Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 94/16715 PCT/US94/01033
21S~ 787
SELECTIVE CELL PROLl~RATION
Cross-Reference to Related Application
This is a co"li"ll~tion-in-part application of U.S. Serial No. 08l009,593, filedJanuary 27, 1993.
Background of the Invention
1. Field of the Invention
This invention relates to methods and compositions for the production and use
of a desired or target cell population. Target cell populations include populations of
stem cells and their differentiated progeny from systems such as the hematopoietic
system which gives rise to lymphoid and myeloid cells, as well as the various organ
systems, tissues, and supporting celhll~r matrices. Target cell populations of the present
invention are useful for cell transpl~nt~tion, as a therapy or prophylaxis against disease
or infection, for recolLsLi~u~ion of a deficient or mi~ing cell population, as a means for
introducing genetic information into the patient, and as a cure for congenital or acquired
genetic defects and other abnormalities.
2. Description of the Related Art
The hematopoietic system is responsible for supplying all of the morphologicallyand functionally recognizable cells of the human blood system. The fully mature cells
of the blood circulatory system inclll~le the erythrocytes, platelets, neutrophilic
granulocytes, monocytes, eosinophilic granulocytes, basophilic granulocytes, B-
lymphocytes and T-lymphocytes. A striking feature of blood cells is their relat*ely brief
life span. This requires the constant regeneration of the total blood pool throughout
life.
The current accepted model of the physiologic system used by the body to
constantly produce the many distinct blood cell types is the stem cell model of
hematopoiesis. In this model, fully mature cells are replaced, on demand, by
morphologically recognizable dividing precursor cells. The precursor cells are of distinct
lineages for respective mature cells such as erythroblasts for the erythrocyte lineage,
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myeloblasts for the promyelocyte lineage, and megakaryocytes for the platelets. The
precursor cells derive from yet more ~lhlliLive cells that have been classified as
progenitor and stem cells. This definition is derived from the functional properties of
the cells that are responsible for the production of the more differentiated cell lineages.
S Some of these cells are the precursor cells of one distinct lineage, such as the
erythrocyte progenitor cell called the burst-forming unit - erythrocyte (BFU-E) and the-
burst forming unit - megakaryocyte (BFU-MK). Others are multi-potential cells such
as the colony-forming unit-granulocyte/macrophage (CFU-GM), responsible for the
granulocyte and macrophage lineage cells, and the colony-forming unit - lymphocyte
(CFU-L) responsible for both the T- and B-lymphocyte lineage cells. A lineage map of
the hematopoietic system is depicted in Figure 1.
As ~;ullelllly viewed, hematopoiesis llltim~tely derives from a pool of
undirrerellliated cells called stem cells. The most ~lil~liLive stem cells are defined by
two functional ~riteri~ the potential to self-renew and thus m~int~in the stem cell pool
and the capacity to give rise to progeny that are the coll~ ed precursors for all single
and multiple hematopoietic lineages. The most primitive stem cells are believed to be
extremely rare, being no more than 1 in 104 in bone marrow cells and most likely rarer
than 1 in 106. Therefore the entire pool of the most ~ ive pluripotent stem cells in
the body is probably no greater than 1 to 2 x 106 cells. This pool of undirr~ren~iated
stem cells gives rise to more differentiated bone marrow stem cells by division and
differentiation as well as the progenitor and precursor cells. A progenitor cell, which
is morphologically indistin~ h~ble from a stem cell, is cl llllllilled to one or more
specific cell lineages and a precursor cell is collllllilled to a specific cell lineage.
Ultimately, through a catenated sequence of proliferation and differentiation, these rare
stem cells regenerate the entire hematopoietic system. This process goes on coll~ lly
throughout life, producing more than 1011 cells per day in the human adult.
Stem and progenitor cells together make up a very small percentage of the total
nncle~ted cells in the bone marrow, spleen, and blood. These cells are about ten times
less frequent in the spleen than in the bone marrow and even less frequent in the blood.
Most of the mononucleated cells (MNC) in the blood, the so-called white blood cells,
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are neutrophils (the majority) and lymphocytes comprising greater than about 92~o of
the MNC. A liter of blood contail~s about 7.5 x 109 white blood cells and about 240 x
109 platelets.
The membranes of cells of the hematopoietic system contain distinct receptor
proteins that have been used to classify and identify different cell types in terms of their
morphological and functional phenotypes. Alarge number of these membrane receptors
have been identified and used as antigens against specific monoclonal antibodies. Many
of these antigens are found in only one specific lineage of cells. In particular, specific
combinations of antigens have been found to be useful to identify specific cells and
lineage of cells in correlation with cell morphology and/or function. One antigen,
CD34, has been found on the most ~ ive progenitor and stem cells found in both
the bone marrow and the blood (U.S. Patent No. 4,714,680). This antigen is
developmental-stage specific and not lineage-dependent. It appears on normal human
bone marrow and blood cells, on colony-forming cells for granulocytes-macrophages
(CFU-GM), on colony-forming cells for erythrocytes (BFU-E), on colony-forming cells
for eosinophils (CFU-Eo), on multi-potent colony-forming cells for granulocyte-
ely~hloid-macrophage-megakaryocyte (CFU-GEMM) and on i"""~ . e lymphoid
precursor cells. In human bone marrow, this antigen appears on about 1.8% of norrnal
marrow cells and on about 0.2~o of normal peripheral blood cells. CD34 does not
appear on normal, mature human lymphoid or myeloid cells. Therefore, the CD34
antigen is useful for the identification of early progenitor and stem cells of the human
hematopoietic system. Some of the antigens used for the diagnosis and c~ ific~fion of
the hematopoietic system are shown below:
Anti~en Distribution Anti~en Distribution
CD1 Thymocytes CD19 ~arly B cells
CD2 T cells CD20 Pan B cells
CD3 Pan T cells CD21 Mature B cells
CD4 Helper T cells, CDæ Pan B cells
monocytes CD23 Activated B cells
CD5 Pan T cells CD33 Earlymyeloidcells
CD7 Pan T cells CD34 Stem cells
CD8 Cytotoxic/ CD38 Activated T cells,
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suppressor T cells plasma cells
CD9 Early hematopoietic cells CD41 Platelets
CD11c Monocytes CD45 Leukocytes
Because hematopoietic progenitor and stem cell populations express the CD34
antigen, anti-CD34 antigen antibody has been used to select subpopulations of
hematopoietic cells for research and therapeutic purposes. It was found that transplants
cont~ining bone marrow cells that lack the CD34 antigen (34-) fail to engraft, whereas
transplants of a small number of CD34+ cells produce engraftment. Other antigensexpressed on cells co~ led to myeloid and lymphoid lineages, together with antigen
specific antibodies have been used to select for a population of cells that is not only
34+, but lineage negative (lin-). The 34+/lin- cell population in bone marrow
comprises less than 0.5% of the total mononuclear cell population and is even a smaller
percentage in peripheral blood. Further, selection of yet a smaller subset of 34+ cells,
by isolating cells failing to express HLA-DR (DR-), CD38 (38-) and CD45 (45-) as well
as failure to stain with the dye Rhod~mine 123 (Rho dull), produces the most ~lh~ ive
stemcells. Thesecellsrepresentabout0.01%to0.1%ofthebonemallowmononuclear
cells, presllm~bly at a lower percentage in the peripheral blood, and morphologically
appear as small hypogranular lymphocytes. Cells with these attributes are generally
found in the Go phase of the cell cycle and display the capacity to differentiate into cells
of recognizable myeloid and lymphoid lineages in in vitro culture, in the severecombined immlmodeficient (SCID) mouse model and in other animal systems.
