitro expansion and
conversion of
non-insulin producing pancreatic cells, such as acinar cells or duct cells,
into insulin
producing cells, would create a new treatment regime for diabetes that avoids
many of the
shortcomings of current diabetes treatments.
[0004] Diabetes mellitus is a disease caused by the loss of the ability to
transport
glucose into the cells of the body, either because not enough insulin is
produced or because
the response to insulin is diminished. In a healthy person, minute elevations
in blood glucose
stimulate the production and secretion of insulin, the role of which is to
increase glucose
uptake into cells, returning the blood glucose to the optimal level. Insulin
stimulates liver
and skeletal muscle cells to take up glucose from the blood and convert it
into the energy
storage molecule glycogen. It also stimulates skeletal muscle fibers to take
up amino acids
from the blood and convert them into protein, and it acts on adipose (fat)
cells to stimulate
the synthesis of fat. In diabetes, the blood stream may be saturated with
glucose, but the
glucose cannot reach the intracellular places where it is needed and utilized.
As a result the
cells of the body are starved of needed energy, which leads to the wasted
appearance of many
patients with poorly controlled insulin-dependent diabetes.
[0005] Prior to the discovery of insulin and its use as a treatment for
diabetes, the
only outcome was starvation followed predictably by death. With insulin
treatment today,
death still occurs with over dosage of insulin resulting in extreme
hypoglycemia and coma
1



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
followed by death unless reversed by the intake of glucose. Death also still
occurs with
major under dosage of insulin leading to ketoacidosis that, if not treated
properly and
urgently will also result in coma and death.
[0006] While diabetes is not commonly a fatal disease thanks to the treatments
available to diabetics today, none of the standard treatments can replace the
body's minute-
to-minute production of insulin and precise control of glucose metabolism. As
a
consequence, the average blood glucose levels in diabetics remain generally
too high. The
chronically elevated blood glucose levels cause a number of long-term
complications over
time. Diabetes is the leading cause of blindness, renal failure, the premature
development of
heart disease or stroke, gangrene and amputation, impotence, and it decreases
the sufferer's
overall life expectancy by one to two decades.
[0007] Diabetes mellitus is one of the most common chronic diseases in the
world. In the United States, diabetes affects approximately 16 million people -
more than
12% of the adult population over 45. The number of new cases is increasing by
about
150,000 per year. In addition to those with clinical diabetes, there are
approximately 20
million people showing symptoms of abnormal glucose tolerance. These people
are
borderline diabetics, midway between those who are normal and those who are
clearly
diabetic. Many of them will develop diabetes in time and some estimates of the
potential
number of diabetics are as high as 36 million or 25-30% of the adult
population over 45
years.
[0008] Diabetes and its complications have a major socioeconomic impact on
modern society. Of the approximately $700 billion dollars spent on healthcare
in the US
today, roughly $100 billion are spent to treat diabetes and its complications.
Since the
incidence of diabetes is rising, the costs of diabetes care will occupy an
ever-increasing
fraction of total healthcare expenditures unless steps are taken promptly to
meet the
challenge. The medical, emotional and financial toll of diabetes is enormous,
and increases
as the numbers of those suffering from diabetes grows.
[0009] Diabetes mellitus can be subdivided into two distinct types: Type 1
diabetes and Type 2 diabetes. Type 1 diabetes is characterized by little or no
circulating
insulin and it most commonly appears in childhood or early adolescence. It is
caused by the
destruction of the insulin-producing beta cells of the pancreatic islets.
There is a genetic
predisposition for Type 1 diabetes with the destruction resulting from an
autoimmune attack
against the beta cells, initiated by some as yet unidentified environmental
event, such as a
2



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
viral infection, or the action of a noninfectious agent (a toxin or a food),
which triggers the
immune system to react to and destroy the patient's beta cells in the
pancreas. The
pathogenic sequence of events leading to Type 1 diabetes is thought to consist
of several
steps. First, it is believed that genetic susceptibility is an underlying
requirement for the
initiation of the pathogenic process. Secondly, an environmental insult
mediated by a virus
or noninfectious agent such as toxin or food triggers the third step, the
inflammatory
response in the pancreatic islets (insulitis) in genetically predisposed
individuals. The fourth
step is an alteration or transformation of the beta cells such that they are
no longer recognized
as "self ' by the immune system, but rather seen as foreign cells or "nonself
'. The last step is
the development of a full-blown immune response directed against the
"targeted" beta cells,
during which cell-mediated immune mechanisms cooperate with cytotoxic
antibodies in the
destruction of the insulin-producing beta cells. Despite this immune attack,
for a period of
time, the production of new beta cells is fast enough to stay ahead of the
destruction by the
immune system and a sufficient number of beta cells are present to control
blood glucose
levels. Gradually, however, the number of beta cells declines. When the number
of beta
cells drops to a critical level (10% of normal), blood glucose levels can no
longer be
controlled and the progression to total failure of insulin production is
almost inevitable. It is
thought that the regeneration of beta cells continues for a few years, even
after functional
insulin production ceases, but that the cells are destroyed as they develop
maturity.
[0010] To survive, people with Type 1 diabetes must take multiple insulin
injections daily and test their blood sugar by pricking their fingers for
blood multiple times
per day. The multiple daily injections of insulin do not adequately mimic the
body's minute-
to-minute production of insulin and precise control of glucose metabolism.
Blood sugar
levels are usually higher than normal, causing complications that include
blindness, heart
attack, kidney failure, stroke, nerve damage, and amputations. Even with
insulin, the average
life expectancy of a diabetic is 15-20 years less than that of a healthy
person.
[0011] Type 2 diabetes usually appears in middle age or later and particularly
affects those who are overweight. Over the past few years, however, the
incidence of Type 2
diabetes mellitus in young adults has increased dramatically. In the last
several years, the
age of onset of Type 2 diabetes has dropped from 40 years of age to 30 years
of age with
those being obese, the new younger victims of this disease. In Type 2
diabetes, the body's
cells that normally require insulin lose their sensitivity and fail to respond
to insulin
normally. This insulin resistance may be overcome for many years by extra
insulin
3



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
production by the pancreatic beta cells. Eventually, however, the beta cells
are gradually
exhausted because they have to produce large amounts of excess insulin due to
the elevated
blood glucose levels. Ultimately, the overworked beta cells die and insulin
secretion fails,
bringing with it a concomitant rise in blood glucose to sufficient levels that
it can only be
controlled by exogenous insulin injections. High blood pressure and abnormal
cholesterol
levels usually accompany Type 2 diabetes. These conditions, together with high
blood sugar,
increase the risk of heart attack, stroke, and circulatory blockages in the
legs leading to
amputation. Drugs to treat Type 2 diabetes include some that act to reduce
glucose
absorption from the gut or glucose production by the liver and others that
stimulate the beta
cells directly to produce more insulin. However, high levels of glucose are
toxic to beta
cells, causing a progressive decline of function and cell death. Consequently,
many patients
with Type 2 diabetes eventually need exogenous insulin. A recent disturbing
finding is the
increase in the estimate from 20% to 40% of the Type 2 diabetics that will
eventually require
insulin treatment.
[0012] Another form of diabetes is called Maturity Onset Diabetes of the Young
(MODY). This form of diabetes is due to a genetic error in the insulin-
producing cells that
restricts its ability to process the glucose that enters this cell via a
special glucose receptor.
Beta cells in patients with MODY cannot produce insulin correctly in response
to glucose,
resulting in hyperglycemia and require treatment that eventually also requires
insulin
inj ections.
[0013] The currently available medical treatments for insulin-dependent
diabetes
are limited to insulin administration and pancreas transplantation either with
whole pancreas
or pancreas segments. Insulin therapy is by far more prevalent than pancreas
transplantation
and entails administration of insulin either conventionally, by multiple
subcutaneous
injections, or by continuous subcutaneous injections. Conventional insulin
therapy involves
the administration of one or two injections a day of intermediate-acting
insulin with or
without the addition of small amounts of regular insulin. The multiple
subcutaneous insulin
injection technique involves administration of intermediate- or long-acting
insulin in then
evening and/or morning as a single dose together with regular insulin prior to
each meal.
Continuous subcutaneous insulin infusion involves the use of a small battery-
driven pump
that delivers insulin subcutaneously to the abdominal wall, usually through a
27-gauge
butterfly needle. With this treatment modality, insulin is delivered at a
basal rate
continuously throughout the day and night, with increased rates programmed
prior to meals.
4



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
In each of these methods, the patient is required to frequently monitor his or
her blood
glucose levels and adjust the insulin dose if necessary. However, controlling
blood sugar is
not simple. Despite rigorous attention to maintaining a health diet, exercise
regimen, and
always injecting the proper amount of insulin, many other factors can
adversely affect a
person's blood-sugar control including: Stress, hormonal changes, periods of
growth, illness
or infection and fatigue. People with Type 1 diabetes must constantly be
prepared for life
threatening hypoglycemic (low blood sugar) and hyperglycemic (high blood
sugar) reactions.
Insulin-dependent diabetes is a life threatening disease which requires never-
ending
vigilance.
[0014) In contrast to insulin administration, whole pancreas transplantation
or
transplantation of segments of the pancreas is known to have cured diabetes in
patients.
However, due to the requirement for life-long immunosuppressive therapy, the
transplantation is usually performed only when kidney transplantation is
required, making
pancreas-only transplantations relatively infrequent operations. Although
pancreas
transplants are very successful in helping people with insulin-dependent
diabetes improve
their blood sugar to the point they no longer need insulin injections and
reduce long-term
complications, there are a number of drawbacks to whole pancreas transplants.
Most
importantly, getting a pancreas transplant involves a major operation and
requires the use of
life-long immunosuppressant drugs to prevent the body's immune system from
destroying
the pancreas that is a foreign graft. Without these drugs, the pancreas is
destroyed in a matter
of days. The risks in taking these immunosuppressive drugs is the increased
incidence of
infections and tumors that can both be life threatening in their own right.
The risks inherent
in the operative procedure, the requirement for life-long immunosuppression of
the patient to
prevent rejection of the transplant and the morbidity and mortality rate
associated with this
invasive procedure, illustrate the serious disadvantages associated with whole
pancreas
transplantation for the treatment of diabetes. Thus, an alternative to both
insulin injections
and pancreas transplantation would fulfill a great public health need.
[0015] Islet transplants are much simpler (and safer) procedures than whole
pancreas transplants and can achieve the same effect by replacing lost beta
cells. Insulin
producing beta cells are found in the islets of Langerhans scattered
throughout the pancreas,
an elongated gland located transversely behind the stomach. The pancreas
secretes between
1.5 and 3 liters of alkaline fluid containing enzymes and pro-enzymes for
digestion into the
common bile duct. Histologically, the pancreas is composed of three types of
functional



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
cells: a) exocrine cells that secrete their enzymes into a lumen, b) ductal
cells that carry the
enzymes to the gut, and c) endocrine cells that secrete their hormones into
the bloodstream.
The exocrine portion is organized into numerous small glands (acini)
containing columnar to
pyramidal epithelial cells known as acinar cells. Acinar cells comprise
approximately 80%
of the pancreatic cells and are responsible for secreting digestive enzymes,
such as amylases,
lipases, phospholipases, trypsin, chymotrypsin, aminopeptidases, elastase and
various other
proteins into the pancreatic duct system. The pancreatic duct system consists
of an intricate,
tributary-like network of interconnecting ducts that drain each secretory
acinus, draining into
progressively larger ducts, and ultimately draining into the main pancreatic
duct. The lining
epithelium of the pancreatic duct system consists of duct cells, a cell type
comprising
approximately 10% of pancreatic cells. Duct cell morphology ranges from
cuboidal in the
fine radicles draining the secretory acini to tall, columnar, mucus-secreting
in the main ductal
system.
[0016] The endocrine portion of the pancreas is composed of about 1 million
small endocrine glands, the islets of Langerhans, scattered throughout the
exocrine pancreas.
Although the islet cells comprise only approximately 2% of the pancreatic
cells, the islet
cells are responsible for the maintenance of blood glucose levels by secreting
insulin
appropriately and are the most important cells in the pancreas. There are
seven types of islet
cells classified according to the type of endocrine hormone secreted. The beta
cells of the
islet produce insulin. As discussed above, when there are insufficient numbers
of beta cells,
or insufficient insulin secretion, regardless of the underlying reason,
diabetes results.
Reconstituting the islet beta cells in a diabetic patient to a number
sufficient to restore normal
glucose-responsive insulin production would solve the problems associated with
both insulin
injection and major organ transplantation.
[0017] The islet transplantation outpatient procedure allows patients to
remain
fully conscious under local anesthesia while the equivalent of a 2-3
milliliters of pure islet
cells is piped through a small catheter to the liver. The patients can return
home or to regular
activities soon after the procedure. Thus, transplanting islets instead of
transplanting the
entire pancreas or segments thereof offers a number of ways around the risks
of the whole
organ transplant. However, the shortage of islet cells available for
transplantation remains an
unsolved problem in islet cell transplantation. Since islets form only about
2% of the entire
pancreas, isolating them from the rest of the pancreas that does not produce
insulin takes
approximately 6 hours. Although an automated isolation method has made it
possible to
6



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
isolate enough islets from one pancreas to transplant into one patient, as
opposed to the 5 or 6
organs previously needed to can y out one transplant, the demand for islets
still exceeds the
currently available supply of organs harvested from cadavers. In the United
States, due to a
combination of low organ donor rates and the increasing occurrence of insulin-
dependent
diabetes, there are only approximately 6,000 pancreases available for
transplantation or islet
cell isolation, while the new cases of insulin-dependent diabetes diagnosed
each year number
approximately 35,000 (Hering, B.J. & Ricordi, C. (1999) Graft 2, 12-27).
(0018] One solution to the problem of severe islet cell shortage is the
genetic
engineering of other cells to produce insulin. Genetically engineering other
cells to produce
insulin has already shown some success in muscle and liver cells in that they
can be modified
to produce proinsulin, the precursor to insulin. However, improving secretion
of the insulin
in these genetically engineered cells will still require considerable
investigative effort and
their low insulin production renders them as yet unsuitable for
transplantation. Another
strategy, xenotransplantation, the transplant of an organ (or tissues or
cells, in the case of
diabetes) from one species to another faces a number of fundamental obstacles
to becoming a
viable alternative to insulin injections of human transplantation. The risks
associated. with
xenotransplantation include transfer of prions such as those causing mad cow
disease (bovine
spongiform encephalopathy or BSE), and transmission of animal retroviruses
such as PoERV
(porcine endogenous retrovirus). Another obstacle is the problem of hyperacute
rejection.
The more distant the two species involved in the transplant are in
evolutionary terms, the
more rapid and severe the rejection process when the organs of one are
transplanted into the
other and the need for stronger and more risky immuno suppression. Strategies
involving the
genetic engineering of animal islets so as to make them less likely to succumb
to immune
system attach and destruction poses the risk of tampering with the silent
human endogenous
retroviral sequences (HERVs) thousands of which are spread throughout the
human genome.
Activation of these sequences by recombination and the ensuing expression of
HERV
proteins may lead to cancer or immune system dysregulation (Romano et al.,
Stem Cells
2000; 18:19-39). Finally, animal and human organs and cells differ in many
ways: In their
anatomy or structure, production of hormones, rates of filtration, secretion
and absorption of
enzymes and other chemicals, in their resistance to disease, and expected
longevity.
[0019] Another strategy to solve the problem of tissue availability for islet
cell
transplantation is the isolation of embryonic or totipotent stem cells.
Totipotent stem cells
are cells that are capable of growing into any other type of cell in the body,
including into an
7



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
entire organism. The problem with using this type of stem cell to grow as many
islets as are
needed to meet the demand for transplants for diabetes lies in their
procurement from
abortions or in vitro fertilizations with inherent ethical and political
risks. Furthermore, the
techniques to differentiate totipotent stem cells into normal insulin-
producing cells has not
been perfected and controlled in terms of their routine differentiation into
insulin-producing
cells in the great quantities that will be needed. Their ability to produce
insulin in response to
increases in glucose concentration that trigger insulin secretion in normal
beta cells,
indicating that they are not behaving as normal islet beta cells (Vogel,
Science, 2001 292:
615-617). Finally, the use of embryonic stem cells for therapeutic purposes in
patients
carries the inherent danger of tumor growth. Mouse embryonic stem cells are
tumorigenic
when injected into adult mice, and human embryonic stem cells also demonstrate
a similar
tumorigenic potential when injected into immune incompetent mice. The
potential use of
embryonic stem cells requires the precise separation of undifferentiated stem
cells from the
desired differentiated progeny, a critical and as yet unattained prerequisite
for clinical
application (Softer and Gearhart, Science 1999, 283: 1468-1470) in order to
prevent potential
tumor formation.
[0020] Thus, there exists a critical unmet medical need for large numbers of
non-
tumorigenic human beta cells to treat millions of diabetic patients worldwide.
A strategy for
the large-scale production of human insulin-producing beta cells from readily
available
starting material such as pancreatic acinar and duct cells that are converted
into clinically
relevant stem cells, would overcome the obstacles faced by the current
approaches.
[0021] In examining the prior art in terms of beginning with primary
pancreatic
cells and converting them to insulin producing cells, the experience
historically falls into
three categories based on the starting cells of interest: either islet cells,
duct cells, or acinar
cells. There are many prior experiences starting with islet cells to grow and
expand the islet
cell mass in vitro. Essentially all of these approaches isolate purified
islets and place them
predominantly into adherent culture systems in which the islets loose their
islet phenotype,
plate out as single cells, and grow to confluence. Most efforts to induce
direct differentiated
islet cell replication in vitro have shown limited capability to proliferate
islet cell mass while
maintaining their differentiated state. The collected experience of these
studies is that in
most circumstances, after a period of culture of these adherent islet cells,
they lose their islet
phenotype and dedifferentiate into a more primitive cell type that is poorly
characterized but
8



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
expands for a time in vitro. Yet, these cells invariably enter into senescence
with the loss of
the cultures.
[0022] It has proved very difficult to redifferentiate these more primitive
cells
back to differentiated islets (Nielson 92, Brelje 93, Bonner Weir 93,
Otonkoski 91,
Otonkoski 94). However, in one approach (Cornelius 97), the islet cultures
from NOD mice
were allowed to plate and then were left without media changes for several
weeks. A few
cells of a poorly identified epithelial cell type was all that survived and
could be, grown out
that demonstrated the ability to proliferate and could be differentiated into
islet cells with
different stages of culture conditions and reagents. The resulting US patents,
5,834,308 and
6,001,647, claim these poorly described epithelial cells as stem cells that
require this method
of culture to isolate, grow, and develop them into functional insulin-
producing cells. While
demonstrating the presence of stem cells by this method of pancreatic cell
adherent culture,
the technique of starvation of the cells to a minimal survival, and growth and
differentiation
into islet cells is problematic. This approach requires extensive growth of
islet cells to reach
the levels required to produce large scale implants for the treatment of
diabetes. There is no
evidence to date that this procedure is applicable to human cells and that
such a scale up is
possible while retaining the differentiated phenotype of these islet cells
required for a clinical
product. Therefore, we have turned to an alternative approach as described in
this invention
that significantly differs in that it does not start with primary islet cells
to form the stem cells
that can be expanded and differentiated to insulin producing cells. Instead,
we start with
non-insulin-producing pancreatic cells, and convert them to stem cells that
expand and then
differentiate into islet cells.
[0023] Others have placed the islet cells into MATRIGEL, collagen, or agarose
rather than the use adherent cultures (Kerr-Conte 96). This results in the
formation of cystic
duct structures with regression of islet tissues and growth and
differentiation of duct
structures and cells of ductal phenotype. The inventors of this application
have also placed
isolated human islets into MATRIGEL and have confirmed the induction of duct
cells that
replace the differentiated islet cell mass. Different matrices can also
convert islet cells to
duct cells, especially in the presence of HGF (Lefrbvre 98), but again fail to
produce islets.
While claims of islet cells forming from these structures have been made, it
is unclear as to
whether their origin is from residual islet tissue present in the starting
cells or new insulin-
producing cells. The duct structures and islet cells may also develop from a
stem cell that
has not as yet been specifically identified.
9



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0024] The next approach that has been explored is to start with pancreatic
duct
cells to determine the ability to form new islet cells. It is based on the
observations in both
developing fetal pancreas as well as adult pancreas induced to damage by
disease or
manipulation where one observes the formation of new islets budding off ductal
structures
that have led to the idea that there is a pancreatic stem cell associated with
the ductal
structures that can be activated by fetal development, or damage or loss to
islet mass in the
adult pancreas.
[0025] Starting from isolated and purified duct structures from mouse and rat
pancreas and not from human pancreas (Fung, US patent 6,326,201), single cells
begin to
form monolayers in vitro that are predominantly a mixture of fibroblasts and
stromal cells.
Eventually some insulin producing cells begin to appear in these adherent
cultures, but
remain at a low level in the monolayers. Addition of a few growth factors
minimally
increased numbers of insulin cells in the monolayer. But, single cells to
groups of cells,
called non-adhering cells (NAC) began to appear floating above the monolayer
cultures that
contain islet hormone cell types. These NAC's could be increased by using
growth factor
pulsing prior to harvesting. They also described pdx 1 positive cells, some
containing with
insulin which is required as a beta cell, and others with pdxl staining only
that they describe
as being progenitor cells. The NAC's were also able to show glucose stimulated
insulin
release. They can also add different growth factors to the monolayers and
induce
proliferation as well as phenotype changes. They describe the use of lectins
to purify these
progenitor cells as they are produced. Thus, their results support the ability
of purified
pancreatic duct cells from large pancreatic ducts to be dedifferentiated into
progenitor cells
that can differentiate into insulin producing cells by the use of their
specific methods. This
invention differs significantly from the Fung work in that our starting
pancreatic cells are
human pancreatic cells and are not isolated from purified duct structures. In
fact, he claims
producing duct cells only from pancreatic duct tissue that he defines as
including the main
pancreatic duct, the accessory pancreatic duct, the dorsal pancreatic duct,
and the ventral
pancreatic duct. He separately defines interlobular ducts and intercalated
ducts as separate
entities that are not included in his definition of pancreatic duct. Our
starting pancreatic
tissue excludes the tissue he defines as pancreatic duct since these larger
structures and parts
of structures are screened out of our preparation during the cell isolation
process and are not
observed in the histologic sections of the starting material. The only
pancreatic duct tissue



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
staining positive for CK19 are the intercalated ducts located within acinar
cell aggregates and
completely surrounded by acinar cells.
[0026] Thus, our starting pancreatic cells are a mixture of acinar cells,
intercalated duct cells surrounded by acinar cells, and stromal cells, that
are harvested after
purifying the islets out of the starting cell mixture, leaving very few islet
cells in the
pancreatic starting cells. In addition, our culturing techniques differ
significantly with the
different modes of culture, the multiple media, as well as the growth factors
that are
significantly different and are described below.
[0027] Another work is that of Bonner-Weir 2000 that also starts with duct
enriched pancreas tissue with the statement that their approach does not
actually work with
the starting pancreatic cells that we are utilizing. Their culture method also
relies on
MATRIGEL that is not the subject of our primary approaches to permit the new
cells to
migrate into and form insulin-producing cells.
[0028] The third approach for developing large quantities of insulin-producing
cells starts with acinar cells. Most of the early work with acinar cells was
to maintain its
phenotype in culture to better understand these cells (Oliver 87, Brannon 88).
Then in
attempting to understand the source of pancreatic cancer cells, attention
turned to duct cells
and the ability of acinar cells to apparently change phenotype to some sort of
duct cell, as it
was described. Culturing acinar cells in collagen gels, Lisle & Losdon 1990
describe the
phenomenon of acinar cells losing their specific cell markers in this culture
and picking up
markers similar to duct cells for 6-12 days of culture, using their own
monoclonal antibodies,
but subsequently reverting back to their original acinar cell markers as the
culture continues.
[0029] Again, interested in pancreatic cancer, Hall & Limoine 1992, describe
the
culture of acinar cells on plastic dishes whereby the cells began to change
over 5-10 days to
begin to express one of the duct cell markers CK19, but die off by 3 weeks.
Arias &
Bendayan 1993 cultured rat and guinea pig acinar cells on MATRIGEL with
maintenance of
their acinar phenotype but loss of the cells by one week. The addition of 2%
DMSO to the
culture of acinar cells in MATRIGEL changed the phenotype to duct-like cells
that began to
form cysts and tubules within the MATRIGEL. In addition, when in the cyst
structures, the
cells began to express CAII, a specific enzyme used by duct cells to release
bicarbonate and
water. Protein inhibitors prohibited the change into a duct-like phenotype. It
appears that the
combination of MATRIGEL and DMSO pushed the dedifferentiated islet cells on
through
the more primitive stage and further differentiated them into mature duct
cells with a
11



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
functional marker and the ability to form three dimensional structures. The
question of
mechanisms was raised as to whether stem cells were involved or whether this
represented
transdifferentiation.
(0030] Then, Bouwens 1994 studied potential duct cell markers in the neonatal
rat
and described that CK7 was a marker for large pancreatic ducts while CK19 was
expressed
in the smaller ducts, the intercalated ducts, and the centroacinar cells of
the acinii. Another
marker unique to the rat, CK20, marked similar cells as CK19. He also noted
that while
proliferation was going on, some cells next to expanding islets also expressed
the CK19 or
CK20. Examining mouse pancreas cells cultured on plates, Vila 1994
demonstrated human
acinar cells express CK18 at the start but changed their expression to CK7 and
CK19 over
time with amylase levels going down. Also mucin 1 expression rose as well as
another duct
cell marker, CFTR, the marker for chloride transporter of duct cells. Again,
the question was
raised as to whether the mechanism of this change represented
transdifferentiation or the
involvement of stem cells. They also found that both HGF and TGFa exposure
caused these
cells to proliferate making the suggestion that a stem cell may be the cause
and may have
bearing in the development of ductal malignancies of the pancreas. But, no
insulin
production was observed.
(0031] Kerr-Conte 1996 demonstrated that placing purified human islets into
MATRIGEL produced cystic duct-like structures that contained islet cells as
small buds. It is
not clear from this work as to what the source of these duct-like cells may be
that could
clearly proliferate, but there was no evidence of proliferation of the islet
cells. Again, as
previously discussed above, the suggestion that these may be dedifferentiating
islet cells into
duct-like cells was made, but the ability of these cells to proliferate while
the differentiated
cells did not proliferate raises the possibility that these cells represent
stem cells. But, no
insulin production was observed.
(0032] Bouwens 1998 compared the possibilities of transdifferentiation versus
the
role of stem cells as causing the proliferation of dedifferentiated cells from
either the duct,
acinar, or islet differentiated cells. While he favored the
transdifferentiation mechanism due
to cell markers showing the expression of the different cell types, his
primary reason was
because definitive stem cell markers for these cells had not yet been
developed so it was not
possible to specifically identify them. Yet, he acknowledged that indirect
evidence can
readily suggest the presence of stem cells and that the specific markers have
simply not as yet
been perfected. Yet again, no insulin production had been observed in his
review.
12



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0033] Kerr-Conte 2000 and in US Patent Application (20020155598) suggests
the presence of "pluripotent pancreatic stem cells" as the primary explanation
of the ability to
change terminally differentiated human pancreas cells to a more primitive cell
type that has
the ability to expand and then be differentiated into another type of specific
cell that is
terminally differentiated. As an accepted marker for this stem cell, she
suggests the duct-like
cells co-expressing CK19 and pdxl, similarly suggested by Fung, are those stem
cells. She
cultured a mixture of human acinar and duct cells in adherent culture showing
the loss of
amylase, the increase of CK19, and the increase of pdxl expressions in the
resulting duct-like
cells that flattened out as monolayers. But, she was not able to show the
conversion to
insulin-producing cells but was able to show the new expression of a
neuroendocrine cell
marker, chromogranin A. In fact, her claim of pdxl and CK19 stained cells as
being
evidence of precursor cells of insulin producing cells agrees with Fung and
ourselves as well
as with their being stem cells. But her claim that these indeed are insulin
producing cells in
her patent application remains unproven by her own data represented in Figures
4 and S that
fails to provide any direct evidence of increased insulin production by these
converted cells.
Thus, she has demonstrated the presence of stem cells but fails to demonstrate
their
differentiation into insulin-producing cells. This is a significant difference
compared to this
invention where we clearly demonstrate the production of insulin-producing
cells. The
methods described in these two publications utilize single pancreas cells
decreased in islet
content, cultured in monolayers to change the acinar phenotype to the duct-
like phenotype
that are called ductal precursors. By her definition, these ductal precursor
cells have the
ability to be differentiated into insulin-producing cells. She attempts the
redifferentiation by
placing the ductal precursor cells into a matrix of MATRIGEL or collagen. She
clearly
demonstrates the ability of the ductal precursor cells to proliferate, but in
the patent
application, does not demonstrate the formation of any new insulin-producing
cells.
[0034] There are significant differences between her techniques and those in
this
invention. The first step of converting the phenotype of non-insulin producing
pancreatic
cells to stem cells in this invention can utilize several different media in
several different
culture modes in addition to adherent culture using several different types of
growth factors.
A stem cell is formed as demonstrated by its ability to undergo replication as
the
intermediary, more primitive cell that carnes the only makers accepted to date
to identify this
stem cell that are duct cell markers like CK19 and pdxl expression in
replicating cells. Her
second step does not produce insulin-producing cells. In our second step,
these stem cells are
13



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
then differentiated into insulin producing cells by a different set of growth
factors and
conditions, again demonstrated in different cell culture modes. Our invention
also utilizes
more complex growth and differentiation factors (Table ???) than described in
her
publication and patent application. The normal histology and function of our
new insulin-
producing cells are also shown below. The definition of the stem cell used in
this invention
is based on the National Library of Medicine's definition that it is a cell
that is not terminally
differentiated that undergoes replication as well as can differentiate into
more than one type
of differentiated cells. Our examples show the starting non-insulin producing
pancreatic
cells are converted under the first set of culture conditions into stem cells
that replicate and
carry the CK19 and pdxl markers. These stem cells can then be differentiated
into hormone
producing islet cells such as insulin or glucagon as well as into duct
structures under separate
differentiating conditions as described below.
Definitions:
[0035] General source of many of these definitions is OMIM, National Center
for
Biotechnology Information, National Library of Medicine, National Institutes
of Health.
[0036] Acinar cells - pancreatic cells that make up 80% of the pancreas and
produce many different enzymes including amylase, lipase, trypsin,
chymotrypsin, elastase,
and many others. Acinar cells can be identified by their enzyme content, by
specific
cytokeratins such as CK18, and by lectins against surface sialoglycoproteins.
Acinar cells
form spherical structural units in the pancreas called acini consisting of
polarized cells that
release their enzyme products into the small, centralized intercalated ducts
located at the
center of each acinus. Many acinar cells contain two nuclei at any time of
examination of
primary cells.
[0037] Duct cells - pancreatic cells making up 10% of the pancreas that define
the larger interlobular and intralobular ducts as well as the smallest,
intercalated ducts, that
drain the pancreatic enzymes from the acini. Duct cells also produce
bicarbonate and water
to dilute the enzymes and alter the intestinal pH upon release into the gut
from these ductal
structures. Duct cells can be identified by cytokeratin subtypes such as CK19
and by the
enzymes responsible for bicarbonate production.
[0038] Islet cells - endocrine cells making up 2% of the pancreas and existing
as
separate cell aggregates called islets that contain different cell types
making different
hormones. Beta cells that are 50-60% of the islet aggregate make insulin that
permits
14



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
glucose entry into most cells of the body. Alpha cells that are 30% of the
islet make
glucagon that is released during fasting to permit glucose delivery from the
liver to maintain
normal blood sugar. Delta cells, 10% of the islet cells, make somatostatin
that fine tunes
glucose levels. Pancreatic polypeptide producing cells (5-10% of the islet
cells) release
their hormone that alters exocrine and gastrointestinal function. In addition
to these major
islet cell types, there are also other islet cell types that make a variety of
other hormones
including GIP, VIP, gastrin, bombesin, and others. In addition, the islets
contain
fenestrated endothelium as a rich capillary bed into which each islet cell to
releases its
hormone product.
[0039] Pancreatic cells - primary pancreatic cells from human donors (or other
mammalian species) that contain acinar, duct, and islet cells types as well as
supportive and
vascular cells.
[0040] Islet-depleted pancreatic cells - the cells remaining after the
isolation of
islets from a suspension of digested pancreatic cells using a discontinuous or
continuous
density gradient. This population is comprised mainly of acinar cells (>90%)
with a small
percentage of intercalated ducts within the acinar aggregates, vascular, and
neuronal tissue,
as well as a residual amount of contaminating islet material.
[0041] Pancreatic Acinus - any of the small spherical acinar cell structures
that
empty their enzyme products into the central acinar area that empties into the
intercalated
pancreatic ducts.
[0042] Intercalated Duct - a duct from a tubule or acinus of the pancreas that
drains into an intralobular duct.
[0043] Intralobular Duct - a duct that collects pancreatic juice from the
intercalated ducts and drains into an interlobular duct.
[0044] Interlobular Duct - a duct that collects pancreatic juice from
intralobular
ducts and drains into pancreatic ducts
(0045] Pancreatic Duct - largest of the ducts that includes the main
pancreatic
duct, the accessory pancreatic duct, the dorsal pancreatic duct, and the
ventral pancreatic duct
[0046] Stem Cell - a cell that is not terminally differentiated that can
undergo
replication and can differentiate into more than one type of differentiated
cell.
[0047] Cell Growth - is the replication of the cellular DNA followed by
cytokinesis that can be demonstrated by BrdU or tritiated thymidine
incorporation or KI67.