Several techniques are used in clinical and research laboratories to identify
classes of cells and to select subsets of cells of the hematopoietic system. One technique
is fluorescent activated cell sorting (FACS). In this technique, a multicolor flow
cytometer is used to detect and separate cells bound with fluorescent conjugatedantibodies to the specific antigens that identify the development stage or lineage stage
of the cells of the hematopoietic system (see Fig. 1). Briefly, detectable fluorescent
signals are generated by hitting the cells with a laser beam as they pass through a flow
sheath. A nonfluorescent forward scatter signal is used to represent volume and a side
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scatter signal detects cellular texture and gr~mll~rity. The color signals of the
fluorochromes used to conjugate with the antibodies detect the cell specific antigens.
FACS analysis is generally able to analyze two or three colors simlllt~neously, and from
this data, computer programs generate contour plots, dot plots, histograms, perform
statistical analyses and the like. Some flow cytometers have sorting capability to isolate
and gate sub-populations of cells physically from the sample being analyzed with greater
than 95% purity. Most flow cytometers are able to analyze particles as small as 0.5 ,um
and to detect cell membrane receptors with a density of about 2000 molecules per cell.
By such means cell populations that are 34+ and subpopulations such as 34 + /DR-/33-
/19-/3-/38- can be detected and analyzed, thereby enabling the i(lentification of
stem/progenitor cell transplants that are rich in the cells most useful for ensuring both
long-term engraftment and short-term hematopoietic recovery.
Other techniques are also used to isolate 34+ and 34+ subpopulations of cells
using the physical separation technique of immnnoseparation. Antibodies against
specific receptor molecules are used in conjunction with immnno~ffinity columns or
incubation containers to bind cells having the specific target receptor while,
theoretically, cells which do not have the target receptor remain unbound. The cells are
physically removed from the antibody complex by employing physical fluid sheàr forces.
With an afflnity column, cells are flushed from the cohlmn into a separate container
while in the case of an incubation device, the non-bound cells are first flushed from the
device and the bound cells are physically removed by fluid shear into a separate device.
By such me~ns, it is possible to select and enrich in concentration specific cell
populations such as 34+ /lin- which are particularly desirable for stem cell transplants.
However, non-specific binding of cells to the affinity antibodies can cause col~L~ tion
by cell populations of undesired cell types and can reduce the efficiency of cell selection.
Stem cell transplants are used to treat a number of diseases and disorders
comprising the groups: hematopoietic m~lign~ncies, m~lign~nt solid tumors of non-
hematopoietic origin, immnne disorders, and diseases resulting from failure/dysfunction
of normal blood cell production/maturation. Among these are congenital disorders,
inclll-ling severe combined immnnodeficiency (SCID), Wiskott-Aldrich syndrome,
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Fanconi's ~nemi~, congenital red cell aplasia, lysosomal storage disease, cartilage-hair
aplasia, thalassemia major, aplastic anemia, leukemias, acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), andother hematologic mel~nom~c, lymphoma, multiple myeloma, hairy cell leukemia,
m~ n~11t histiocytosis, myelodysplastic syndromes, solid tumors such as breast
carcinomas, and neuroblastomas.
Three sources of hematopoietic cells are used for therapeutic stem cell
transplants, bone marrow, peripheral blood, and cord blood. The most commorily used
transplants are bone marrow transplants for the restoration of hematopoiesis in cancer
patients who receive high-dose chemotherapy and/or ablative radiation therapy or in
patients who have defective hematopoiesis. Peripheral blood transplants are now being
advanced as an alternative, less costly, less invasive technique to bone marrow
transplants.
Cord blood has also recently been used succeccflllly for the hematopoietic
reconstitution of children with lethal disorders of hematopoiesis, as an alternative to
marrow-derived cells. Cord blood is obtained from the umbilical cord and placental
blood of newborns which is otherwise discarded. Cord blood conlail s about the same
number of progenitor stem cells as does adult bone marrow per unit number of
mononucleated cells (U.S. Patent No. 5,004,681).
Three types of stem/progenitor cell tr~ncpl~nt.c are used, syngeneic (identical
twin) transplants, allogenic transplants, and autologous transplants using the recipient's
own cells. Syngeneic transplants are not rejected and do not cause graft-versus-host-
disease (GVHD). Allogeneic grafts, taken usually from a human leukocyte antigen
(HLA)-identical donor, are the most common form of transplant used by about 30-405~
of patients having a suitable donor. Patients that receive transplants from a
phenotypically-matched identical parent or related donor but micm~t~hed for orlly an
HLA-A, -B, or -D locus have transplant results comparable to those receiving
transplants from an HLA-identical sibling. Transplants are less succescflll for recipients
micm~t-~hed for two or more HLA loci. A smaller number of patients receive
transplants from unrelated, HLA-identical donors. In North America, less than 30% of
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patients are able to find an HLA-matched sibling and only 3-5~o have a one-locusmi~m~trhed relative. Therefore, most patients using an allogeneic transplant must find
an HLA-unrelated volunteer. Recent studies on unrelated donor transplants have shown
that even a single base-pair mi~m~trh in the DNA coding for the HLA loci can lead to
clinical complications. Polymerase chain reaction (PCR) assay methods are now being
investigated as a possible means to determine true, proper HLA-m~tclling~. Autologous
bone marrow transplants are sometimes used to treat patients who do not have an HLA-
identical sibling or cannot find a suitable HLA-matched donor.
The traditional bone marrow transplant (BMT) was developed initially as a
treatment for disorders of the bone marrow, inclll~linp leukemia and aplastic anemia.
At one time, bone marrow was thought to be the only tissue where the pluripotent stem
cells reside and hence bone marrow transplant was necessary to replace these cells when
the marrow was lethally ablated by radiation therapy and/or high-dose chemotherapy.
It is now known that these stem cells are also present in the peripheral blood.