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0048] Cell Expansion - used to define numbers of cells that have gone through
cell division and are increasing their numbers and overall mass, rather than
simply enlarging
by hypertrophy.
[0049] Proliferation - rapid and repeated production of new parts or of
offspring
(as in a mass of cells by a rapid succession of cell divisions).
[0050] Cell Hypertrophy - used to define enlarging cells that have increased
their cell volume, rather than growing by cell division.
[0051] Cell Cycle - cell growth cycle. Cells that are in cell cycle have left
the
resting state (Go phase) and are replicating their contents and dividing in
two.
[0052] Differentiation - is used to declare that a cell has passed from a
progenitor level or more basic or generalized function to one of more specific
function.
(0053] Transdifferentiation - is uses to declare that a cell has changed from
a
level of defined function to another.
[0054] Dedifferentiation - is used to declare that a cell has passed from a
level
of defined function to one of less defined function or to a basic cell.
[0055] Totipotent - capable of developing into a complete organism or
differentiating into any of its cells or tissues.
[0056] Pluripotent - 1 : not fixed as to developmental potentialities : having
developmental plasticity such as a pluripotent cell or pluripotent embryonic
tissue. 2
capable of affecting more than one organ or tissue.
[0057] Growth Factors (GF) - include a number of compounds that may induce
cell replication. There are general GF's such as Epidermal GF (EGF) and
Vascular
Endothelial GF (VEGF). There are also GF's that are more specific in their
action. (e.g. the
action of Insulin-like GF 1 (IGF1) on islets, or erythropoietin on red blood
cell progenitors).
[0058] Differentiation Factors (DF) - include a number of compounds that may
induce cell type specific differentiation. There are specific differentiation
factors for islet
cells, for acinar cells, and for duct cells. An example for acinar cells is
dexamethasone.
[0059] Dedifferentiation Factors (DDF) - include a number of factors for islet
cells, for acinar cells, and for duct cells that permit the cell to lose
differentiated function and
change to a level of function that is lower in the lineage.
[0060] Matrix or Matrices - used to define hydrogels or polymerizable
materials
that hold cells in place for culture under different conditions. These include
MATRIGEL,
collagen, alginate, and others.
16



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0061] Tissue Culture Flask, Dish or Plate Substrates - used to define
specific
types of plastic or glass surfaces that are configured either in tissue
culture flasks, petri dishes
or culture plates that are used to grow cells. These surfaces are prepared
such that they either
promote or discourage adherent or non-adherent cell growth.
[0062] Coated Culture Flask, Dish, or Plate Surfaces - a cell culture dish
coated with a thin layer of a compound.
[0063] Suspension culture - cells suspended in tissue culture medium in the
absence of any support from a thin layer of a compound or any matrix.
[0064] Alpha-tocopherol - any of several fat-soluble vitamins that are
chemically tocopherols, are essential in the nutrition of various vertebrates
in which their
absence is associated with infertility, degenerative changes in muscle, or
vascular
abnormalities, are found especially in wheat germ, vegetable oils, egg yolk,
and green leafy
vegetables or are made synthetically, and are used chiefly in animal feeds and
as
antioxidants.
[0065] Apotransferrin - protein produced by oligodendricytes that is necessary
for cell survival and involved in cell differentiation.
[0066] Atrial Natriuretic Peptide - A potent natriuretic and vasodilatory
peptide
or mixture of different-sized low molecular weight peptides derived from a
common
precursor and secreted by the heart atria. All these peptides share a sequence
of about 20
amino acids.
[0067] Biotin - a colorless crystalline growth vitamin ClpH~6N2O3S of the
vitamin
B complex found especially in yeast, liver, and egg yolk.
[0068] BSA - (bovine) serum albumin is a monomeric protein that comprises
about one-half of the blood's serum proteins. In vivo, it plays a role in
stabilizing
extracellular fluid volume and functions as a Garner for steroids, fatty
acids, and some
hormones.
[0069] C natriuretic peptide (CNP) - A 22-amino acid peptide that is a member
of the natriuretic peptide family. It is from endothelial and renal cell
origin with selective
cardiovascular actions.
[0070] CAII - carbonic anhydrase type II, the enzyme used by duct cells to
produce bicarbonate that is secreted into the pancreatic ducts to neutralize
the acid in the
duodenum generated by the stomach.
17



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0071] Calcium pantothenate - a white powdery salt C1gH32CaNZO~o made
synthetically and used as a source of pantothenic acid.
[0072] Carnitine - a quaternary ammonium compound C~H~SN03 present
especially in vertebrate muscle and involved in the transfer of fatty acids
across
mitochondria) membranes.
[0073] Catalase - enzyme that consists of a protein complex with hematin
groups
and catalyzes the decomposition of hydrogen peroxide into water and oxygen
[0074] CCK - cholecystokinin is a brain and gut peptide. In the gut, it
induces the
release of pancreatic enzymes and the contraction of the gallbladder. It has
the capacity to ,
stimulate insulin secretion. CCK peptides exist in multiple molecular forms
(e.g., sulfated
CCKB, unsulfated CCKB, and CCK4), each resulting from distinct
posttranslational
processing of the CCK gene product.
[0075] CFTR - cystic fibrosis transmembrane conductance regulator (CFTR)
functions as a chloride channel. Mutations in the CFTR gene have been found to
cause cystic
fibrosis. Mutations in CFTR effect the exocrine function of the pancreas,
intestinal glands,
biliary tree, bronchial glands and sweat glands.
[0076] CGRP alpha, (Calcitonin Gene Related Peptide) - A test that measures
the amount of the hormone calcitonin in the blood.
[0077] Cholera Toxin B Subunit - The nontoxic subunit B of Cholera Toxin is
important to the protein complex as it allows the protein to bind to cellular
surfaces via the
pentasaccharide chain of ganglioside GM1.
[0078] CK19 - cytokeratin 19 is the smallest known (40-kD) acidic keratin, one
of a family of water-insoluble intermediate filaments. Different cytokeratins
can be used as
markers to identify certain types of epithelia and epithelial tumors. CK19
keratin is found in
many types of epithelial cells, including numerous ductal and glandular
epithelia. In the
pancreas, it is present in ductal epithelia and absent in endocrine and
exocrine cells.
[0079] CK19+ cells - cytokeratin 19 is expressed in epithelial cells in
culture, in
particular, in "intermediary" or transdifferentiating cells from pancreatic
tissues.
[0080] Corticosteroid - any of various adrenal-cortex steroids (as
corticosterone,
cortisone, and aldosterone) that are divided on the basis of their major
biological activity into
glucocorticoids and mineralocorticoids.
[0081] Corticosterone - a colorless crystalline corticosteroid Cz~H3o0a of the
adrenal cortex that is important in protein and carbohydrate metabolism.
18



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0082] Corticosterone (Reichstein's substance H) - a colorless crystalline
corticosteroid CZ1H3o0a of the adrenal cortex that is important in protein and
carbohydrate
metabolism
(0083] C-peptide - the c-peptide ("connecting" peptide) is a short polypeptide
released after the conversion of proinsulin to mature insulin. Its molecular
weight is 3,582
Da.
[0084] Cyclodextran - 2-hyrdroypropyl-beta-cyclodextrin. A tissue culture
medium additive that facilitates solubilization of hydrophobic substances.
[0085] DIF-1/Differanisole A - Differentiation-inducing factor-1 (DIF-1) is a
chlorinated hexaphenone isolated from Dictyostelium. DIF-1 exhibits antitumor
activity in
several types of mammalian tumor cells, although the underlying mechanisms
remain
unknown. The structure of morphogen of Dictyostelium discoideum, DIF-1, is
closely similar
to that of differanisole A which had been isolated from the metabolites of a
simple eukaryote,
Chaetomium, as the differentiation-inducer of murine and human
undifferentiated tumor
cells.
[0086] DL-alpha-tocopherol acetate - a tocopherol C29HSO02 with high vitamin
E potency.
[0087] DMF (n-n-dimethylformamide) - affects cellular differentiation.
Suppression of acidification rate is likely due to decreased metabolic acid
production.
Alterations in H+ production and transport contribute its effects on cellular
differentiation.
[0O88J DMSO - dimethyl sulfoxide (CH3)ZSO - that is an agent known to
induce cell differentiation, also a solvent, also a cryoprotectant for
freezing living cells, also
an anti-inflammatory agent for the treatment of interstitial cystitis
[0089] DMSO (dimethylsulfoxide) - an anti-inflammatory agent (CH3)ZSO used
in the treatment of interstitial cystitis
[0090] EGF - epidermal growth factor is a potent mitogenic factor for a
variety of
cultured cells of both ectodermal and mesodermal origin and has a profound
effect on the
differentiation of specific cells in vivo. Mature EGF is a single-chain
polypeptide consisting
of 53 amino acids and having a molecular mass of about 6,000.
[0091] Endothelia 1 - any of several polypeptides consisting of 21 amino acid
residues that are produced in various cells and tissues, that play a role in
regulating
vasomotor activity, cell proliferation, and the production of hormones, and
that have been
implicated in the development of vascular disease
19



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[0092] Ethanolamine - a colorless liquid amino alcohol C2H7N0 used especially
as a solvent for fats and oils, -- called also monoethanolamine.
[0093] Exendin 4 - a long acting analog of GLP-1
[0094] FACS - fluorescence activated cell sorting
[0095] FCS - fetal calf serum. Blood serum recovered from an unborn cow.
[0096] FGF - The FGF superfamily consists of 23 known members, all of which
contain a conserved 120 amino acid region. The FGFs were originally recognized
to have
proliferative activities; they are now considered to play substantial roles in
development,
angiogenesis, hematopoiesis, and tumorigenesis. Almost all of the FGFs
isoforms have the
ability to activate other isoform's receptors. This accounts for similar
effects generated by
different FGF subtypes.
[0097] FGF2 - fibroblast growth factor 2 (FGF-basic) is a wide-spectrum
mitogenic, angiogenic, and neurotrophic factor that is expressed at low levels
in many tissues
and cell types. FGF2 has been implicated in a multitude of physiologic and
pathologic
processes, including limb development, angiogenesis, wound healing, and tumor
growth.
(0098] Galactose - an optically active sugar C6H1z06 that is less soluble and
less
sweet than glucose and is known in dextrorotatory, levorotatory, and racemic
forms.
[0099] GLP-1 - Glucagon like-peptide 1 is a 30 amino acid peptide derived from
the preproglucagon molecule. GLP-1 enhances glucose secretion and synthesis.
It renders
pancreatic beta-cells 'glucose-competent' and may be useful in the treatment
of noninsulin-
dependent diabetes mellitus.
[00100] GLP-2 - GLP-2 is a 33-amino acid proglucagon-derived peptide. GLP-2
maintains the integrity of the intestinal mucosal epithelium via effects on
gastric motility and
nutrient absorption, crypt cell proliferation and apoptosis, and intestinal
permeability.
[00101] Glucose - an optically active sugar C6H1zO6 that has an aldehydic
carbonyl group. The breakdown of carbohydrates, particularly glucose, is a
major source of
energy for all plant and animal cells. In diabetes, there is a diminished
ability to transport
glucose into the cells of the body. Blood glucose levels are abnormally high
(hyperglycemia). Elevated blood glucose can lead to ketoacidosis, resulting in
coma and
death. Milder hyperglycemia causes long-term complications affecting the eyes,
kidneys,
nerves, and blood vessels.
[00102] Glutathione - a peptide C~oH»N306S that contains one amino acid
residue each of glutamic acid, cysteine, and glycine, that occurs widely in
plant and animal



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
tissues, and that plays an important role in biological oxidation-reduction
processes and as a
coenzyme.
[00103] Growth hormone - growth hormone (GH) is synthesized by the anterior
pituitary gland. Human growth hormone has a molecular mass of 22,005 and
contains 191
amino acid residues with 2 disulfide bridges. The principal biological role of
growth
hormone is the control of postnatal growth. It's affect is mediated largely by
insulin-like
growth factors.
[00104] GRP (Gastrin Releasing Peptide) - The gastrin-releasing peptide
receptor (GRP-R) can cause the proliferation of many, but not all, cells in
which it is
expressed.
[00105] Hb9 - Homeo box-9 is one of a family of proteins that bind DNA in a
sequence-specific manner and are implicated in the control of gene expression
in both
developing and adult tissues.
[00106] HGF - hepatocyte growth factor (also scatter factor or hepatopoietin
A)
has a spectrum of targets including endothelial cells and melanocytes in
addition to epithelial
cells such as hepatocytes. It affects diverse tissues, mediating placental
growth
developmental determining liver and muscle development in the embryo and B-
cell
proliferation and growth.
[00107] HNF3a - hepatocyte nuclear factor 3, alpha. A member of the forkhead
class of transcription factors. Both HNF3A and HNF3B are expressed in tissues
of
endodermal origin, i.e., stomach, intestines, liver, and lung. All members of
the HNF3
family as well as HNF4G and HNF6 are expressed in pancreatic beta cells
[00108] HNF6 - During mouse development, Hnf6 is expressed in the epithelial
cells that are precursors of the exocrine and endocrine pancreatic cells. In
hnf6-null embryos,
the exocrine pancreas appeared to be normal but endocrine cell differentiation
was impaired.
The expression of neurogenin-3, a transcription factor that is essential for
determination of
endocrine cell precursors, was almost abolished. Later in life, the number of
endocrine cells
increased but the architecture of the islets was perturbed, and the beta cells
were deficient in
glucose transporter-2 expression. Adult hnf6-null mice were diabetic. This
suggests that
Hnf6 controls embryonic pancreatic endocrine differentiation at the precursor
stage and
positively regulates the proendocrine gene ngn3.
[00109] HuSA - human serum albumin - see BSA (bovine serum albumin).
21



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00110] IBMX - 3-isobutyl-1-methylxanthine A compound that inhibits cyclic
AMP phosphodiesterase, which causes beta cells to release insulin.
[00111] IGFl - Insulin-like growth factor-I. Both IGF1 and IGF2 have a
striking
structural homology to proinsulin.
[00112] IGF2 - Insulin- like growth factor 2. Both IGF1 and IGF2 have a
striking
structural homology to proinsulin.
[00113] Johe's N2 - a serum free medium formulated for the support of multi-
potential CNS stem cells is supplemented with various growth and
differentiation factors
[00114] KGF - keratinocyte growth factor or FGF-7: a 28 kDa, single chain,
secreted glycoprotein that has a target specificity restricted to epithelium.
Adult cells known
to express FGF-7 include fibroblasts, T cells, smooth muscle cells, and
ovarian theca cells. In
the embryo, KGF is found at many stages of development throughout the
mesenchyrne.
[00115] Ki67 - a cell proliferation marker. This protein of unknown function
is
expressed during G1 of the cell cycle; it has a half life of 60-90 minutes.
[00116] Lactogen - any hormone (as prolactin) that stimulates the production
of
milk.
[00117] Laminin - a glycoprotein that is a component of connective tissue
basement membrane and that promotes cell adhesion.
[00118] Leu-Enkephalin - A natural peptide neurotransmitter. Natural opiate
pentapeptides isolated originally from pig brain. Leu-enkephalin (YGGFL) and
Met-
enkephalin (YGGFM) bind particularly strongly to d -type opiate receptors.
[00119] Linoleic acid - a liquid unsaturated fatty acid C~gH3202 found
especially
in semidrying oils (as peanut oil) and essential for the nutrition of some
animals -- called also
linolic acid.
[00120] Linolenic acid - a liquid unsaturated fatty acid C~gH3o0z found
especially
in drying oils (as linseed oil) and essential for the nutrition of some
animals.
[00121] Met-Enkephalin - A natural peptide neurotransmitter. Natural opiate
pentapeptides isolated originally from pig brain. Leu-enkephalin (YGGFL) and
Met-
enkephalin (YGGFM) bind particularly strongly to d-type opiate receptors.
[00122] Muc 1 - mucin type 1, the main type of mucoprotein normally secreted
by
special pancreatic duct cells.
22



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00123] Myoinositol - a biologically active inositol that is not optically
active, that
is a component of the vitamin B complex and a lipotropic agent, and that
occurs widely in
plants, microorganisms, and higher animals including humans -- called also
mesoinositol
[00124] N2 - Johe's N2 medium.
[00125] Neuro - neurobasal medium, a neural cell culture medium.
[00126] NGF - Nerve growth factor is a 12.5 kDa, nonglycosylated polypeptide
120 as residues long. It is synthesized as a prepropeptide; its processed form
is a 120 as
segment. The typical form for NGF is a 25 kDa, non-disulfide linked homodimer.
Nerve
growth factor is known to regulate growth and differentiation of sympathetic
and certain
sensory neurons.
[00127] Nicotinamide - niacinamide (nicotinic acid amide) a bitter crystalline
basic amide C6H6NZ0 that is a member of the vitamin B complex and is formed
from and
converted to niacin in the living organism, that occurs naturally usually as a
constituent of
coenzymes, and that is used similarly to niacin.
[00128] PCNA+ cells - cells that label with an anti proliferating cell nuclear
antigen. Proliferating cell nuclear antigen was originally correlated with the
proliferative
state of the cell. More recent evidence shows that PCNA may also be correlated
with DNA
repair.
[00129] PDGF - platelet derived growth factor. A factor released from
platelets
upon clotting was shown to be capable of promoting the growth of various types
of cells.
This factor was subsequently purified from platelets and given the name
platelet-derived
growth factor (PDGF). PDGF is now known to be produced by a number of cell
types
besides platelets and it has been found to be a mitogen for almost all
mesenchymally-derived
cells, i.e., blood, muscle, bone/cartilage, and connective tissue cells.
[00130] pdx-1 - Pancreatic duodenal homeobox factor-1, PDX-1, is required for
pancreas development, islet cell differentiation, and the maintenance of beta
cell function.
Also called insulin promoter factor-1 (IPF1) or IDX1 or somatostatin
transcription factor-1
(STF1). PDX-1 appears to serve as a master control switch for expression of
both the
exocrine and endocrine pancreatic developmental programs, as revealed by gene
disruption
studies in which targeted deletion of the gene leads to a'null pancreas
phenotype. PXDX-1
is initially expressed in both exocrine and endocrine cells; as pancreatic
morphogenesis
proceeds, it restricted to some duct cells and beta and delta cells of the
islets. PDX-1 also
plays a role in adult cells, regulating the insulin and somatostatin genes.
Mutations in the
23



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
PDXl gene can cause pancreatic agenesis, maturity-onset diabetes of the young,
and possibly
type II diabetes.
[00131] Placental lactogen - This peptide hormone is structurally,
immunologically, and functionally similar to pituitary growth hormone It is
synthesized by
the placental syncytiotrophoblast.
[00132] Progesterone - a female steroid sex hormone CZ~H3o0z that is secreted
by
the corpus luteum to prepare the endometrium for implantation and later by the
placenta
during pregnancy to prevent rejection of the developing embryo or fetus and
that is used in
synthetic forms as a birth control pill, to treat menstrual disorders, and to
alleviate some
cases of infertility.
[00133] Proinsulin - the precursor of insulin. Insulin is derived from a
folded,
one-chain precursor that is linked by 2 disulfide bonds. Proinsulin is
converted to insulin by
the enzymatic removal of a segment that connects the amino end of the A chain
to the
carboxyl end of the B chain.
[00134] Prolactin - A growth factor with strong structural similarity to
growth
hormone.
[00135] PTFl - see PDX-1
[00136] PTHRP - parathyroid related protein.
[00137] Putrescine - a crystalline slightly poisonous ptomaine C4H12N2 that is
formed by decarboxylation of ornithine, occurs widely but in small amounts in
living things,
and is found especially in putrid flesh.
[00138] Regl - regenerating islet-derived protein Laos known as pancreatic
stone
protein
[00139] Retinoic Acid (Vitamin A) - a local regulator of cellular
differentiation.
It has many functions in the developing limb, regulates key events in limb
regeneration in
lower vertebrates.
[00140] Retinyl acetate - a derivative of vitamin A.
[00141] Selenium (Selenious Acid) - a nonmetallic element that resembles
sulfur
and tellurium chemically, causes poisoning in range animals when ingested by
eating some
plants growing in soils in which it occurs in quantity, and occurs in
allotropic forms of which
a gray stable form varies in electrical conductivity with the intensity of its
illumination and is
used in electronic devices.
24



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00142] Sonic Hedgehog (mouse, recombinant) - plays important roles in the
development of many cell types including the brain, bone, skin, gonads, and
lungs.
[00143] Soybean Trypsin Inhibitor (type I-S) - A high-molecular-weight protein
(approximately 22,500) containing 198 amino acid residues. Soybean trypsin
inhibitor
suppress proteolytic but not elastolytic activity.
[00144] Substance P - Substance P is the predominant neuropeptide released at
primary afferent-second order neuron synapses upon high-intensity stimulation
of
nociceptive afferents. Via activation of NK1 receptors (see table in chapter
nociception)
substance P produces slow, long-lasting depolarizations of second order
neurons . This leads
to potentiation of the post-synaptic response to nociceptor stimulation and
thereby functions
as an intensity-coding mechanism for nociceptive transmission.
[00145] Superoxide Dismutase (SOD) - a metal-containing antioxidant enzyme
that reduces potentially harmful free radicals of oxygen formed during normal
metabolic cell
processes to oxygen and hydrogen peroxide.
[00146] TGF alpha and beta - Transforming growth factors (TGFs) are
biologically active polypeptides that reversibly confer the transformed
phenotype on cultured
cells. Alpha-TGF shows about 40% sequence homology with epidermal growth
factor. TGF
beta is a multifunctional peptide that controls proliferation,
differentiation, and other
functions in many cell types. TGFB acts synergistically with TGFA in inducing
transformation. It also acts as a negative autocrine growth factor.
Dysregulation of TGFB
activation and signaling may result in apoptosis. Many cells synthesize TGFB
and almost all
of them have specific receptors for this peptide. TGFB1, TGFB2, and TGFB3 all
function
through the same receptor signaling systems.
[00147] TGF beta sRII (soluble receptor type 2) - TGF-beta regulates growth
and proliferation of cells, blocking growth of many cell types. The TGF-beta
receptor
includes type 1 and type 2 subunits that are serine-threonine kinases and that
signal through
the SMAD family of transcriptional regulators. Defects in TGF-beta signaling,
includes
mutation in SMADs, have been associated with cancer in humans.
[00148] Transcription Factors (TF) - Transcription factors bind to specific
regulatory sequences in DNA and modulate the activity of RNA polymerase. This
is the key
step that regulates the process whereby genes coded in DNA are copied or
transcribed into
messenger RNA. Normally, the interactions of many different transcription
factors
determine the specific phenotype of different cell types. TF's can be positive
or negative



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
regulators of gene expression. PDX1, neurogenin 3 (ngn3), Pax4, Pax6, and
others are
examples of those TF's that are involved in pancreatic development and
differentiation.
(00149] Transferrin - a beta globulin in blood plasma capable of combining
with
ferric ions and transporting iron in the body.
[00150] Triiodothyronine - a crystalline iodine-containing hormone C~SH~2I3N04
that is an amino acid derived from thyroxine and is used especially in the
form of its soluble
sodium salt in the treatment of hypothyroidism and metabolic insufficiency --
called also
liothyronine, T3.
[00151] Triiodothyronine (T3) - a crystalline iodine-containing hormone
C~SHIZI3N04 that is an amino acid derived from thyroxine and is used
especially in the form
of its soluble sodium salt in the treatment of hypothyroidism and metabolic
insufficiency.
[00152] Trolox (soluble Vitamin E) - A cell-permeable, water-soluble
derivative
of vitamin E with potent antioxidant properties. Prevents peroxynitrite-
mediated oxidative
stress and apoptosis in rat thymocytes.
[00153] Vasoactive Intestinal Peptide (VIP) - A test that measures the amount
of
VIP in serum.
(00154] VEGF - vascular endothelial growth factor - VEGF is a heparin-
binding glycoprotein that is secreted as a homodimer of 45 kDa. One of the
most important
growth and survival factors for endothelium. It is structurally related to
platelet-derived
growth factor. VEGF induces angiogenesis and endothelial cell proliferation
and it plays an
important role in regulating vasculogenesis. Most types of cells, but usually
not endothelial
cells themselves, secrete VEGF.
[00155] Zinc sulphate - Zinc is an important trace mineral and is required for
the
enzyme activities necessary for cell division, cell growth, and wound healing.
Zinc is also
involved in the metabolism of carbohydrates. Beta cells of the pancreas have a
high zinc
content.
Summar5r of the Invention
[00156] In one embodiment, the invention is drawn to a method of converting
differentiated non-hormone producing pancreatic cells into differentiated
hormone-producing
cells, including the steps of: a) culturing the differentiated non-hormone
producing
pancreatic cells in a first cell culture system with a first cell culture
medium including a basal
medium, with or without serum, and with or without growth factors, under
conditions which
26



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
provide for converting the differentiated non-hormone producing pancreatic
cells into stem
cells; and b) culturing the stem cells in a second cell culture system with a
second cell
culture medium, including at least one compound selected from Group A and at
least one
compound selected from Group B, where Group A includes the following
compounds:
Betacellulin, Activin A, BMP-2, TGF-(3 SRII, DMSO, Sonic Hedgehog, Laminin,
Met-
Enkephalin, DMF, and Cholera Toxin A; and where Group B includes the following
compounds: Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone
Morphogenic Protein
(BMP-2), Bone Morphogenic Protein (BMP-4), C natriuretic peptide (CNP),
Caerulein,
Calcitonin Gene Related Peptide (CGRP-a), Cholecystokinin (CCKB-amide),
Cholecystokinin octapeptide (CCKB-sulfated), Cholera Toxin B Subunit,
Corticosterone
(Reichstein's substance H), Dexamethasone, DIF-1, Differanisole A,
Dimethylsulfoxide
(DMSO), EGF, Endothelin 1, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I,
Gastrin
Releasing Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1), Glucose, Growth
Hormone,
Hepatocyte Growth Factor (HGF), IGF-1, IGF-2, Insulin, KGF, Lactogen, Laminin,
Leu-
Enkephalin, Leukemia Inhibitory Factor (LIF), Met-Enkephalin, n Butyric Acid,
Nerve
Growth Factor ((3-NGF), Nicotinamide, n-n-dimethylformamide (DMF), Parathyroid
Hormone Related Peptide (Pth II RP), PDGF AA + PDGF BB MIX, PIGF (Placental
GF),
Progesterone, Prolactin, Putrescine Dihydrochloride Gamma-Irradiated Cell
Culture, REG1,
Retinoic Acid, Selenium, Selenious Acid, Sonic Hedgehog, Soybean Trypsin
Inhibitor,
Substance P, Superoxide Dismutase (SOD), TGF-a, TGF-~3 sRII, TGF-X31,
transferrin,
Triiodothyronine (T3), Trolox, Vasoactive Intestinal Peptide (VIP), VEGF,
Vitamin A, and
Vitamin E, under conditions which provide for differentiating the stem cells
into hormone-
producing cells.
[00157] In a preferred embodiment, the second cell culture medium includes at
least two compounds selected from Group A and at least two compounds selected
from
Group B.
[00158] In a more preferred embodiment, the second cell culture medium
includes
at least three compounds selected from Group A and at least three compounds
selected from
Group B.
[00159] In a yet more preferred embodiment, the second cell culture medium
includes at least four compounds selected from Group A and at least four
compounds
selected from Group B.
27



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00160] In a yet more preferred embodiment, the second cell culture medium
includes at least five compounds selected from Group A and also at least five
compounds
selected from Group B.
[00161] In a yet more preferred embodiment, the second cell culture medium
includes at least six compounds selected from Group A and at least six
compounds selected
from Group B.
[00162] In one embodiment, the invention is drawn to a method of culturing
stem
cells into differentiated hormone-producing cells, including culturing the
stem cells in a cell
culture system with a cell culture medium where the stem cells are
differentiated into
hormone-producing cells and where the culture medium includes basal medium
without
serum and also includes at least one compound selected from Group A and at
least one
compound selected from Group B, where Group A includes the following
compounds:
Betacellulin, Activin A, BMP-2, TGF-(3 SRII, DMSO, Sonic Hedgehog, Laminin,
Met-
Enkephalin, DMF, and Cholera Toxin A; and Group B includes the following
compounds:
Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone Morphogenic Protein
(BMP-2),
Bone Morphogenic Protein (BMP-4), C natriuretic peptide (CNP), Caerulein,
Calcitonin
Gene Related Peptide (CGRP-a), Cholecystokinin (CCKB-amide), Cholecystokinin
octapeptide (CCKB-sulfated), Cholera Toxin B Subunit, Corticosterone
(Reichstein's
substance H), Dexamethasone, DIF-l, Differanisole A, Dimethylsulfoxide (DMSO),
EGF,
Endothelin 1, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I, Gastrin
Releasing
Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1), Glucose, Growth Hormone,
Hepatocyte
Growth Factor (HGF), IGF-1, IGF-2, Insulin, KGF, Lactogen, Laminin, Leu-
Enkephalin,
Leukemia Inhibitory Factor (LIF), Met-Enkephalin, n Butyric Acid, Nerve Growth
Factor ((3-
NGF), Nicotinamide, n-n-dimethylformamide (DMF), Parathyroid Hormone Related
Peptide
(Pth II RP), PDGF AA + PDGF BB MIX, PIGF (Placental GF), Progesterone,
Prolactin,
Putrescine Dihydrochloride Gamma-Irradiated Cell Culture, REGl, Retinoic Acid,
Selenium,
Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor, Substance P,
Superoxide
Dismutase (SOD), TGF-a, TGF-~3 sRII, TGF-(31, transferrin, Triiodothyronine
(T3), Trolox,
Vasoactive Intestinal Peptide (VIP), VEGF, Vitamin A, and Vitamin E.
[00163] In a preferred embodiment, the cell culture medium includes at least
two
compounds selected from Group A and at least two compounds selected from Group
B.
28



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00164] In a yet more preferred embodiment, the cell culture medium includes
at
least three compounds selected from Group A and at least three compounds
selected from
Group B.
[00165] In a yet more preferred embodiment, the cell culture medium includes
at
least four compounds selected from Group A and at least four compounds
selected from
Group B.
[00166] In a yet more preferred embodiment, the cell culture medium includes
at
least five compounds selected from Group A and at least five compounds
selected from
Group B.
[00167] In a yet more preferred embodiment, the cell culture medium includes
at
least six compounds selected from Group A and at least six compounds selected
from Group
B.
Brief Description of the Drawings
[00168] Fig.l. Insulin release from cells cultured in alginate in the presence
of
growth and differentiation factors in a combinatorial array. Donors #2212,
#2278, and #3023.
[00169] Fig. 2. Stimulation index of insulin release from cells cultured in
alginate
in the presence of growth and differentiation factors in a combinatorial
array. Donors #2212,
#2278, and #3023.
[00170] Fig. 3. Insulin release from cells cultured in alginate in the top
eight
growth and differentiation factor combinations (A-H): Donor #2212
[00171] Fig. 4. Stimulation indices of insulin release from cells cultured in
alginate
in the top eight growth and differentiation factor combinations (A-H): Donor
#2212.
[00172] Fig. 5. Insulin release from cells cultured in alginate in the top
eight
growth and differentiation factor combinations (A-H): Donor #2278
[00173] Fig. 6. Stimulation indices of insulin release from cells cultured in
alginate
in the top eight growth and differentiation factor combinations (A-H): Donor
#2278
[00174] Fig. 7. Insulin release from cells cultured in alginate in the top
eight
growth and differentiation factor combinations (A-H): Donor #3023
[00175] Fig. 8. Stimulation indices of insulin release from cells cultured in
alginate
in the top eight growth and differentiation factor combinations (A-H): Donor
#3023
[00176] Fig. 9. Insulin release from cells cultured in alginate in the top
eight
growth and differentiation factor combinations (A-H): Donor #3036
29



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00177] Fig. 10. Stimulation indices of insulin release from cells cultured in
alginate in the top eight growth and differentiation factor combinations (A-
H): Donor #3036
[00178] Fig. 11. Stimulation indices of c-peptide release from cells cultured
in
alginate in the top eight growth and differentiation factor combinations (A-
H): Donor #3036
[00179] Fig. 12. C-peptide release from cells cultured in adherent culture in
the top
four growth and differentiation factor combinations (I-L)
[00180] Fig. 13. Numbers of proinsulin positive cells per well of cells
cultured in
adherent culture in the top six growth and differentiation factor combinations
determined in a
second tier 30 factor screen.
Detailed Description Of The Invention and Preferred Embodiment
[00181] In one embodiment, the invention is drawn to a method for producing a
hormone producing cell from a differentiated cell type that does not produce a
hormone.
Preferably, the differentiated cell type is a pancreatic cell. Preferably, the
cells are islet-
depleted pancreatic cells. More preferably, the differentiated cell type is a
non-hormone
producing pancreatic cells cell.
[00182] The hormone-producing cell produced in one aspect of the present
invention preferably produces one or more of the hormones produced by islet
cell. More
preferably, the hormone-producing cell produces insulin.
[00183] Accordingly, a preferred aspect of the invention are methods and
compositions for the large scale expansion of non-hormone producing pancreatic
cells and
the large scale transformation of non-hormone producing pancreatic cells into
hormone-
producing cells. Preferably, the hormone produced is insulin but other
hormones are also
encompassed within the invention, particularly hormones from islet cells.
[00184) In another preferred embodiment, the invention provides compositions
useful for the method of converting pancreatic non-hormone producing
pancreatic cells into
hormone-producing cells.
[00185] Tables 5 and 6 list factors which may be added to the culture media
which
include potential growth factors and potential differentiation factors. For
purposes of this
disclosure, the terms "factor", "component" and "supplement" may be used
interchangeably.
[00186] These components, factors and supplements include but are not limited
to
Activin A, Atrial Natriuretic Peptide, Betacellulin, Bone Morphogenic Protein
(BMP-2),