Bone marrow is collected by an invasive surgical procedure requiring general
anesthesia. About one to two liters of bone marrow is extracted with a large-bore
needle inserted into a large bone, almost always the pelvis in an area just distal to the
iliac crest. One hundred or more separate aspirations are usually required. The
marrow is then purified to remove most of the extraneous cells and blood. After
purification the marrow is frozen and stored for future BMT reinfil~inn.
In the case of bone marrow transplants for the treatment of m~lign~ncies, after
the extraction procedure the patient is subjected to ablative chemotherapy and/or
ablative radiation therapy in an attempt to rid the patient's body of all tumor cells. The
cells are often treated with cytotoxic chemicals, selected to kill preferentially any tumor
cells. In this procedure, the patient's bone marrow cells are essentially killed leaving
the patient hematopoietically deficient. The patient's own marrow is then thawed and
reinfused. In the case of an allogeneic transplant, the donor's own marrow is used and
infused. The marrow cells then engraft, usually within 3 to 4 weeks, and establish
hematopoiesis. During this time period the patient is required to undergo intensive and
expensive hospital care requiring repetitive platelet tr~n~ ns to ~r~vell~ episodes of
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bleeding caused by cytothrombopenia. In addition, during this period, the patient is
susceptible to serious and sometimes fatal infections because of neutropenia.
Serious complications often arise from transplantation therapy, in addition to side
effects, that lead to patient morbidity and mortality. In the case of bone marrow
transplant, about 200 ml of whole marrow are infused into the patient's circulatory
system. This large volume causes side effects in patients including hemoglobinuria, red
urine, nausea, elevated blood pressure, fever, chills, vol~liLhlg and tachycardia. Early
complications of bone marrow transplants include chemotherapy and radiation toxicity,
graft rejection, GVHD, immnne deficiency, infections, and interstitial pneumonitis.
Interstitial pnenmonitic is also caused by drug and radiation toxicity, as well as by
opportunisticinfections causedbypost-transpl~n~tion immnnodeficiency. Opportunistic
infections from bacterial and fungal org~nicmc are also a complication. The major
concern in autologous transplants is the possible reintroduction of m~lign~nt cells with
the wyo~reserved marrow. Different methods are used to attempt to purge tumor cells
from the marrow prior to infusion, inclll-ling density cellL~irllg~tion~ monoclonal
antibodies, and ph~rm~cologic techniques.
Morbidity and mortality from GVHD are major problems. GVHD occurs when,
in an allogeneic transplant, the donor's T-cells recognize the recipient's cells as foreign
and attack them. Post-transplant immnnosuppressive treatment with drugs
(methotrexate, cyclosporine A) is ~(lminictered to prevent or ",i"i",i,e GVHD. T-cell
depletion ex vivo before transpl~nt~tir~n is sometimes used to prc;vell~ GVHD. While
this approach decreases the risk of GVHD, it is associated with increased graft failure
and recurrent lellk~mi~
Instead of infllcing whole marrow, therapeutic approaches have recently been
developed to separate stem/progenitor cells from extracted bone marrow. In this case,
only the much smaller population of stem/progenitor cells is infused in a volume of
about 10 ml. Using this technique the amount of reinfused cells can be reduced by as
much as 100-fold, greatly reducing the morbidity associated with whole-marrow
transplantation.
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In peripheral blood transpl~nt~tiQn, stem cell isolation is also used to enrich the
transplant with stem/progenitor cells. Peripheral blood stem cell transplantation is less
invasive to the patient than the bone marrow aspiration. Cells are collected from a
patient by leukaphoresis during which the patient is connected to a ce~ll, irllg~tion device
used to separate the white blood cells and return the red blood cells and platelets
continuously to the patient. Peripheral blood is sometimes also enriched in
stem/progenitor cells by mobilizing progenitor cells in the patient's blood using
cytokines such as G-CSF and GM-CSF, prior to extraction of the blood. By these
means, it is possible to provide progenitor stem cell levels in the peripheral blood about
equal to levels found in bone marrow. Benefits of primed peripheral blood stem cell
transplants with progenitor stem cell separation/enrichment include the ability to screen
out suppressor T-cells in allogeneic transplants to reduce GVHD. In addition, since it
is believed that all or most tumor cells are not part of the stem/progenitor cell
population, by using positive selection of progenitor stem cells there is also the benefit
of fill~loved purging of tumor cells.
In the clinical ellviro~ ent, the level of progenitor stem cells is usually assayed
using colony-forming assays for lineage progenitor cells, the most common assays being
colony-forming units-granulocyte macrophage (CFU-GM) and burst-forming units-
erythrocytes (BFU-E). CFU-GM and BFU-E assays are used to correlate the number
of progenitor stem cells required to obtain succes~ l engraftment of transplants. The
review of the literature shows that sncces~ll engr~*Tnent correlates against CFU-GM
(U.S. Patent No. 5,004,681). The range of CFU-GM per transplant is 2-425 x 104 per
kg body weight. An acceptable Illillillllllll level is greater than 10 x 104 CFU-GM per
kg body weight per transplant.
Extensive studies have been conducted to expand hematopoietic stem and
progenitor cells, the CD34+ cells, with various degrees of success. The basic approach
in most of these studies involves (a) enrichment of CD34+ cell by positive and/or
negative absorption to a purity of greater than 80~o, (b) culture of cells in basal
medium, for example, RPMI-1640 or Iscove's modified DMEM cont~inin~; fetal calf
serum and on a stromal layer as feeder cells, and (c) supplement of a cocktail of
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cytokines which include IL-1, IL 3, IL-6, colony stim~ ting factors (CSFs),
elylhlol~oietin (EPO), and stem cell factor (SCF). More recently, some studies have
been carried out in serum-free medium without stromal cells which achieved comparable
results in cell expansion.
Based on these studies, several observations have been made. First, there is
expansion of CD34+ and progenitor cells. Up to a 100-fold cell ~Yp~n~inn has been
reported for 12 to 14 days. It is likely that the variation in cell expansion is donor-
dependent. Second, during culture, cell differentiation towards the myeloid lineage is
indicated, le~(ling to a mainly monocyte and macrophage population in long-term
culture. Finally, the percentage of CD34+ cells in the total cell population drops
precipitously to less than one percent in about 14 to 21 days of culture. These results
indicate that there is presently a limited capacity, using current culture conditions, to
expand CD34+ cells, which differentiate toward myeloid lineage.
Summary of the Invention
The present invention overcomes the problems and disadvantages associated with
current strategies and designs and provides a new method for rapidly producing adesired or target cell population.
Using the method of the present invention, from a very small sample of cells or
tissue, large populations of target cells can be easily, inexpensively, and rapidly
produced. Target cells can be m~int~ined in culture, expanded, stored for later use, or
stim~ ted to further differentiate as needed. The procedures described herein are
short, straiglllrol w~rd, and applicable to many dirrerell~ cell types. Target cell
populations are useful in transpl~nt~tinn therapy and provide a number of advantages
over ~;ullell~ procedures. Multiple pools of donor cells are not necessary, nor are the
extens*e separation procedures presently required to enrich an inoculum with
stem/progenitor cell populations. Moreover, large numbers of stem cells, progenitor
cells, and precursor cells for nearly any particular tissue can be produced, as it is these
cells which are most useful in transpl~nt~tinn therapy.
As broadly described herein, the present invention is directed to a method for
producing an expanded cell culture co~ lg an enriched fraction of a desired target
~ WO 94/16715 ~ PCT/US94/01033
population of cells and further, to the expanded target cell population produced thereby.
The cell culture is enriched in the target cell fraction by culturing the cells in the
presence of a balanced selective factor u~i~ure (BSFM) which provides a balance of
stim~ tory and inhibitory effects that favors the proliferation of the target cell
population. The target population comprises a population of plill~ilive stem cells,
progenitor cells, precursor cells, cells in intermediate stages of differentiation, terminally
differentiated cells or ll~i~LLu~es thereof. These target cells are subsequently useful in
transplantation therapy.
The present invention also is directed to the BSFM composition comprising a
llli~ule of cell factors having a balance of stim~ tory and inhibitory effects which favors
the proliferation of a desired cell population. The composition is produced by treating
a cell population with an inducing agent which preferably comprises a mitogen. Useful
mitogens inclllde plant lectins such as phytohem~ ;l,ill (PHA) or co~c~n~valin A(ConA), T-cell mitogens such as TPA or mezerein, or a T-cell monoclonal antibody such
as O~I 3. The BSFM can be selectively modified by removing or adding specific factors
to favor the proliferation of a dirreren~ target cell population. Alternatively, a BSFM
can be prepared from a variety of different starting cell populations, thereby creating
a BSFM-1, a BSFM-2, and so on, each specific for a dirrerell~ target cell(s) population.
In another embodiment, the present invention is directed to a method of
transplantation therapy wherein primary m~mm~ n cells (e.g. from blood, other bodily
fluids, or tissues) are cultured in vitro with a BSFM, according to the above-described
process. The resulting expanded target cell poplll~tion~ are m~int~ined or ~;lyopreserved
for later use or can be immediately introduced into a patient for transplantation therapy
or for other therapeutic or prophylactic uses. Target cells may be used to repair or
create new organs or organ systems to be transplanted, and can be utilized to
supplement existing diseased or damaged organs, tissues, and cell systems. Alternatively,
target cells can be employed to produce useful cell products such as hemoglobin from
erythrocytes.
In a further embodiment, the present invention provides a method of gene
therapy. Target cells are made by the method of the invention and directly transfected
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with a genetic sequence or infected with recombinant viral vectors co~ illg a genetic
sequence. Cells which have integrated and properly expressed the sequence of interest
are selected, isolated, and cultured in vitro. These recombinant cells are then
reintroduced into the patient. Useful genes for gene therapy inrhl-le genes whose
expression products are absent or defective in the patient, and genes and other genetic
sequences whose expression provide a beneficial effect to the patient.
One further embodiment of the present invention provides a method of
transplantation therapy or prophylaxis wherein a patient is treated with BSFM in vivo
to selectively proliferate a particular target population of the body. In vivo BSFM
therapy can be employed to create or repair organs or cell systems which have become
or are expected to become diseased or injured. This form of therapy is relatively non-
invasive and ;ullelllly unavailable for such a wide variety of cells.
Brief Description of the Drawin~s
Figure 1 The hematopoietic chain and its correlation with developmental-
specific antigens.
Figure 2 Diagr~mm~tic representation of the methods which may be usedto produce various populations of target cells with a BSFM.
Figure 3 A represe"L~live micrograph showing the colony-forming units of
a BSFM treated PBMNC culture.
Figure 4 A representative micrograph showing the burst-forming units of a
BSFM treated PBMNC culture.
Figure 5 A representation micrograph of unfractionated cells one day after
BSFM treatment (10x).
Figure 6 A representative micrograph of unfractionated cells three days
after BSFM tre~tment (10x).
Figure 7 A represellLa~ive rnicrograph of fractionated cells one day after
BSFM treatment (10x).
Figure 8 A representative micrograph of fractionated cells three days after
BSFM treatment (10x).
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Figure 9 A representative FACS analysis of CD34+ CD38 DR cells from
a unfractionated PBMNC culture after six days of incubation.
- Figure 10 A representative FACS analysis of CD3 + CD33CD34 cells from an
unfractionated PBMNC culture after five days of incubation.
Description of the Invention
Current techniques of stem cell transplants require invasive and costly methods
to provide sufficient quantities of stem/progenitor cells to recolLslilule fully the patient's
depleted or mi~ing cell system. Using the method of the present invention, in a very
short time and at low cost, large numbers of cells can be produced allowing for not only
the complete reconstitution of the depleted or mi~ing cell system, but also the flexibility
of having sufficient cells to permit multiple or cyclic tre~tment~ if more than one is
deemed necessary. Also, current techniques typically require the purification of cells
from very large pools of biologic~l fluid. Using the method of the present invention,
large numbers of cells can be produced from very small samples of biological fluid. This
saves the expense and time of using inefficient separation techniques to enrich a target
cell population, uvercollles the risk that a co~ nt exists in one of the many samples
used to create the pool from which the relatively few stem cells are collected, and even
more importantly, provides a consistent source which one of ordinary skill in the art can
modif~r to provide the highest level of a desired product.
1. Target Cell Selection
The method of the present invention is broadly applicable to the selective
expansion of an extremely diverse population of cell types. Accordingly, the first step
in this method comprises the selection of a desired target cell or ",.~Llule of target cells.
In the practice of the method described herein, one or more cell types present in an
original cell population can be preferentially expanded to enrich the fraction of the
target cell(s) in the expanded cell population. Thus, a cell type present at very low
levels in normal cell samples can be selectively proliferated to increase its fraction in
the expanded population. During this selective expansion and enrichment, the non-
target cells in the population can be allowed to die off, to remain unexpanded or to fall
in number and/or proportion in the expanded culture. One of the important clinical
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advantages of the method of this invention is that cell populations co,,l~illil,g a high
fraction of the selected target cell(s) can be produced simply and in many cases without
need for separation or purification steps or the addition of separate and expensive
cytokines. In some irstances, the desired .-Yp~ncled cell population will inclll~le a
number of cell types whose fraction, relative to physiological cell samples, is increased
or the ratios of these targets cells changed to f~cilit~te a particular utility. Where, for
example, hematopoietic reconstitution is the objective, a cell population substantially
enriched in stem cells and progenitor cells to provide long-term engraftment may also
inchl(le specifically tailored fractions of precursor cells or terminally differentiated cells
to provide for short-term positive effects.