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Bone Morphogenic Protein (BMP-4), C natriuretic peptide (CNP), Caerulein,
Calcitonin
Gene Related Peptide (CGRP-a), Cholecystokinin (CCKB-amide), Cholecystokinin
octapeptide (CCKB-sulfated), Cholera Toxin B Subunit, Corticosterone
(Reichstein's
substance H), Dexamethasone, DIF-1, Differanisole A, Dimethylsulfoxide (DMSO),
EGF,
Endothelin l, Exendin 4, FGF acidic, FGF2, FGF7, FGFb, Gastrin I, Gastrin
Releasing
Peptide (GRP), Glucagon-Like Peptide 1 (GLP-1), Glucose, Growth Hormone,
Hepatocyte
Growth Factor (HGF), IGF-1, IGF-2, Insulin, KGF, Lactogen, Laminin, Leu-
Enkephalin,
Leukemia Inhibitory Factor (LIF), Met-Enkephalin, n-Butyric Acid, Nerve Growth
Factor
((3-NGF), Nicotinamide, n-n-dimethylformamide (DMF), Parathyroid Hormone
Related
Peptide (Pth II RP), PDGF AA + PDGF BB MIX, PIGF (Placental GF), Progesterone,
Prolactin, Putrescine Dihydrochloride Gamma-Irradiated Cell Culture, REG1,
Retinoic Acid,
Selenium, Selenious Acid, Sonic Hedgehog, Soybean Trypsin Inhibitor, Substance
P,
Superoxide Dismutase (SOD), TGF-a, TGF-[i sRII, TGF-(31, transferrin,
Triiodothyronine
(T3), Trolox, Vasoactive Intestinal Peptide (VIP), VEGF, Vitamin A, and
Vitamin E.
[00187] For Activin A, a preferred concentration is 0.125 - 1.5 ng/ml; yet
more
preferred concentration is 0.25 - 1 ng/ml; yet more preferred concentration is
0.375 - 0.75
ng/ml; yet more preferred concentration is 0.45 - 0.6 ng/ml; and most
preferred concentration
is 0.5 ng/ml.
[00188] For Atrial Natriuretic Peptide, a preferred concentration is 38.25 -
459
ng/ml; yet more preferred concentration is 76.5 - 306 ng/ml; yet more
preferred concentration
is 114.75 - 229.5 ng/ml; yet more preferred concentration is 137.7 - 183.6
ng/ml; and most
preferred concentration is 153 ng/ml.
[00189] For Betacellulin, a preferred concentration is 1.25 - 15 ng/ml; yet
more
preferred concentration is 2.5 - 10 ng/ml; yet more preferred concentration is
3.75 - 7.5
ng/ml; yet more preferred concentration is 4.5 - 6 ng/ml; and most preferred
concentration is
ng/ml.
[00190] For Bone Morphogenic Protein (BMP-2), a preferred concentration is
1.25
- 15 ng/ml; yet more preferred concentration is 2.5 - 10 ng/ml; yet more
preferred
concentration is 3.75 - 7.5 ng/ml; yet more preferred concentration is 4.5 - 6
ng/ml; and most
preferred concentration is S ng/ml.
(00191] For Bone Morphogenic Protein (BMP-4), a preferred concentration is
0.125 - 1.5 ng/ml; yet more preferred concentration is 0.25 - 1 ng/ml; yet
more preferred
31



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
concentration is 0.375 - 0.75 ng/ml; yet more preferred concentration is 0.45 -
0.6 ng/ml; and
most preferred concentration is 0.5 ng/ml.
[00192] For C natriuretic peptide (CNP), a preferred concentration is 27.4625 -

329.55 ng/ml; yet more preferred concentration is 54.925 - 219.7 ng/ml; yet
more preferred
concentration is 82.3875 - 164.775 ng/ml; yet more preferred concentration is
98.865 -
131.82 ng/ml; and most preferred concentration is 109.85 ng/ml.
[00193] For Caerulein, a preferred concentration is 7.5 - 90 ng/ml; yet more
preferred concentration is 15 - 60 ng/ml; yet more preferred concentration is
22.5 - 45 ng/ml;
yet more preferred concentration is 27 - 36 ng/ml; and most preferred
concentration is 30
ng/ml.
[00194] For Calcitonin Gene Related Peptide (CGRP-D), a preferred
concentration
is 47.625 - 571.5 ng/ml; yet more preferred concentration is 95.25 - 381
ng/ml; yet more
preferred concentration is 142.875 - 285.75 ng/ml; yet more preferred
concentration is
171.45 - 228.6 ng/ml; and most preferred concentration is 190.5 ng/ml.
[00195] For Cholecystokinin (CCK8-amide), a preferred concentration is 6.25 -
75
ng/ml; yet more preferred concentration is 12.5 - SO ng/ml; yet more preferred
concentration
is 18.75 - 37.5 ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml;
and most preferred
concentration is 25 ng/ml.
[00196] For Cholecystokinin octapeptide (CCKB-sulfated), a preferred
concentration is 1.425 - 17.1 ng/ml; yet more preferred concentration is 2.85 -
11.4 ng/ml;
yet more preferred concentration is 4.275 - 8.55 ng/ml; yet more preferred
concentration is
5.13 - 6.84 ng/ml; and most preferred concentration is 5.7 ng/ml.
[00197] For Cholecystokinin octapeptide (CCKB-sulfated), a preferred
concentration is 3.125 - 37.5 ng/ml; yet more preferred concentration is 6.25 -
25 ng/ml; yet
more preferred concentration is 9.375 - 18.75 ng/ml; yet more preferred
concentration is
11.25 - 1 S ng/ml; and most preferred concentration is 12.5 ng/ml.
[00198] For Corticosterone (Reichstein's substance H), a preferred
concentration is
0.5 - 6 ng/ml; yet more preferred concentration is 1 - 4 ng/ml; yet more
preferred
concentration is 1.5 - 3 ng/ml; yet more preferred concentration is 1.8 - 2.4
ng/ml; and most
preferred concentration is 2 ng/ml.
[00199] For Dexamethasone, a preferred concentration is 0.5 - 6 ng/ml; yet
more
preferred concentration is 1 - 4 ng/ml; yet more preferred concentration is
1.5 - 3 ng/ml; yet
more preferred concentration is 1.8 - 2.4 ng/ml; and most preferred
concentration is 2 ng/ml.
32



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00200] For DIF-1, a preferred concentration is 75 - 900 ng/ml; yet more
preferred
concentration is 150 - 600 ng/ml; yet more preferred concentration is 225 -
450 ng/ml; yet
more preferred concentration is 270 - 360 ng/ml; and most preferred
concentration is 300
ng/ml.
[00201] For Differanisole A, a preferred concentration is 75 - 900 ng/ml; yet
more
preferred concentration is 150 - 600 ng/ml; yet more preferred concentration
is 225 - 450
ng/ml; yet more preferred concentration is 270 - 360 ng/ml; and most preferred
concentration
is 300 ng/ml.
[00202] For Dimethylsulfoxide (DMSO), a preferred concentration is 0.25 - 3
ng/ml; yet more preferred concentration is 0.5 - 2 ng/ml; yet more preferred
concentration is
0.75 - 1.5 ng/ml; yet more preferred concentration is 0.9 - 1.2 ng/ml; and
most preferred
concentration is 1 ng/ml.
[00203] For EGF, a preferred concentration is 1.25 - 15 ng/ml; yet more
preferred
concentration is 2.5 - 10 ng/ml; yet more preferred concentration is 3.75 -
7.5 ng/ml; yet
more preferred concentration is 4.5 - 6 ng/ml; and most preferred
concentration is 5 ng/ml.
[00204] For Endothelin 1, a preferred concentration is 125 - 1500 ng/ml; yet
more
preferred concentration is 250 - 1000 ng/ml; yet more preferred concentration
is 375 - 750
ng/ml; yet more preferred concentration is 450 - 600 ng/ml; and most preferred
concentration
is 500 ng/ml.
[00205] For Exendin 4, a preferred concentration is 5.25 - 63 ng/ml; yet more
preferred concentration is 10.5 - 42 ng/ml; yet more preferred concentration
is 15.75 - 31.5
ng/ml; yet more preferred concentration is 18.9 - 25.2 ng/ml; and most
preferred
concentration is 21 ng/ml.
[00206] For FGF acidic, a preferred concentration is 0.625 - 7.5 ng/ml; yet
more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00207] For FGF2, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00208] For FGF7, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
33



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00209] For FGFb, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00210] For Gastrin I, a preferred concentration is 0.008038 - 0.09645 ng/ml;
yet
more preferred concentration is 0.016075 - 0.0643 ng/ml; yet more preferred
concentration is
0.024113 - 0.048225 ng/ml; yet more preferred concentration is 0.028935 -
0.03858 ng/ml;
and most preferred concentration is 0.03215 ng/ml.
[00211] For Gastrin Releasing Peptide (GRP), a preferred concentration is
35.75 -
429 ng/ml; yet more preferred concentration is 71.5 - 286 ng/ml; yet more
preferred
concentration is 107.25 - 214.5 ng/ml; yet more preferred concentration is
128.7 - 171.6
ng/ml; and most preferred concentration is 143 ng/ml.
[00212] For Glucagon-Like Peptide 1 (GLP-1), a preferred concentration is 8.25
-
99 ng/ml; yet more preferred concentration is 16.5 - 66 ng/ml; yet more
preferred
concentration is 24.75 - 49.5 ng/ml; yet more preferred concentration is 29.7 -
39.6 ng/ml;
and most preferred concentration is 33 ng/ml.
[00213] For Glucose, a preferred concentration is 270 - 3240 ng/ml; yet more
preferred concentration is 540 - 2160 ng/ml; yet more preferred concentration
is 810 - 1620
ng/ml; yet more preferred concentration is 972 - 1296 ng/ml; and most
preferred
concentration is 1080 ng/ml.
[00214] For Growth Hormone, a preferred concentration is 6.25 - 75 ng/ml; yet
more preferred concentration is 12.5 - 50 ng/ml; yet more preferred
concentration is 18.75 -
37.5 ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml; and most
preferred
concentration is 25 ng/ml.
[00215] For Hepatocyte Growth Factor (HGF), a preferred concentration is 0.625
-
7.5 ng/ml; yet more preferred concentration is 1.25 - 5 ng/ml; yet more
preferred
concentration is 1.875 - 3.75 ng/ml; yet more preferred concentration is 2.25 -
3 ng/ml; and
most preferred concentration is 2.5 ng/ml.
[00216] For IGF-1, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
34



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00217] For IGF-2, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
(00218] For Insulin, a preferred concentration is 2375 - 28500 ng/ml; yet more
preferred concentration is 4750 - 19000 ng/ml; yet more preferred
concentration is 7125 -
14250 ng/ml; yet more preferred concentration is 8550 - 11400 ng/ml; and most
preferred
concentration is 9500 ng/ml.
[00219] For KGF, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
(00220] For Lactogen, a preferred concentration is 12.5 - 150 ng/ml; yet more
preferred concentration is 25 - 100 ng/ml; yet more preferred concentration is
37.5 - 75
ng/ml; yet more preferred concentration is 45 - 60 ng/ml; and most preferred
concentration is
50 ng/ml.
(00221] For Laminin, a preferred concentration is 562.5 - 6750 ng/ml; yet more
preferred concentration is 1125 - 4500 ng/ml; yet more preferred concentration
is 1687.5 -
3375 ng/ml; yet more prefer ed concentration is 2025 - 2700 ng/ml; and most
preferred
concentration is 2250 ng/ml.
[00222] For Leu-Enkephalin, a preferred concentration is 0.75 - 9 ng/ml; yet
more
preferred concentration is 1.5 - 6 ng/ml; yet more preferred concentration is
2.25 - 4.5 ng/ml;
yet more preferred concentration is 2.7 - 3.6 ng/ml; and most preferred
concentration is 3
ng/ml.
[00223] For Leukemia Inhibitory Factor (LIF), a preferred concentration is
0.625 -
7.5 ng/ml; yet more preferred concentration is 1.25 - 5 ng/ml; yet more
preferred
concentration is 1.875 - 3.75 ng/ml; yet more preferred concentration is 2.25 -
3 ng/ml; and
most preferred concentration is 2.5 ng/ml.
[00224] For Met-Enkephalin, a preferred concentration is 0.75 - 9 ng/ml; yet
more
preferred concentration is 1.5 - 6 ng/ml; yet more preferred concentration is
2.25 - 4.5 ng/ml;



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
yet more preferred concentration is 2.7 - 3.6 ng/ml; and most preferred
concentration is 3
ng/ml.
[00225] For n-Butyric Acid, a preferred concentration is 1135 - 13620 ng/ml;
yet
more preferred concentration is 2270 - 9080 ng/ml; yet more preferred
concentration is 3405
- 6810 ng/ml; yet more preferred concentration is 4086 - 5448 ng/ml; and most
preferred
concentration is 4540 ng/ml.
[00226] For Nerve Growth Factor (~-NGF), a preferred concentration is 0.625 -
7.5 ng/ml; yet more preferred concentration is 1.25 - 5 ng/ml; yet more
preferred
concentration is 1.875 - 3.75 ng/ml; yet more preferred concentration is 2.25 -
3 ng/ml; and
most preferred concentration is 2.5 ng/ml.
[00227] For Nicotinamide, a preferred concentration is 152500 - 1830000 ng/ml;
yet more preferred concentration is 305000 - 1220000 ng/ml; yet more preferred
concentration is 457500 - 915000 ng/ml; yet more preferred concentration is
549000 -
732000 ng/ml; and most preferred concentration is 610000 ng/ml.
[00228] For n-n-dimethylformamide (DMF), a preferred concentration is 0.25 - 3
X 10-~ percent; yet more preferred concentration is 0.5 - 2 X 10-6 percent;
yet more preferred
concentration is 0.75 - 1.5 X 10-6 percent; yet more preferred concentration
is 0.9 - 1.2 X 10-6
percent; and most preferred concentration is 1 X 10-6 percent.
[00229] For Parathyroid Hormone Related Peptide (Pth II RP), a preferred
concentration is 51.5 - 618 ng/ml; yet more preferred concentration is 103 -
412 ng/ml; yet
more preferred concentration is 154.5 - 309 ng/ml; yet more preferred
concentration is 185.4
- 247.2 ng/ml; and most preferred concentration is 206 ng/ml.
[00230] For PDGF AA + PDGF BB mix, a preferred concentration is 1.25 - 15
ng/ml; yet more preferred concentration is 2.5 - 10 ng/ml; yet more preferred
concentration is
3.75 - 7.5 ng/ml; yet more preferred concentration is 4.5 - 6 ng/ml; and most
preferred
concentration is S ng/ml.
[00231] For PIGF (Placental GF), a preferred concentration is 0.625 - 7.5
ng/ml;
yet more preferred concentration is 1.25 - 5 ng/ml; yet more preferred
concentration is 1.875
- 3.75 ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most
preferred
concentration is 2.5 ng/ml.
[00232] For Progesterone, a preferred concentration is 0.75 - 9 ng/ml; yet
more
preferred concentration is 1.5 - 6 ng/ml; yet more preferred concentration is
2.25 - 4.5 ng/ml;
36



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
yet more preferred concentration is 2.7 - 3.6 ng/ml; and most preferred
concentration is 3
ng/ml.
[00233) For Prolactin, a preferred concentration is 0.3 - 3.6 ng/ml; yet more
preferred concentration is 0.6 - 2.4 ng/ml; yet more preferred concentration
is 0.9 - 1.8 ng/ml;
yet more preferred concentration is 1.08 - 1.44 ng/ml; and most preferred
concentration is 1.2
ng/ml.
[00234] For Putrescine Dihydrochloride Gamma-Irradiated Cell Culture, a
preferred concentration is 0.025 - 0.3 ng/ml; yet more preferred concentration
is 0.05 - 0.2,
ng/ml; yet more preferred concentration is 0.075 - 0.15 ng/ml; yet more
preferred
concentration is 0.09 - 0.12 ng/ml; and most preferred concentration is 0.1
ng/ml.
[00235] For REG1, a preferred concentration is 8.1375 - 97.65 ng/ml; yet more
preferred concentration is 16.275 - 65.1 ng/ml; yet more preferred
concentration is 24.4125 -
48.825 ng/ml; yet more preferred concentration is 29.295 - 39.06 ng/ml; and
most preferred
concentration is 32.55 ng/ml.
[00236] For Retinoic Acid, a preferred concentration is 6.25 - 75 ng/ml; yet
more
preferred concentration is 12.5 - 50 ng/ml; yet more preferred concentration
is 18.75 - 37.5
ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml; and most preferred
concentration
is 25 ng/ml.
[00237] For Selenium (Selenious Acid), a preferred concentration is 6.25 - 75
ng/ml; yet more preferred concentration is 12.5 - 50 ng/ml; yet more preferred
concentration
is 18.75 - 37.5 ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml;
and most preferred
concentration is 25 ng/ml.
[00238] For Sonic Hedgehog, a preferred concentration is 6.25 - 75 ng/ml; yet
more preferred concentration is 12.5 - 50 ng/ml; yet more preferred
concentration is 18.75 -
37.5 ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml; and most
preferred
concentration is 25 ng/ml.
[00239] For Soybean Trypsin Inhibitor, a preferred concentration is 250 - 3000
ng/ml; yet more preferred concentration is 500 - 2000 ng/ml; yet more
preferred
concentration is 750 - 1500 ng/ml; yet more preferred concentration is 900 -
1200 ng/ml; and
most preferred concentration is 1000 ng/ml.
[00240] For Substance P, a preferred concentration is 1250 - 15000 ng/ml; yet
more preferred concentration is 2500 - 10000 ng/ml; yet more preferred
concentration is
37



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
3750 - 7500 ng/ml; yet more preferred concentration is 4500 - 6000 ng/ml; and
most
preferred concentration is 5000 ng/ml.
[00241] For Superoxide Dismutase (SOD), a preferred concentration is 2.5 - 30
IU/ml; yet more preferred concentration is 5 - 20 IU/ml; yet more preferred
concentration is
7.5 - 15 ILJ/ml; yet more preferred concentration is 9 - 12 ILJ/ml; and most
preferred
concentration is 10 IU/ml.
[00242] For TGF-a, a preferred concentration is 0.25 - 3 ng/ml; yet more
preferred
concentration is 0.5 - 2 ng/ml; yet more preferred concentration is 0.75 - 1.5
ng/ml; yet more
preferred concentration is 0.9 - 1.2 ng/ml; and most preferred concentration
is 1 ng/ml.
[00243) For TGF-/3 sRII, a preferred concentration is 1.25 - 15 ng/ml; yet
more
preferred concentration is 2.5 - 10 ng/ml; yet more preferred concentration is
3.75 - 7.5
ng/ml; yet more preferred concentration is 4.5 - 6 ng/ml; and most preferred
concentration is
ng/ml.
[00244] For TGF-(31, a preferred concentration is 0.125 - 1.5 ng/ml; yet more
preferred concentration is 0.25 - 1 ng/ml; yet more preferred concentration is
0.375 - 0.75
ng/ml; yet more preferred concentration is 0.45 - 0.6 ng/ml; and most
preferred concentration
is 0.5 ng/ml.
[00245] For transferrin, a preferred concentration is 687.5 - 8250 ng/ml; yet
more
preferred concentration is 1375 - 5500 ng/ml; yet more preferred concentration
is 2062.5 -
4125 ng/ml; yet more preferred concentration is 2475 - 3300 ng/ml; and most
preferred
concentration is 2750 ng/ml.
[00246] For Triiodothyronine (T3), a preferred concentration is 8.375 - 100.5
ng/ml; yet more preferred concentration is 16.75 - 67 ng/ml; yet more
preferred concentration
is 25.125 - 50.25 ng/ml; yet more preferred concentration is 30.15 - 40.2
ng/ml; and most
preferred concentration is 33.5 ng/ml.
[00247] For Trolox, a preferred concentration is 156.25 - 1875 ng/ml; yet more
preferred concentration is 312.5 - 1250 ng/ml; yet more preferred
concentration is 468.75 -
937.5 ng/ml; yet more preferred concentration is 562.5 - 750 ng/ml; and most
preferred
concentration is 625 ng/ml.
[00248] For Vasoactive Intestinal Peptide (VIP), a preferred concentration is
16.625 - 199.5 ng/ml; yet more preferred concentration is 33.25 - 133 ng/ml;
yet more
38



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
preferred concentration is 49.875 - 99.75 ng/ml; yet more preferred
concentration is 59.85 -
79.8 ng/ml; and most preferred concentration is 66.5 ng/ml.
[00249] For VEGF, a preferred concentration is 0.625 - 7.5 ng/ml; yet more
preferred concentration is 1.25 - 5 ng/ml; yet more preferred concentration is
1.875 - 3.75
ng/ml; yet more preferred concentration is 2.25 - 3 ng/ml; and most preferred
concentration
is 2.5 ng/ml.
[00250] For Vitamin A, a preferred concentration is 6.25 - 75 ng/ml; yet more
preferred concentration is 12.5 - 50 ng/ml; yet more preferred concentration
is 18.75 - 37.5
ng/ml; yet more preferred concentration is 22.5 - 30 ng/ml; and most preferred
concentration
is 25 ng/ml.
[00251] For soluble Vitamin E, a preferred concentration is 156.25 - 1875
ng/ml;
yet more preferred concentration is 312.5 - 1250 ng/ml; yet more preferred
concentration is
468.75 - 937.5 ng/ml; yet more preferred concentration is 562.5 - 750 ng/ml;
and most
preferred concentration is 625 ng/ml.
[00252] In one embodiment, stem cells are cultured with a mode of suspension,
adherent or matrix in a cell culture medium, with or without serum, containing
compounds
listed in any column of Table 1. More preferably, the culture mode is
MATRIGEL, collagen,
hydrogel, or other crosslinkable gel matrix. More preferably, the culture mode
is a hydrogel
matrix. Most preferably, the culture mode is an alginate matrix.
[00253] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column A, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
A, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column A, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column A, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
A, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column A, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column A, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column A,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column A, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column A, Table 1. More preferably, the culture
medium contains
39



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
at least ten of the factors and supplements listed in Column A, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column A, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column A, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column A, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column A, Table
1. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column A, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column A, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column A, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column A, Table
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column A, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column A, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column A, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column A, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column A, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column A, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column A, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column A, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column A, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column A, Table
1. Most
preferably, the culture medium contains all the factors and supplements listed
in Column A,
Table 1.
[00254] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column B, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
B, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column B, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column B, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
B, Table 1.



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column B, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column B, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column B,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column B, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column B, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column B, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column B, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column B, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column B, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column B, Table
1. More
preferably, the culture medium contains at least 1 S of the factors and
supplements listed in
Column B, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column B, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column B, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column B, Table
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column B, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column B, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column B, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column B, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column B, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column B, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column B, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column B, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column B, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column B, Table
1. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column B, Table 1. More preferably, the culture medium contains at least 30 of
the factors
41



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
and supplements listed in Column B, Table 1. More preferably, the culture
medium contains
at least 31 of the factors and supplements listed in Column B, Table 1. Most
preferably, the
culture medium contains all the factors and supplements listed in Column B,
Table 1.
[00255] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column C, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
C, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column C, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column C, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
C, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column C, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column C, Table 1. Mare preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column C,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column C, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column C, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column C, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column C, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column C, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column C, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column C, Table
1. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column C, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column C, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column C, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column C, Table
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column C, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column C, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column C, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
42



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Column C, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column C, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column C, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column C, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column C, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column C, Table 1. Most preferably, the
culture medium
contains all the factors and supplements listed in Column C, Table 1.
[00256] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column D, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
D, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column D, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column D, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
D, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column D, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column D, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column D,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column D, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column D, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column D, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column D, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column D, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column D, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column D, Table
1. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column D, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column D, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column D, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column D, Table
43



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column D, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column D, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column D, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column D, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column D, Table 1. Most preferably, the culture
medium contains
all the factors and supplements listed in Column D, Table 1.
[00257] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column E, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
E, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column E, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column E, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
E, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column E, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column E, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column E,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column E, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column E, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column E, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column E, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column E, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column E, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column E, Table
1. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column E, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column E, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column E, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column E, Table
44



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column E, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column E, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column E, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column E, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column E, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column E, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column E, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column E, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column E, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column E, Table
1. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column E, Table 1. More preferably, the culture medium contains at least 30 of
the factors
and supplements listed in Column E, Table 1. More preferably, the culture
medium contains
at least 31 of the factors and supplements listed in Column E, Table 1. More
preferably, the
culture medium contains at least 32 of the factors and supplements listed in
Column E, Table
1. Most preferably, the culture medium contains all the factors and
supplements listed in
Column E, Table 1.
[00258) In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column F, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
F, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column F, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column F, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
F, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column F, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column F, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column F,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column F, Table 1. More preferably, the culture medium contains at least nine
of the factors



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
and supplements listed in Column F, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column F, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column F, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column F, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column F, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column F, Table
1. More
preferably, the culture medium contains at least 1 S of the factors and
supplements listed in
Column F, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column F, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column F, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column F, Table
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column F, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column F, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column F, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column F, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column F, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column F, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column F, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column F, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column F, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column F, Table
1. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column F, Table 1. More preferably, the culture medium contains at least 30 of
the factors
and supplements listed in Column F, Table 1. More preferably, the culture
medium contains
at least 31 of the factors and supplements listed in Column F, Table 1. More
preferably, the
culture medium contains at least 32 of the factors and supplements listed in
Column F, Table
1. Most preferably, the culture medium contains all the factors and
supplements listed in
Column F, Table 1.
46



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
[00259] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column G, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
G, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column G, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column G, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
G, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column G, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column G, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column G,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column G, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column G, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column G, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column G, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column G, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column G, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column G, Table
1. More
preferably, the culture medium contains at least 1 S of the factors and
supplements listed in
Column G, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column G, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column G, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column G, Table
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column G, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column G, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column G, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column G, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column G, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column G, Table 1. More
preferably, the
47



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
culture medium contains at least 25 of the factors and supplements listed in
Column G, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column G, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column G, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column G, Table
1. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column G, Table 1. Most preferably, the culture medium contains all the
factors and
supplements listed in Column G, Table 1.
[00260] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column H, Table 1.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
H, Table 1.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column H, Table 1. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column H, Table 1. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
H, Table 1.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column H, Table 1. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column H, Table 1. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column H,
Table 1. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column H, Table 1. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column H, Table 1. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column H, Table 1. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column H, Table
1. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column H, Table 1. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column H, Table 1. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column H, Table
1. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column H, Table 1. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column H, Table 1. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column H, Table 1. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column H, Table
48



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
1. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column H, Table 1. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column H, Table 1. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column H, Table
1. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column H, Table 1. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column H, Table 1. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column H, Table 1. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column H, Table
1. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column H, Table 1. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column H, Table 1. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column H, Table
1. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column H, Table 1. More preferably, the culture medium contains at least 30 of
the factors
and supplements listed in Column H, Table 1. More preferably, the culture
medium contains
at least 31 of the factors and supplements listed in Column H, Table 1. More
preferably, the
culture medium contains at least 32 of the factors and supplements listed in
Column H, Table
1. More preferably, the culture medium contains at least 33 of the factors and
supplements
listed in Column H, Table 1. Most preferably, the culture medium contains all
the factors
and supplements listed in Column H, Table 1.
[00261] In one embodiment, stem cells are cultured with a mode of suspension,
adherent or matrix in a cell culture medium, with or without serum, containing
compounds
listed in any column of Table 2. More preferably, the culture mode is
adherent. Most
preferably, the culture mode is an alginate adherent.
[00262] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column I, Table 2.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
I, Table 2.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column I, Table 2. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column I, Table 2. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
I, Table 2.
More preferably, the culture medium contains at least five of the factors and
supplements
49



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
listed in Column I, Table 2. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column I, Table 2. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column I,
Table 2. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column I, Table 2. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column I, Table 2. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column I, Table 2. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column I, Table
2. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column I, Table 2. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column I, Table 2. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column I, Table
2. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column I, Table 2. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column I, Table 2. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column I, Table 2. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column I, Table
2. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column I, Table 2. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column I, Table 2. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column I, Table
2. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column I, Table 2. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column I, Table 2. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column I, Table 2. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column I, Table
2. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column I, Table 2. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column I, Table 2. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column I, Table
2. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column I, Table 2. More preferably, the culture medium contains at least 30 of
the factors
and supplements listed in Column I, Table 2. More preferably, the culture
medium contains



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
at least 31 of the factors and supplements listed in Column I, Table 2. More
preferably, the
culture medium contains at least 32 of the factors and supplements listed in
Column I, Table
2. Most preferably, the culture medium contains all the factors and
supplements listed in
Column I, Table 2.
[00263] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column J, Table 2.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
J, Table 2.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column J, Table 2. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column J, Table 2. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
J, Table 2.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column J, Table 2. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column J, Table 2. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column J,
Table 2. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column J, Table 2. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column J, Table 2. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column J, Table 2. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column J, Table
2. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column J, Table 2. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column J, Table 2. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column J, Table
2. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column J, Table 2. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column J, Table 2. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column J, Table 2. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column J, Table
2. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column J, Table 2. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column J, Table 2. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column J, Table
2. More
51



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column J, Table 2. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column J, Table 2. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column J, Table 2. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column J, Table
2. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column J, Table 2. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column J, Table 2. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column J, Table
2. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column J, Table 2. More preferably, the culture medium contains at least 30 of
the factors
and supplements listed in Column J, Table 2. More preferably, the culture
medium contains
at least 31 of the factors and supplements listed in Column J, Table 2. Most
preferably, the
culture medium contains all the factors and supplements listed in Column J,
Table 2.
[00264] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column K, Table 2.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
K, Table 2.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column K, Table 2. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column K, Table 2. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
K, Table 2.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column K, Table 2. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column K, Table 2. More preferably, the
culture medium
contains at least seven of the factors and supplements listed in Column K,
Table 2. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column K, Table 2. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column K, Table 2. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column K, Table 2. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column K, Table
2. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column K, Table 2. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column K, Table 2. More preferably, the
culture medium
52



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
contains at least 14 of the factors and supplements listed in Column K, Table
2. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column K, Table 2. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column K, Table 2. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column K, Table 2. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column K, Table
2. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column K, Table 2. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column K, Table 2. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column K, Table
2. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column K, Table 2. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column K, Table 2. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column K, Table 2. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column K, Table
2. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column K, Table 2. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column K, Table 2. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column K, Table
2. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column K, Table 2. More preferably, the culture medium, contains at least 30
of the factors
and supplements listed in Column K, Table 2. Most preferably, the culture
medium contains
all the factors and supplements listed in Column K, Table 2.
(00265] In one embodiment, stem cells are cultured in a cell culture medium,
with
or without serum, containing compounds listed in Column L, Table 2.
Preferably, the culture
medium contains at least one of the factors and supplements listed in Column
L, Table 2.
More preferably, the culture medium contains at least two of the factors and
supplements
listed in Column L, Table 2. More preferably, the culture medium contains at
least three of
the factors and supplements listed in Column L, Table 2. More preferably, the
culture
medium contains at least four of the factors and supplements listed in Column
L, Table 2.
More preferably, the culture medium contains at least five of the factors and
supplements
listed in Column L, Table 2. More preferably, the culture medium contains at
least six of the
factors and supplements listed in Column L, Table 2. More preferably, the
culture medium
53



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
contains at least seven of the factors and supplements listed in Column L,
Table 2. More
preferably, the culture medium contains at least eight of the factors and
supplements listed in
Column L, Table 2. More preferably, the culture medium contains at least nine
of the factors
and supplements listed in Column L, Table 2. More preferably, the culture
medium contains
at least ten of the factors and supplements listed in Column L, Table 2. More
preferably, the
culture medium contains at least 11 of the factors and supplements listed in
Column L, Table
2. More preferably, the culture medium contains at least 12 of the factors and
supplements
listed in Column L, Table 2. More preferably, the culture medium contains at
least 13 of the
factors and supplements listed in Column L, Table 2. More preferably, the
culture medium
contains at least 14 of the factors and supplements listed in Column L, Table
2. More
preferably, the culture medium contains at least 15 of the factors and
supplements listed in
Column L, Table 2. More preferably, the culture medium contains at least 16 of
the factors
and supplements listed in Column L, Table 2. More preferably, the culture
medium contains
at least 17 of the factors and supplements listed in Column L, Table 2. More
preferably, the
culture medium contains at least 18 of the factors and supplements listed in
Column L, Table
2. More preferably, the culture medium contains at least 19 of the factors and
supplements
listed in Column L, Table 2. More preferably, the culture medium contains at
least 20 of the
factors and supplements listed in Column L, Table 2. More preferably, the
culture medium
contains at least 21 of the factors and supplements listed in Column L, Table
2. More
preferably, the culture medium contains at least 22 of the factors and
supplements listed in
Column L, Table 2. More preferably, the culture medium contains at least 23 of
the factors
and supplements listed in Column L, Table 2. More preferably, the culture
medium contains
at least 24 of the factors and supplements listed in Column L, Table 2. More
preferably, the
culture medium contains at least 25 of the factors and supplements listed in
Column L, Table
2. More preferably, the culture medium contains at least 26 of the factors and
supplements
listed in Column L, Table 2. More preferably, the culture medium contains at
least 27 of the
factors and supplements listed in Column L, Table 2. More preferably, the
culture medium
contains at least 28 of the factors and supplements listed in Column L, Table
2. More
preferably, the culture medium contains at least 29 of the factors and
supplements listed in
Column L, Table 2. Most preferably, the culture medium contains all the
factors and
supplements listed in Column L, Table 2.
EXAMPLES
54