It is also possible, using the method of the present invention, to produce a cell
population that includes a substantial fraction of cell types not present in the starting
cell population. This can occur by providing conditions that favor the differentiation or
dedifferentiation of cells along their known dirrert;-lliation pathways.
The target cell can be selected from any m~mm~ n cell and preferably a
primary mzlmm~ n cell and more preferably a primary human cell. A target cell
population or the m~mm~ n cell population from which it is selected may comprisean entire cell lineage, organ, or organ system incl~ ing the stem, progenitor, and
precursor cells of that system. Examples of tissues and organs from which such cell
populations can be selected include hematopoietic cells, liver cells, pancreas cells, kidney
cells, brain cells, nerve cells, heart cells, lymph node cells, thymus cells, spleen cells,
bone marrow cells, bone cells, cartilage cells, muscle cells, endothelial cells, epithelial
cells and the like. The preferred target cells are hematopoietic cells. Where the
llltim~te use for expanded cell populations involves tissue repair or regeneration, it will
be appreciated that mixed populations of cell types norm~lly found in such tissue can
be the desired target cell ll~i~lule or can be prepared separately and combined in vivo
or in vitro to facilitate such an objective. In a preferred approach, the target cell
population incllllles stem cells and/or progenitor cells which can, either during the
preparation of the expanded cell population or during its use, provide diL~el-ellLiated cells
in the pathway of the stem/progenitor cell lineage.
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- In the hematopoietic system, the desired target cells can include pluripotent stem
cells of the most p~ iLive type as well as progenitor cells, precursor cells, cells at
intermediate stages of differentiation and terminally differentiated cells or mixtures
thereof. These cells include myeloid progenitors such as CFU-G (granulocyte), CFU-
GM (granulocyte/macrophage), CFU-GEMM
(granulocyte/erythrocyte/macrophage/monocyte), CFU-E (erythrocyte), CFU-MK
(monokaryocyte), CFU-M (mac~ophage), CFU-Eo (eosinophil), CFU-Ba (basophil),
BFU-E (erythrocyte), and BFU-MK (megakaryocyte), and lymphoid progenitors such
as CFU-L (lymphocyte), CFU-B (B cell) and CFU-T (T cell). Also useful are target cell
populations comprising T lymphocytes, B lymphocytes and their co~ Led precursors.
Myeloid lineage cells such as megakaryocytes (platelets), erythrocytes, granulocytes,
macrophages, monocytes, basophils, eosinophils and their committed precursors are also
desired targets alone or as mixtures with other target cells.
Of particular interest from the standpoint of use in hematopoietic reconstitution
are cells expressing the CD34 antigen. CD34+ cells can incllltle plillliLive CD34+1in-
cells as well as those more ~ lliLive cells characterized as CD34+CD38-CD45-DR-
RH0123(Dull). Also of interest in treating immllne disorders and diseases are cells
e~ressillg T cell antigens such as CD3, CD4 (helper T cells) and CD8 (suppressor T
cells). Erythrocytes are also a desirable target cell both for use of the cell population
in transplant therapy and for use in the production of hemoglobin.
One skilled in the art will appreciate which target cells and target cell mixtures
are useful for the applications described herein and other applications employing
specifically tailored cell populations.
Cells and cell populations made by the method of the invention are analyzed
morphologically, antigenically, and functionally. First, preferentially expanded cell
populations are ~x~mined microscopically for size, characteristics such as the presence
or absence of granules, and overall appearance. Stem cells and other relatively
undifferentiated cells are small with well defined membranes, whereas, more
differentiated cells are generally larger. Second, stem cells express characteristic
antigens which are detectable in assays such as an ELISA or a RIA using antigen-
WO 94tl6715 PCT/US94/01033
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specific monoclonal antibodies and are sortable by FACS techniques. Results ~romthese antigen-binding assays provide a developmental map of cell lineages. Lastly, cells
are also analyzed functionally by their ability to form colonies and/or to perform other
biological functions in vitro.
2. Selection of a Starting C: ell Population
In the plerelled embodiment of the present invention, a first cell population isselected and induced to produce a BSFM which comprises a ll~LLlu~e of cell factors
obtained from the first cell population and having a predetermined balance of
stim~ tory and inhibitory effects which preferentially favor the expansion and
enrichment of the desired target cell(s). These first cell populations comprise primary
cells of the blood, bone marrow, body tissues of hnm~n~ or nonhnm~n~, preferablym~mm~ n tissues, or established cell lines. This first cell population can be of the
same cell type as the desired target cell, contain cells of the target cell type, contain
cells that differentiate to cells of the desired target cell type or cells that are of a
completely dirrerenl type from the desired target cell type. In one embodiment of this
invention, this first cell population can be the cell population that is llltim~tely expanded
as described below.
It is generally preferred, however, to employ a separate cell population of the
same type to be nltim~tely expanded. Thus, for the hematopoietic cell expansion, a
blood-based cell sample can be couve', ,ently employed. Often it is desirable for the first
cell population to comprise a portion or subpopulation of the cell population that will
be eYrl~n~led as described below. It is contemplated, however, that tissue cells of non-
hematopoietic origin may be expanded by in~ ing a first population of hematopoietic
cells to produce a BSFM that will selectively expand the target cells in the tissue.
With reference to the hematopoietic system, the first cell population useful to
produce a particular target-specific BSFM preferably is selected from peripheral blood
cells, cord blood cells and bone marrow cells. Due to numerous clinical advantages, the
selection of peripheral blood cell populations is preferred. First, peripheral blood is
easy to obtain requiring a relatively noninvasive procedure (needle stick). No hospital
stay is required, the risk of infection is very small, and the procedure can be performed
WO 94/1671~ PCT/US94/01033
'
~5
-17- d',~
- by most health care workers. Second, peripheral blood samples can be taken with little
regard to the health of the individual because blood loss to the patient has little to no
systemic impact. Third, peripheral blood is already used as a source for a number of
medical assays. Standard procedures and supplies are presently available in every
medical office.
The peripheral blood cell populations useful as the starting first cell population
can include whole peripheral blood (e.g., an as-removed blood sample) and peripheral
blood mononucleated cells (PBMNC). The source of PBMNC can inchl-le the product
of known collection and separation techniques. For example, the buffy coat fraction
from a coarse centri~lg~l separation can be employed as well as finer separationfractions from specific gravity-based systems such as FICOLL. It is also possible to
employ as the first cell population highly purified PBMNCs or a specific subpopulation
thereof having a particular cell type (e.g., T-lymphocytes) by employing affinity-based
separation techniques. As described below, it is often useful to employ PBMNCs that
have undergone minim~l separation procedures as they may cont~in useful accessory
cells and be less damaged than highly processed cells. The first population of cells can
be used shortly after harvest and separation or can be kept refrigerated or frozen for
future use.
3. Inducing the First Cell Population
An important aspect of the invention is the ability selectively to expand and
enrich target cells in a cell population in the presence of a BSFM. Production of this
BSFM is plefelably accomplished by inflncing the first cell population described above
to produce this target-specific ~ Lule of factors. Thus, the in~llcing step should be
controlled to produce the desired BSFM. The inducing step can be performed on the
first cell population in a separate step or as a part of the target cell expansion step when
the second cell population described below is the same as the first cell population. In
a preferred embodiment the first cell population is induced by an added in(lll~ing agent
and also may be further induced during culture by factors produced by the cells, e.g., in
a cascade fashion.
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-18-
In the preferred embodiment, the target cell(s)-specific endpoint directing nature
of the BSFM is influenced by a combination of physical, ~hemi~ l and biological
parameters. One such parameter of particular cignific~nce is the nature of the added
inducing agent, as described below. Another parameter that can be varied to change
the target cell(s) is the nature of the first cell pop~ tion~ as described above.
Applicants have found for example, that, in the context of hematopoietic cells, relatively
unprocessed, coarsely separated PBMNCs such as those obtained from a leukaphoresis
treatment produce BSFMs that are targeted to stem/progenitor cells such as CD34+cells. Other factors that can influence the BSFM inducing process include culture
conditions, e.g., medium, temperature, time, pH and the like.
In one preferred embodiment the in-lnring step is carried out by culturing a
selected first cell population in a medium which supports leukocyte growth, to which has
been added an in~ cing agent. The medium preferably can be serum-free and does not
require stromal cell involvement. Suitable medium types in~ le ISCOVES
modification of DMEM, RPMI 1640, and CCM-2 produced by Verax Corporation of
Lebanon, New Hampshire.
The added in~ çing agents that are useful according to the ylefelled
embodiment in general comprise m~teri~l~ that have a mitogenic effect on the cell ~pes
of the first cell population. Mitogens are known for various cell types and the effects
of such mitogens in inducirlg various factors have been observed for hematopoietic cells.
T-cell mitogens are often used for this process.
Among the classes of mitogens that are useful to induce or f~rilit~te the
induction of BSFMs from hematopoietic cells are plant lectins, T-cell mitogens, and
monoclonal antibodies. Particular plant lectins that have the desired mitogenic activity
in~llltle those derived from the following:
WO 94/16715 ~: - . PCT/US94/01033
~ , ?. .
19 21 S~ 78 7
Lectin Source
Phaseolus vulgaris (PHA, phytohem~gl~ ;")
Dolichos biflorus
Sol~nllm tuberosum
Sophora japonica
Maclura pomifera
Pisum sativum
Ulex europeus (UEA-I, U. europeus ~g~
Ulex europeus (UEA-II, U. europeus agslll~i
Arachis hypogaea
Glycine max
Canavalia ensiforrnis (Con A, conc~n~valin A)
Triticum vulgaris (WGA, wheat germ ~g~ 1 l i " i ")
Ricinus colllllllllli~ (RCA-I, R. colllllllllli~ i"ill I)
Lycopersicon esculentum
Phytolacca americana (PWM, pokeweed mitogen)
Listeria monocytogenes (LPS, lipopolysaccharide)
A particularly useful group of plant-derived mitogens includes PHA, ConA,
mezerein (MZN) and TPA (and related diterpene esters). TPA and some of its related
compounds are set forth below:
Phorbol 12-myristate-13-acetate (TPA)
Phorbol (4-0-methyl) 12-myristate-13-acetate
Phorbol (20-oxo-20-deoxy) 12-myristate-13-acetate
Phorbol 12-monomyristate
Phorbol 12, 13-didecanoate
Phorbol 12,13-dibutyrate
Phorbol 12,13-dibenzoate
Phorbol 12,13-diacetate
Mitogens of non-plant origin can also be useful such as Staphylococcal
enterotoxin A (SEA), Streptococcal protein A, gal~ct~ce l~xirl~e and T-cell antibodies
such as OKT3. Interferon-alpha (IFN~) and IFN,~ can also be used as in~ cing agents
in some circumstances as well as stem cell proliferation factor (SCPF), stem cell factor
(SCF), any of the growth factors and the bone morphogenic proteins (BMPs).
In the preferred induction process, the first cell population selected as described
above is cultured in the presence of an enh~ncing (i.e. potenti~ting) agent prior to the
actual indllcing step. Agents useful to enhance the induction inchlcle TPA (or related
WO 94/16715 PCT/US94/01033
.. 2~ 8~ -20-
phorbol esters) MZN, IFN~ and IFN,~. This step can then be followed by addition of
an above-described inclllcer to the culture. Most prefelled combinations involve the use
of IFNa and IFN,~, and MZN as an enh~n~er, and PHA, ~onA or OKT3 as an inducer.
Selection of an inducer and/or enhancer from the above-listed agents or ltheir
known equivalents can be made without concern for their cytotoxic or tumorigenicproperties since the BSFM is preferably separated, as a supern~t~nt from the treated
cells. This step can be perfQrmed in a way to ensure that the BSFM is not
co~ ,i"~ted with any harmful residual mitogens or by-products thereof. In the case
where intlll~ing or enh~ncing agents such as INFs and T-cell antibodies are employed,
the separation of BSFM from cells or cell products/debris may not be necessary.
The amount of in~lllcin~ agent used and the length of treatment can be
empirically determined for each specific cell culture treated. The preferred amount of
indllçing agent used is from about 5 ug/ml of medium to about 100 ug/ml of medium.
More preferably, for the described hematopoietic cell in~ tion process, the in(lllçing
agent can be added in an amount of from about 5 ug/ml to 50 ug/ml, most preferably
about 20 ug/ml to 50 ug/ml when treating first cell populations of whole or fractionated
blood. In those cases where a potentiator/enh~ncer is employed, it can be added to the
medium at a level of from about 5 ng/ml to 500 ng/ml, preferably 5 ng/ml to 50 ng/ml
and most preferably 10 ng/ml to 20 ng/ml. Tre~tmen~ times can be from hours to days,
preferably from about 1 day to 10 days and most preferably 2 days to 5 days.
~ltPrn~tively, treatment may involve multiple treatments over a longer period of time,
or more intense treatments over a shorter period of time.
One skilled in the art can select the desired induction conditions, agents and
starting cell populations based on the te~rhinp;c cont~ined herein and simple
experiments to correlate changes of initial con~litiQns with target cell effects. The
present invention is based at least in part on the discovery that employing an induction
factor ~ LLule (BSFM) having a balance of both positive and negative effects permits
the controlled selective expansion of any number of desired target cell populations.