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Example 1
Sequential culture of pancreatic cells in alginate followed b~pension culture
[00266] Pancreatic cells were cultured for 6-12 days in 1.6% alginate in a
medium consisting of a mixture of DMEM and Ham's F12 nutrient mixture
supplemented
with 10% FBS, insulin, transferrin, selenium and EGF resulting in the
generation of stem
cells. Stem cells were harvested from the alginate beads by depolymerization
and cultured in
suspension in ultra low adherence plates (Costar) for 11 days in basal medium
supplemented
with combinations of 60 growth and differentiation factors in a 120
combinatorial array. At
the end of the culture period cells were subjected to a 24 hr challenge with
basal glucose
medium (SmM glucose), 20 mM glucose or 20 mM glucose + IBMX. Supernatants were
harvested and analyzed for insulin content using an ELISA. Cells were washed
and lysed and
the DNA content per well determined using a picogreen assay The "insulin
difference" was
calculated by the subtraction of the insulin content in wells stimulated with
basal medium
from the insulin content in the supernatants in wells after stimulation with
glucose alone or in
combination with IBMX. Insulin difference of supernatants generated after
stimulation with
glucose alone ranged from 0.007-0.9908 ng/well and from 0.0098-1.1523 ng/well
after
stimulation with glucose and IBMX. Many wells produced low levels of insulin
as calculated
by the insulin difference. A few wells produced significant amounts of insulin
compared to
control wells assayed prior to the addition of factors in the combinatorial
array as well as
control wells cultured in basal medium without additional growth and
differentiation factors.
[00267] These data show that selecting specific growth and differentiation
factors, in combination, can be used with different culture modes in order to
promote the
differentiation of a pancreatic stem cell into an insulin producing cell.
Example 2
Sequential culture of stem cells in alginate followed by adherent culture.
[00268] Pancreatic cells were cultured for 6-12 days in 1.6% alginate in a
medium
consisting of a mixture of DMEM and Ham's F12 nutrient mixture supplemented
with 10%
FBS, insulin, transfernn, selenium and EGF resulting in the generation of stem
cells. Stem
cells were harvested from the alginate beads by depolymerization, and cultured
in adherent
culture, on collagen coated plates for 8 days in basal medium supplemented
with
combinations of 60 growth factors in a 120 combinatorial array. At the end of
the culture



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
period cells were subjected to a 24 hr challenge with basal glucose medium
(SmM glucose),
20 mM glucose or 20 mM glucose + 1mM IBMX. Supernatants were harvested and
analyzed
for insulin content using an ELISA. Cells were washed and lysed and the DNA
content per
well determined using a picogreen assay. The "insulin difference" was
calculated by the
subtraction of the insulin content in wells stimulated with basal medium from
the insulin
content in the supernatants in wells after stimulation with glucose alone or
in combination
with IBMX. Insulin difference of supernatants generated after stimulation with
glucose alone
ranged from 0.0019-0.9714 ng/well and from 0.0052-0.9524 ng/well after
stimulation with
glucose and IBMX. Many wells produced low levels of insulin as calculated by
the insulin
difference. A few wells produced significant amounts of insulin compared to
control wells
assayed prior to the addition of factors in the combinatorial array as well as
control wells
cultured in basal medium without additional growth and differentiation
factors.
[00269] These data show that selecting specific growth and differentiation
factors,
in combination, can be used with different culture modes in order to promote
the
differentiation of a pancreatic stem cell into an insulin producing cell.
Example 3
Culture of stem cells in alginate culture.
[00270] Pancreatic cells were cultured for 6-12 days in 1.6% alginate in a
medium
consisting of a mixture of DMEM and Ham's F12 nutrient mixture supplemented
with 10%
FBS, insulin, transfernn, selenium and EGF resulting in the generation of stem
cells. Stem
cells were harvested from the alginate beads by depolymerization, and recast
into 1.2%
alginate beads and cultured for an additional 7-11 days in basal medium
supplemented with
combinations of 60 growth factors in a 120 combinatorial array. At the end of
the culture
period cells were subjected to a 24 hr challenge with basal glucose medium
(SmM glucose),
20 mM glucose or 20 mM glucose + 1mM IBMX. Supernatants were harvested and
analyzed
for insulin and C-peptide content using an ELISA. Alginate beads were
depolymerized and
the cells were washed and lysed and the DNA content per well determined using
a picogreen
assay
[00271] Insulin and c-peptide data from 4 replicate experiments using material
from four separate human donors were examined with results examined from
duplicate wells.
Wells that showed consistent stimulation of insulin release were identified by
comparison of
the level of insulin or-peptide induced by incubation in the presence of
glucose or glucose
and IBMX to that produced by wells incubated in basal medium. Insulin assays
were
56



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
performed on all wells to determine which well combinations of growth and
differentiation
factors produced significant stimulated insulin release: The results of these
assays are plotted
in Figure 1. These plots show insulin content following either basal glucose,
high glucose or
high glucose plus IBMX for each well in the combinatorial array. Many of these
show wells
with very little insulin, some wells show high basal levels of insulin as well
as high
stimulation and others with significant stimulated release. To aid in the
picking of the best
well combinations, a stimulation index was calculated for the high glucose
divided by the
basal or IBMX divided by the basal. These results are shown in figure 2. These
clearly show
several candidates for best wells using these results. Several additional
analyses were done to
determine which were the eight best wells that were selected.
[00272] Examining four different experiments using cells from four different
donors show that there was donor to donor variation in these experiments. The
eight best
wells were determined by the analyses from all donors. A comparison of each of
these
individual donors follows. In donor #2212 the insulin release from basal
versus IBMX
stimulation are shown in figure 3. Compared with day 0, each of the best wells
had
significant increase in stimulated insulin except for wells A, D and E. All of
the wells from
this donor had somewhat high basal insulin. Expressing the results as
stimulation index
(Figure 4) show that best wells B, C, F, G and H had the highest responses.
Examining the
results of donor #2278 the insulin release from basal, high glucose or IBMX
show a
significant difference over the control wells at day 0, 7 and 14 (Figure S).
This donors best
wells all had very high basal insulin for reasons that were unclear resulting
in a low
stimulation index for all the best wells (Figure 6). Examining the results of
donor #3023, the
insulin release from basal, hi8gh glucose or glucose and IBMX from the best
wells were
compared with day 0, 7 and 14 controls (Figure 7). With lower basal insulin
release for this
donor, essentially all the best wells had significant stimulated insulin
release. Calculating the
stimulation index (Figure 8) these also showed significant release from the
best wells.
However, there were differences in the insulin release with the best wells,
each with different
factor combinations. Some showed IBMX release higher than glucose (expected)
and others
showed glucose higher than glucose plus IBMX. This suggests the different
combinations in
these wells result in insulin producing cells with different capabilities.
Examining the results
for the fourth donor (3036), the basal insulin levels were low with
significant stimulated
insulin release after glucose or glucose plus IBMX challenge. (Figure 9).
Looking back at the
wells in figure 2, these best wells are clearly better than most of the
responses, as is the case
57



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
from the other donors. Examination of the stimulation index (Figure 10) show
that the insulin
producing cells generated from this donor gave higher responses with glucose
plus IBMX
compared to glucose alone. These supernatants were assayed for c-peptide
content as shown
in figure 11 as stimulation indices for c-peptide release.
[00273] In summary, these results demonstrate marked differences between wells
containing different combinations of growth and differentiation factors as
well as donor to
donor variation. The selected best wells are not the final answer and
additional studies are
required in order to define the optimal combinations.
Table 1 shows the growth factor composition of these "best wells
58



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Table 1
Composition of Media Resulting in Best Insulin Production
Substance c~"~. A B C D E F G H
(~g~ml)


Activin A 0.0005


Atrial Natriuretic Peptide 0.1530


Betacellulin 0.0050


Bone Morphogenic Protein (BMP-2) 0.0050


Bone Morphogenic Protein (BMP-4) 0.0005


C natriuretic peptide (CNP) 0.1099


Caerulein 0.0300


CCK8 0.0057
.


CCK8 (26-33), amide, 0.0250


CGRP alpha 0.1905


Cholera Toxin B Subunit 0.0125


Corticosterone 0.0020


Dexamethasone 0.0020


DIF-1/Differanisole A 0.3000


DMF (n n dimethylformamide) 0.0000


DMSO (dimethylsulfoxide) 0.0010


EGF 0.0050


Endothelin 1 0.5000


Exendin 4 0.0210


FGF acidic (aFGF = FGF1) 0.0025


FGF7 (KGF) 0.0025


FGFb (=FGF2) 0.0025


Gastrin I Human 0.0000


GLP-1 (7-36) amide, human (Glucagon-Like0.0330
Peptide 1)


Glucose 1.0800


Growth Hormone (somatotropin) 0.0250


GRP (Gastrin Releasing Peptide) 0.1430


Hepatocyte Growth Factor (HGF) 0.0025


IGF-1, recombinant human 0.0025


IGF-2, recombinant human 0.0025


Insulin 9.5000


Lactogen, from human placenta 0.0500


Laminin 2.2500


Leu-Enkephalin 0.0030


LIF, human (leukemia inhibitory 0.0025
factor)


Met-Enkephalin 0.0030


n Butyric Acid, Sodium Salt 4.5400


Nerve Growth Factor, human (beta 0.0025
NGF)


Nictotinamide 610


D11/~L' A A 1 D71!'_C DD AATV
0.0050


59



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
PIGF (Placental GF, human) 0.0025


Progesterone 0.0030


Prolactin 0.0012


pT II RP (Parathyroid Hormone Related0.2060
Peptide)


Putrescine Dihydrochloride Gamma-Irradiated0.0001
Cell Culture


REG1, Novocell Peptide Mimetic 0.0326


Retinoic Acid (Vitamin A) 0.0250


Selenium (Selenious Acid, Na salt) 0.0250


Sonic Hedgehog (mouse, recombinant)0.0250


Substance P (full length) (H1875 5
is frag 1-4)


Superoxide Dismutase (SOD) 5 IU/ml


TGF alpha 0.0010


TGF B 1 0.0005


TGF beta sRII (soluble receptor 0.0050
type 2)


transferrin 2.7500


Triiodothyronine (T3) 0.0335


Trolox (soluble Vitamin E) 0.6250


Trypsin Inhibitor, soybean (type 0.5000
I-S)


Vasoactive Intestinal Peptide (VIP)0.0665


VEGF 0.0025


Example 4
Culture Media Analysis of cells cultured in al ig nate
[00274] Statistical analysis of the insulin content of the supernatants
generated by
3 donors, produced in example 3 from the combinatorial array, resulted in a
list of positive
and negative effectors influencing insulin production and cell growth, as well
as, consistently
good combinations.
[00275] Growth and differentiation factors that had a potential positive
effect on
the conversion of stem cells into insulin producing cells, as identified by
this combinatorial
system, are: Betacellulin, BMP-2, Caerulein, CCKB sulfated, Cholera Toxin B
Subunit,
CNP, Corticosterone, DMF, DMSO, EGF, Exendin 4, FGF-1, Glucose, GRP, IGF-1,
IGF-2,
Insulin, KGF, Laminin, Leu-Enkephalin, Met-Enkephalin, NGF beta,
Nictotinamide, PDGF
AA.BB, pTHRP, Selenium, SHH, Substance P, TGF beta sRII, Transferrin, vEGF,
VIP.
[00276] Growth and differentiation factors that had a potential negative
effect on
the conversion of stem cells into insulin producing cells, as identified by
this combinatorial
system, are: Activin A, ANP, BMP-4, CCK8 amide, CGRP alpha, Dexamethasone, DIF-
l,
Endothelia l, FGF-2, Gastrin I, GH, GLP-1, HGF, Lactogen, LIF, n Butyric Acid,
P1GF,



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Progesterone, Prolactin, Putrescine, REG-l, Retinoic Acid, SOD, Soybean
Trypsin Inhibitor,
T3, TGF alpha, TGF beta 1, Trolox
Example 5
Sequential culture of stem cells in adherent followed by adherent culture.
[00277] Stem cells, generated by a 6-12 day adherent culture on collagen
coated
plates in PCM, were cultured on collagen coated plates for an additional 8
days in basal
medium supplemented with combinations of 60 growth factors in a 120
combinatorial array.
Alternatively cells were removed from the collagen coated plates after the
first culture period
and replated onto fresh culture plates then cultured for an additional 8 days
in basal medium
supplemented with combinations of 60 growth factors in a 120 combinatorial
array. At the
end of the culture period cells were subjected to a 24 hr challenge with basal
medium or 20
mM glucose. Supernatants were harvested and analyzed for insulin or C-peptide
content
using an ELISA. Cells were washed and lysed and the DNA content per well
determined
using a picogreen assay
[00278] Data from wells that constitutively produced insulin or induced to
produce
insulin glucose stimulation from 3 independent preparations were subjected to
statistical
analysis and "best wells" identified. The composition of growth factors
present in the top
four "best wells" is shown in Table 2.
Table 2
Composition of Media Resulting in Best Insulin Production
Substance cor,~. I J K L
(wglml)


Activin A 0.0005


Atrial Natriuretic Peptide 0.1530


Betacellulin 0.0050 ~


Bone Morphogenic Protein (BMP-2) 0.0050


Bone Morphogenic Protein (BMP-4) 0.0005


C natriuretic peptide (CNP) 0.1099 ~


Caerulein 0.0300 ~


CCK8 sulphated 0.0057


CCK8 (26-33), amide, 0.0250 '~


CGRP alpha 0.1905 ~


Cholera Toxin B Subunit 0.0125 ~


Corticosterone I 0.0020 ~


61



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Dexamethasone 0.0020~


DIF-1/Differanisole A 0.3000~


DMF (n n dimethylformamide) 0.0000


DMSO (dimethylsulfoxide) 0.0010'~


EGF 0.0050


Endothelin 1 0.5000


Exendin 4 0.0210


FGF acidic (aFGF=FGF1) 0.0025


FGF7 (KGF) 0.0025~'


FGFb (=FGF2) 0.0025


Gastrin I Human 0.0000


GLP-1 (7-36) amide, human (Glucagon-Like0.0330'~
Peptide 1)


Glucose 1.0800


Growth Hormone (somatotropin) 0.0250


GRP (Gastrin Releasing Peptide) 0.1430


Hepatocyte Growth Factor (HGF) 0.0025


IGF-1, recombinant human 0.0025~


IGF-2, recombinant human 0.0025~'


Insulin 9.5000~


Lactogen, from human placenta 0.0500


Laminin 2.2500~


Leu-Enkephalin 0.0030~


LIF, human (leukemia inhibitory 0.0025~
factor)


Met-Enkephalin 0.0030~


n Butyric Acid, Sodium Salt 4.5400~


Nerve Growth Factor, human (beta 0.0025~
NGF)


Nictotinamide 610


PDGF AA + PDGF BB MIX 0.0050


PIGF (Placental GF, human) 0.0025~


Progesterone 0.0030~


Prolactin 0.0012


pT II RP (Parathyroid Hormone Related0.2060~
Peptide)


Putrescine Dihydrochloride Gamma-Irradiated0.0001
Cell Culture


REG1, Novocell Peptide Mimetic 0.0326~


Retinoic Acid (Vitamin A) 0.0250~


Selenium (Selenious Acid, Na salt) 0.0250


Sonic Hedgehog (mouse, recombinant)0.0250~


Substance P (full length) (H1875 5
is frag 1-4)


Superoxide Dismutase (SOD) 5 ICJ/ml~


TGF alpha 0.0010~


TGF B 1 0.0005


TGF beta sRII (soluble receptor 0.0050~
type 2)


transferrin 2.7500~


Triiodothyronine (T3) 0.0335


Trolox (soluble Vitamin E) 0.6250


Trypsin Inhibitor, soybean (type 0.5000
I-S)


62



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Vasoactive Intestinal Peptide (VIP) ~ 0.0665
VEGF 0.0025
(00279] Figure 12 presents the c-peptide results from this experiment showing
release from basal, glucose and glucose plus IBMX stimulations showing
positive responses.
Ranges of insulin and DNA concentration were detected in the samples harvested
from
individual wells demonstrating that this is a feasible method for screening
growth and
differentiation factor combinations for their effect in the growth and
differentiation of
pancreatic cell derived stem cells
Example 6
Further optimization of the 120 combinatorial array.
[00280] Data presented in previous examples identified "best wells", in terms
of
induced insulin production, or total insulin production. The ingredients
present in the "best
media" can then undergo a second tier screen to simplify and better define a
minimal number
of factors that induce the production of insulin-producing cells from stem
cells.
Alternatively, the positive effectors (Example 4) can undergo a second tier
screening to
achieve the same result.
[00281] In this example, thirty components of media "L" were arrayed into a 60
factor array. Stem cells, generated by a 7 day adherent culture on collagen
were placed into
screening conditions for an additional 3, 5 orl0 days. At each time point,
cells were fixed and
processed for immunohistochemistry using a proinsulin-specific antibody. The
number of
proinsulin-positive cells was counted using automated image analysis. The
number of pro-
insulin positive cells using media M, N, O, P, and Q on days 3, S and 7 is
shown in figure 13.
These media are the most promising of the second tier screen. In the figure,
they are
compared to media "L", a promising media from the 60 factor array. In
conclusion, this
example shows that the 60 factor combinatorial array can be refined and
improved.
Example 7
Gene-chip studies (DNA oligo microarray)
[00282) The use of a "gene chip" (BD Atlas array) allows us to measure the
relative expression levels (mRNA levels) of 8,000 genes. This method can be
used to
"fingerprint" or identify cell types. The analysis of mRNA expression in
differentiating
pancreatic cells potentially identifies genes that are involved in the
transdifferentiation
63



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
process. This type of comparison will allow us to compare starting pancreatic
cells to the
intermediary stem cell, intermediary stem cells to hormone-producing cells,
and this final
product to normal human pancreatic islets.
[00283] The utilization of such technology to produce a "forger print" of the
gene
expression patterns of the different cell types found in the human pancreas
would serve two
critical functions. Perhaps the most important function of such an analysis
would be to
clearly define the gene expression profile of cells generated during the
transdifferentiation
process and thus define, on the molecular level, the unique characteristics of
these cells. The
second function of such an analysis would be to provide tools to improve our
research
methods. The analysis would give us insights into the mechanisms of how the
insulin-
producing phenotype is regulated. Knowledge of cell surface markers would
facilitate rapid
cell identification as well as provide the means to sort desirable cells from
"undesirable"
cells. Information of the cell signaling molecules and transcription factors
present on these
cells will facilitate the identification of growth factors that may be
required to more
efficiently complete expansion and transdifferentiation of the starting
material into cells
capable of producing insulin in a similar manner to naturally occurring beta
cells. While
there is some information on gene expression and phenotype of pancreatic cells
available in
publicly available literature and reports, much of it relates to non-human
animal models, or
embryonic development. These gene chip studies are specific to our
applications and
discoveries.
[00284) Tables 3 and 4 show the result of two islet-depleted, human pancreatic
cell
preparations that were compared after 7 days of culture in adherent culture in
PCM. RNA
was isolated by standard methods and screened in comparative micro arrays.
While the two
preparations were cultured under identical conditions, one preparation was
judged to be
"excellent", while the other was judged to be "OK" (by the criteria of its
subsequent ability to
produced c-peptide). Most of the genes expressed in these cultures will be the
same, but there
will be some genes that are differentially expressed. Some differences will be
donor specific
(e.g. differences in MHC markers), while others may give us insights into the
genes that are
determinative in "excellent" versus "OK" results.
[00285] Examination of the 8,000 genes expressed by each of the different
preparations result in an extensive list that is too long to include. Table 3
summarizes those
genes that we believe may be particularly useful to our studies and objectives
for obtaining
new insulin-producing cells. Some of these genes are mechanistically important
to the
64



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
differentiation process, while others are correlative and possibly predictive
of successful stem
cell formation. Table 4 is a compilation of about 90 "strongly expressed"
messages (signal
strength of 10-100% of maximal). The strongly expressed messages may be
particularly
useful in identifying surface markers that can be used to identify and sort
the different cell
populations (acinar vs islet or successfully differentiated vs poorly
differentiated). Again, the
complete list of "strongly expressed" genes is extensive and an abbreviated
version is
presented.



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Table 3
A summary of important/useful genes expressed at comparatively different
levels in the
"excellent" prep (2071) versus the "OK" prep (2078).
Genes
expressed
at a
higher
level
in the
"excellent"
prep.


Line Gene Ratio Genbank Short summary
# prep


2078:2071


574 hairless Down AF039196A transcription factor, found
(mouse) 5.4 in many different tissues


homolog with highest expression in
brain. May function as a


specific repressor. Difference
between two preps is


reat, hi her ex ression in
" ood" re .


1237 sine oculisDown NM-005413A homeobox gene that has been
2.6 studied in eye


homeobox development; also expressed
in adult (fully


(Drosophila) differentiated eye tissues).
Activates Pax6 expression!


homolog (Pax6 is a mature beta cell
3 marker). No publications


re ardin sine oculis in ancreatic
literature.


16 7-60 proteindown IVM-007346Receptor for opioid growth
2.5 factor, Met(5)-enkephalin


(a factor present in the MFA).
Ligand is an inhibitory


peptide that modulates cell
proliferation and tissue


organization during development,
cellular renewal,


cancer, wound healing, and
an io enesis.


234 CDC37 (celldown U63131 Positive regulator of cell
2.8 cycle progression through


division interactions with CDK4. May
37, S. also be a component of


cerevisiae, a complex that regulates NF-kappa
B.


homology


Genes
expressed
at a
higher
level
in the
"OK"
prep
(2078)


Line Gene Ratio Genbank Short summary
# prep


2078:2071


866 neurogenic up 2.6 NM-006160A helix-loop-helix transcription
factor known to


differentiation mediate neuronal differentiation.
2 Closely related to


(ND2) NeuroDl. (aka "Beta cell E-box
transactivator" or


"Beta 2"). Role for ND1 well
established in mouse


model, role of ND2 not determined.
Is ND2


predominant acinar form? Predominant


transdifferentiatin form?


935 pancreatitis-up 4.1 D 13510 An acinar protein. Abundance
is normally very low,


associated but very high in pancreatitis.
protein It is also a marker for


some liver cancers. Function?
Is expression induced


durin transdifferentiation?


489 G protein-coupledup 3.1 NM-005682Has similarity to some secretin-like
receptors and has


receptor a mucin-like domain. Present
56 in a wide range of


tissues. Highest levels in
the smaller, more actively


secreting follicles of human
thyroid. Marker for


undifferentiated acinar?


1115 retinoic up 3.2 NM-002889Retinoids exert potent growth
acid inhibitory and cell


receptor differentiation activities.
responder These effects are mediated


(tazarotene by specific nuclear receptor
proteins that are members


induced) of the steroid and thyroid
2 hormone receptor


superfamily of transcriptional
regulators. Marker for


undifferentiated acinar?


66



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Table 4
A summary of potentially important and/or useful genes expressed at high
levels in both cell
preparations after 7 days in culture.
Enzymes
& cofactors


Line # Gene Genbank Short summary


122 ATPase Ca++L20977 Membrane Ca++ pump, highly restricted
tissue distribution, well


transporting characterized in cochlear outer hair
cells and spiral ganglion.


plasma membrane Expression is strong. Good acinar
membrane marker?


2


387 Dual-specf.NM_004714Regulates nuclear functions? Implicated
Tyr. in postembryonic-


Phosph regulated neurogenesis. Also, enables colon
carcinoma cells to survive


rotein under certain stress conditions


657 Inhib of NM-003639Kinase that activates the enhancer
kappa of NF-kappa-B activation; it


light polypeptide would play a role in activating the
response to inflammatory


enhancer c tokines. Perha s a role in differentiatin
kinase cells?


659 Inositol NM 001567INPPL or SHIP2 may play a significant
role in regulation of


polyphosphate PI3K signaling by growth factors
and insulin. Primary defect in


phosphatase-like KO mouse shows that SHIP2 is a potent
negative regulator of


insulin signaling and insulin sensitivity
in vivo. Important


re ulator of rowth factor si nalin
in our s stem?


824 MAP kinase NM-004579Found in many tissues, participates
in B-cell differentiation.


kinase kinase


kinase 2


1228 Sialyl transferaseNM_003034Modifies NCAM with polysialic acid.
Involved in modifying cell


8 adhesion molecules in our s stem?


67



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
DNA,
transcription
factors
and
developmental
genes.


196 Cardiac-specificNM 004387aka NKX2-5. Homeobox-containing genes
are essential for


homeo box tissue differentiation, as well as
determining the temporal and


spatial patterns of development. This
one has been characterized


in terms of heart formation. Mouse
pancreas researchers have


focused on nloc2.2 and nla6.1


200 Cartilage NM 006982Function unknown in humans. In mouse,
paired necessary for survival


class of the forebrain mesenchyme. Mutations
lead to acrania and


homeo rotein meroanence hal .


213 C/EBP alphaU34070 Regulates differentiation in a number
of cell types. Has also


been shown to inhibit cyclin-dependent
kinases and cause growth


arrest.


214 C/EBP beta NM_005194Regulates differentiation in a number
of cell types. Required for


a normal roliferative res onse.


933 Paired NM 002653Bicoid class of homeodomain proteins.
Members of this family


homeodomain are involved in organ development,
left-right asymmetry. Also


transcription acts as a transcriptional regulator
in some adult tissues (e.g.


factor prolactin gene). In developmental
models, Pitx2 is directly


initiated by Nodal signaling and is
subsequently maintained by


Nkx2. If it is maintained by Nkx2,
may be present in maturing


al ha and beta cells.


995 POU domain,NM 002699AKA Oct-6. Involved in nerve development
and regeneration;


class 3 other developmental roles? Oct 4 plays
a role in mouse


transcription pancreatic development.


factor


1285 Spi NM-003120Related to ets. Essential for the
development of myeloid and B-


1 hoid cells


1347 TranscriptionNM 003206A basic helix-loop-helix transcription
factor, In adults, expressed


factor 2 1 in lung, kidney, heart, placenta and
pancreas. In embryos,


essential for the development of the
coronary vasculature and


organs containing epithelial-lined
tubular structures. May


represent a point of regulatory convergence
between a number of


transcri tion factors.


Growth factor and related genes


90 Anti MullerianNM_000479Anti-Mullerian hormone is a member
of the TGF-beta and


Hormone inhibin gene family. Mediates male
sexual differentiation:


Causes the regression of Mullerian
ducts which would otherwise


differentiate into the uterus and
fallopian tubes. Unknown


function in adults.


935 Pancreatitis-D13510 Expression is low in normal acinar
cells but very high in


associated pancreatitis. Also expressed by epithelial
protein cells of the small


intestine and some liver cancers.
Function?


197 CardiotropinNM-001330Family of cytokines that includes
1 LIF, ciliary neurotrophic factor


(CNTF), oncostatin M, interleukin
6 and 11 Required for


motorneuron develo ment, romotes motorneuron
survival.


808 Midkine NM 002391Exhibits neurite outgrowth-promoting
neurite activity and plays a role in


growth promoting nervous system development and/or
maintenance. Expression


factor 2 believed to be very low, except for
a short period during


develo ment


68



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Receptor signal transduction related
and


81 AngiotensinIVM-004835Angiotensin is an important effector
controlling blood pressure


receptor and volume in the cardiovascular system.
1B These receptors are


also found in the exocrine, endocrine
and vascular cells of the


pancreas. Immunostaining to AR is
predominantly in the


endothelia of the blood vessels and
the epithelia of the pancreatic


ductal s stem and weakl in ascini.


550 Growth hormoneNM_005310Diverse family important in tyrosine
kinase signaling.


receptor-bound Homologous to ras-GAP. In some models,
involved in metastatic


rotein 7 ro ression


554 Growth hormoneNM 004122GRS and GH releasing factor have the
reciprocal effect of


secretagogue somatostatin on growth hormone release
from the pituitary (see


receptor next). This G-protein coupled receptor
can also bind ghrelin.


Would have ex ected this marker on
endocrine cells.


1277 SomatostatinNM 001051Somatostatin acts at many sites to
inhibit the release of many


receptor 3 hormones and other secretory proteins.
The biological effects of


somatostatin are probably mediated
by G protein-coupled


receptors that are expressed in a
tissue-specific manner. SSTR3


is ex ressed in hi best levels in
brain and ancreatic islets.


1056 Protein NM 002850Receptor-type PTP. A signaling molecule
tyrosine that may regulate


phosphatase growth and differentiation. This PTP
has been also implicated in


the control of adult nerve re air.


426 Ephrin AS U26403 Binds to members of the EPH group
of receptor tyrosine kinases.


May be involved in axon guidance.
Ephrin and its receptor may


shift the cellular res onse from re
ulsion to adhesion.


172 Butyrate X79067 Induced by various agents: phorbol
response ester TPA, EGF, etc. May


factor 1 mediate rapid degradation of cytokine
(EGF (AU-rich) mRNA. Marker


res onse for activation/differentiation?
factor
1


69



CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734
Cell ace or structural genes
surf


47 AdenomatousNM 005883Located in both the membrane/cytoskeletal
and the nuclear


polyposis fraction, ubiquitously expressed.
coli-like APC interacts with catenins,


and through these, with E-cadherin.
May regulate transmission of


the contact inhibition signal into
the cell, or may regulate


adhesion. The former is more consistent
with mutated APC's


early role in tumorigenesis. Depending
on what proteins are


members of the APC complex, APC may
participate in cell cycle


progression, developmental pathways
cell morphology or


neuronal function.


95 Aquaporin NM-001651Aquaporins are water channel proteins.
5 Aq5 is known to play a


role in the generation of saliva,
tears and pulmonary secretions.


Marker for duct a ithelium?


220 CD 151 antigenNM 004357Member of the transmembrane 4 superfamily,
aka tetraspanin


family. A cell surface glycoprotein
that is known to complex


with integrins and other tetraspanins.
The complexes are cell-


attachment sites for binding to basement
membranes. The


proteins mediate signal transduction
events in the regulation of


cell develo ment, activation, growth
and motili


221 CD3 E antigenNM-000733CD3 epsilon is one of the T-cell antigen
receptor complex


subunits. CD3-E is a signal transducing
component that may be


particularly important in instructing
pre T Cell lineage


commitment. Role in ancreatic lines
a commitment?


303 Cofilin NM 005507A widely distributed intracellular
actin-modulating protein that


depolymerizes filamentous actin and
inhibits the polymerization


of monomeric actin.


465 Ficolin 1~1M_003665Characterized as a serum protein.
Has calcium-independent


lectin activity but unlike other family
members, it does not bind


fibronectin or elastin.


669 Integrin, NM 002204Cell surface adhesion molecule, integral
alpha 3 membrane protein;


interacts with man extracellular-matrix
roteins


690 Junction M23410 A major cytoplasmic protein that occurs
in a soluble and a


plakoglobin membrane-associated form and in adhering
junctions


desmosomes and intermediate 'unctions


694 Keratin x03212 Expression patterns of CK19 have been
7 very useful for us


Keratin NM_000422already; the other keratins may also
17 be useful.


Keratin M34225
8


872 Neuronal NM 014486NTP is one of the proteins expressed
thread during growth and


protein sprouting of neuronal cells. Expressed
during


transdifferentiation to neuroendocrine
cells?


1008 Profilin NM 005022A ubiquitous actin monomer-binding
1 protein. Regulates actin


of erization in res onse to extracellular
si nals.


1187 S 100 Ca++ NM 005620Family of proteins localized in the
cytoplasm and/or nucleus of a


binding wide range of cells. Involved in the
protein regulation of cell cycle


progression and differentiation. Remarkably
elevated in


colorectal cancers com ared to normal
mucosa.


1299 Stratifin AF029082 Diffusely distributed in the cytoplasm.
Most abundant in tissues


enriched in stratified keratinizing
epithelium. Mediates signal


transduction by binding to phosphoserine-containing
proteins.


Induced in response to DNA damage,
and causes cells to arrest in


G2 AKA 14-3-3-si ma


1335 Thymosin M92381 An actin-sequestering protein
beta


1435 Wishot AldrichNM-003387Involved in transduction of signals
from receptors on the cell


interactin surface to the actin c oskeleton.
rotein Induces actin of erization.





CA 02487858 2004-11-29
WO 03/100026 PCT/US03/16734



0


~.
Y



(~ 4~
~~ U


_
U
~L
~


0
0 w
,,~



~ x


~ ~
~
b
'


o


.o


Y
Y


G
o


o a~


~
f~


b
~'


~,
U



H


a a~
~,
~.



00 0,


0
0


y


U



'U
Y


,
O



N


N



Representative Drawing

Sorry, the representative drawing for patent document number 2487858 was not found.

Administrative Status

For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee  and Payment History  should be consulted.

Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2003-05-28
(87) PCT Publication Date 2003-12-04
(85) National Entry 2004-11-29
Dead Application 2008-05-28

Abandonment History

Abandonment Date Reason Reinstatement Date
2007-05-28 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2004-11-29
Application Fee $400.00 2004-11-29
Maintenance Fee - Application - New Act 2 2005-05-30 $100.00 2004-11-29
Registration of a document - section 124 $100.00 2005-03-08
Maintenance Fee - Application - New Act 3 2006-05-29 $100.00 2006-05-12
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
NOVOCELL, INC.
BECTON, DICKINSON AND COMPANY
Past Owners on Record
COUTTS, MARGARET
HAALAND, PERRY
HEIDARAN, MOHAMMAD
LATTA, PAUL P.
MCINTYRE, CATHERINE
PRESNELL, SHARON C.
SCHARP, DAVID
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
Documents

To view selected files, please enter reCAPTCHA code :



To view images, click a link in the Document Description column. To download the documents, select one or more checkboxes in the first column and then click the "Download Selected in PDF format (Zip Archive)" or the "Download Selected as Single PDF" button.

List of published and non-published patent-specific documents on the CPD .

If you have any difficulty accessing content, you can call the Client Service Centre at 1-866-997-1936 or send them an e-mail at CIPO Client Service Centre.


Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Claims 2004-11-29 4 140
Abstract 2004-11-29 1 62
Description 2004-11-29 71 3,976
Drawings 2004-11-29 13 259
Cover Page 2005-03-11 2 44
PCT 2004-11-29 3 113
Assignment 2004-11-29 3 97
PCT 2004-11-29 2 100
Correspondence 2005-03-09 1 26
Correspondence 2005-03-21 1 32
Assignment 2005-03-08 9 295
Correspondence 2005-03-08 2 46
Assignment 2005-03-24 1 28
Fees 2006-05-12 1 40