The induction step has been described in the prefelled in vitro mode, but it is
possible to perform this step in vivo in certain ~;h~;ull~ ces. Thus, in the case where
WO 94/16715 PCTIUS94/01033
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-21- ~ S
a cell population (either circ~ ting or static) can be treated with an int11lcin~ agent that
is not otherwise harmful to the patient, the BSFM can be generated in vivo and either
collected from the patient for further use ex vivo or left in the patient to mediate the
selective target cell expansion in vivo, e.g., at the site of tissue repair or regeneration.
An intllll~ing agent lltili7e~1 in vivo may be injected or infused in the patient, taken orally,
or directly placed on the cells to be treated by, for example, transdermal patch, and may
involve a single treatment or multiple treatments.
4. The Balanced Selective Factor Mixture
The present invention provides a family of BSFMs, each clesigned to act on a
particular second cell population in a specfflc way to cause expansion and enrichment
of the desired target cell(s). All of these BSFMs share the characteristics of providing
a mixture of cell factors that have a balance of stim~ tory and inhibitory effects which
preferentially favor the proliferation of the selected target cell(s). Prior approaches
have generally focused on the use of defined proliferative (i.e. stimlll~ting) factors in
connection with nallowly selected cell types to determine the positive effect of these
factors or combinations of factors. The methodology of the present invention is based
on the use of a balance of complex positive and negative effects and feedback loops
similar to those employed in nature. As a result this method is at the same time both
powerful and simple.
By way of non-limiting exemplification, the BSFM which has been shown to
~l~ferellLially expand CD34+ cells in a PBMNC population can, according to a
preferred embodiment, be prepared by in(lllçing a leukaphoresis fraction of peripheral
blood with MZN and ConA. This BSFM has been partially char~cteri7ed and found tocont~in the following known cell factors:
Cytokine Cell Source
IL 1 Macrophages
IL-2 T-lymphocytes
IL-3 T-lymphocytes
IL-4 T-lymphocytes
IL-5 T-lymphocytes
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IL-6 - T-lymphocyte, macrophages,
lymphoid cells, monocytes
IL-8 Monocytes
CSFs (GM-CSF, G-CSF) T-lymphocytes, B-lymphocytes,
macrophages, monocytes
INF- y T-lymphocytes
TNFs (TNF-~x, TNF-,~) T-lymphocytes, macrophages,
neutrophils
The concentrations of some of these cytokines have been determined. For
example, the concentration of IFN- y and IL-2 are about 10,000-20,000 IU/ml and about
2000-5000 IU/ml, respectively. The concentration of the other listed cytokines can be
readily determined, for example, by ELISA assays, using co_mercially available assay
kits and/or methodologies. Examples of additional cytokines include EPO, SCPF, SCF,
any of the growth factors and the BMPs.
Given the te~hing of this invention it is possible for one of ordinary skill in the
art to characterize fully the factors cont~ined in this BSFM and determine the o~ulhllu
nature and amount of known active factors (both stim~ tory and inhibitory) which are
required for expanding the desired CD34+ cells. Accordingly, the synthesis of such
o~ e~l, defined BSFM's is within the scope of this invention. In addition, this
invention provides a means to identify and isolate new cytokine(s) which are useful or
important for e~Ypansion of a target cell and/or m~inten~nce of the target cell type. For
~Y~mplç, after removing all the known cytokines from the preparation using
immlmoafflnity techniques, the rem~inin~ components can then be tested biologically
for their ability to support the eY~pansion and/or dirrelellliation of target cells. In
addition, further tests can be conducted by reconslilulillg the known components to
prepare a BSFM of known composition and testing the ability of this BSFM to produce
the result of the induced BSFM. If it does not, those skilled in the art could determine
the mi~ing factors.
The isolated "unknown" component(s) can also be analyzed and purified by
col,ve,,lional techniques. Thus, the amino acid sequence of proteinaceous component(s)
can be determined and the genes for these component(s) can be isolated and sequenced
WO 94/16715 'C~ PCT/U594/011)3:~
-23-
by collvel I lional recombinant techniques. Consequently, these proteins can be produced
recombinantly.
Another embodiment of this invention involves the modification of one BSFM
composition by adding or subtracting positive or negative factors to produce a second
BSFM which favors preferential expansion of a different target cell(s). Several
approaches can be used to obtain BSFM preparations which favor the e~z3n~ion andenri~llment of different or additional target cells. First, one or more of the factors in
the BSFM preparation described above can be removed, preferably by afflnity
chromatography. Affinity matrices with antibodies against specific cytokines arecommercially available. For example, if BSFM is allowed to pass through an afflnity
coh1mn to remove IL-2, the resultant BSFM will no longer favor the expansion andenrichment of lymphoid lineage cell types. Also, macrophages can be removed by
adherence to plastic surfaces, thereby resnltin~ in a cell population which produces little
or no IL-1 or CSFs in the BSFM. Further, known factors within BSFM can be
selectively inactivated without isolation using factor-specific monoclonal or polyclonal
antibodies. By using a positive- and/or negative-affinity approach, modified forms of
BSFM can be obtained to favor expansion of a desired target cell (see Fig. 2).
Alternatively, based on the kinetics of cytokine induction, BSFM ~rc~ tion with
various compositions can be obtained by harvesting at di~rerelll times after induction.
For example, IL-2 is induced ahead of IFN-y. IFN-y con~ çntration is higher in BSFM
preparation harvested at 96 hours than that in BSFM harvested at 48 hours, while IL-2
concçntration will be the same in both BSFM ~repal~tions.
5. Expansion of Target Cells
The next step in the process of the present invention involves the selective
expansion of target cells of a second cell population by culturing this cell population in
the presence of the BSFM that selects for the desired target cell. As indicated above,
the second cell population can be of a dirrerellL cell type or the s~me cell type as the
first population. In many cases it will be desirable to employ an original cell population
that is divided into ffrst and second identical subpopulations for the generation and use
of the BSFM. Under certain ~;h~;u~l~Lallces the ffrst and second cell populations may
WO 94116715 PCT/US94/01033
-24-
be the same population, i.e., the same cells are first induced and then expanded, with
or without separation steps.
Selection of the desired second cell population depends on the desired target cell.
The target cell should be among the cell types in this population or derivable from it,
e.g., by differenti~tion or de-lirrerellLiation. In general, the same kinds of cell types
described above for the first cell population can be used for the second population.
The selective target cell(s) expansion can be controlled by varying the nature and
amount of the BSFM, and the culture con~lition~. Selection criteria for BSFM[s are
discussed above. The culture step is plerelably carried out in an a~l~lo~liate basal
medium, which can be supplemented with defined cytoldne(s). Culture conditions for
individual cell types may vary, but standard tissue culture conditions form the basis of
culture tre~tment Typically, cells are incubated in 5% CO2 incubators at 37C inmedia. Specific chemic~l agents, proteins, media components such as insulin or plasma,
and certain growth or colony stimnl~ting factors (CSFs) may be required for the
m~inten~nce of certain cell types. These requirements are either known in the subject
field or can be determined by one of ordinary skill in the art. By way of a non-limiting
example, the BSFM that favors CD34+ cell expansion can be cultured in serum-freemedia such as CCM-2.
The BSFM can be added to the medium in an amount sufflcient to obtain the
desired exI-~n~ion/enrichment of the target cell(s). Additive nlounts will vary
depending on the nature of the BSFM, the make-up of the second cell populahon and
the culture conditions. In practice, this ~dllition can be from about 1~ to 10% and
preferably is about 2% to 5%.
The length of the culture steps can be varied to assist further in the selectiveproliferation of the target cell. When the cell population involves cells on or induced
to enter a di~erellLiation pathway, the final target cell enrichment may depend on when
the culture is terminated. Typically, for example, a population enriched in CD34 + cells
can optimally be cultured for about 5 to 20 days and preferably for about 5 to 10 days,
depending on the extent of enrichment desired.
wo 94116715 , j~o~
Once the desired expanded target cell population is achieved it can be used at
that time, or refrigerated or frozen for future use. In an alternative embo~liment, the
- target cell population can be modified to change the population to one enriched in a
second target cell(s). For example, a CD34+ rich/lymphoid preferring rnixture can be
converted into a population enriched in T lymphocytes by further culturing the
population in the presence of known factors that promote the differentiation of T-cell
progenitors down the lymphoid line. Also, the target cells can be incubated for longer
periods of time, or subjected to varied culture media, such as media supplemented -with
factors to drive the target cells to the desired population and/or the desired stage of
differentiation. Examples of factors include IL-2, granulocyte colony-stim~ ting factor
G-CSF, macrophage colony-stim1-1~ting factor M-CSF, elyLhropoietin (EPO), interferon
(IFN), retinoic acid, SCPF, SCF, BMP and any of the growth factors such as nervegrowth factor (NGF), epidermal growth factor (EGF), fibroblast-derived growth factor
(FGF), and platelet-derived growth factor (PDGF). During this step cell cultures may
or may not be m~int~ined in BSFM-co~ ;11i11g media as conditions w~rlant.
Apart from obtaining target cells for various clinical applications, one added
advantages of this invention is the inherent purging of tumor cells during cell e~al~ion.
It has been reported that the number of some chronic myeloid leukemic cells rapidly
declines in long-term culture. The purging of tumor cells during expansion accoldillg
to this invention can be f~ilitzlted by the presence of cytokines and cells, for example,
IFN-y, TNFs, and activated macrophages which have antiproliferative and/or cytotoxic
effects on tumor cells.
As in the case of the in~ cement step, the e~allsion step can be carried out in
vivo or in vitro. In vivo applications involve introducing the BSFM into the bloodstream
for circn1~ting cell populations or into specific tissue cells. ~ltern~tively, cells can be
combined with or treated by the BSFM and then reintroduced into the body to effect
repair, reconstitution or regeneration of systems, tissue or organs.
According to one embodiment, this invention is directed to the establi~hment of
a hemostasis for a given cell culture by mimicking the "natural" control of in vivo
biological events. The definition of hemostasis is limited to the dyn~mic equilibrium
wo 94/1671~ PcTrusg4/0l033
2~5 47 ~ -26-
established as a result of cell/cell and cell/factor interactions under specific culture
conditions. Hemostasis can be attained by providing a balanced ~ lule of factors and
an array of various cell types in the culture. The balanced l)l~xlule of factors is added
exogenously and additional factors can be induced de novo during culture. For example,
addition of IFN~ and/or TNF-cY to the culture can induce the production of CSFs from
monocytes and macrophages. As a result of the specific cell/cell and cell/signalinteractions, a balanced stim~ tory and inhibitory ellvhol~ erlt is created to favor the
population's expansion of and/or differentiation towards a target cell type. This
dynamic equilibrium may change as the cell population changes as a result of expansion
and enrichment of a target cell type. Accoldillgly, the initial balanced stim~ tory and
inhibitory envirol~llent can be modified in order to m~int~in a co~ le~l expansion of
the target cell.
6. The Expanded Cell Population
The expanded cell population of the present invention can have both a greater
number of target cells (expansion) and a higher percentage of target cells (enriçhment)
in the final pop~ tion, as colllpaled to the original population. For example, cells
present in very low numbers and fr~ction~, such as stem cells, can be expanded and
enriched to give populations having greater than about 5%, preferably greater than
about 20% and most ~lefe~bly greater than about 50% of said target cells. ~or cells
origin~lly present in larger numbers or for applications requiring high fractionpopulations (e.g., erythrocytes for hemoglobin production), expanded populationscol~l~illillg at least about 50% target cells and preferably at least 80% target cells can
be produced according to the present invention. In these new populations, the target
cell can be expanded to at least about 500-fold, preferably at least about l,000-fold, and
most ~lererably at least about 10,000-fold.
As an example, one enriched cell population according to the present invention
is the hematopoietic cell population comprising about 30% CD34+ cells. This
population also has about 7.5% of CD34 + CD38-CD45-DR-Rhol23 (Dull) cells. Thesecell populations are enriched for CD34+ cells by at least about 1,500 fold over normal
peripheral blood samples.
WO 94/16715 PCT/US94/01033
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7. Uses of the Expanded Cell Population
Expanded target cell populations made by the method of the present invention
are useful for cell transplantation, tissue regeneration inçlll~ing the regeneration of
organs or cell systems such as bone marrow replacement, supplementation therapy, such
as specific cell infusion, and as a means for treating or preve~ g an infection or a
disease.
Preferably, target cells of the present invention are stem cells, progenitor cells,
or precursor cells. Stem cells, progenitor cells, and precursor cells are more easily
utilized to create more differentiated cells, whereas, the reverse is often not true. In
one embodiment, target cells are used for the regeneration or replacement of an entire
cell system, such as the hematopoietic system or of cells of only the lymphoid or myeloid
lineages. Erythroid target cell populations can be stockpiled or stored and used for
blood transfusions. Alternatively, elylhloid cells can be used for the generation of large
amounts of red blood cell expression products such as hemoglobin. Lymphoid cells can
be used in the treatment of diseases such as Bruton's ~g~mm~globlllinemi~ (B cell
deficiency), DiGeorge's syndrome (thymic hypoplasia), SCID (adenosine (3e~Tnin~edeficiency), thrombocytopenia such as Wiscott-Aldrich syndrome, and other
immlln-~logical deficiency diseases.
In general, peripheral blood stem cell transpl~nt~tinn therapy, according to this
invention, can be substituted for ~;ullen~ly used transplantation procedures such as bone
marrow transplants and peripheral blood transplants. With either of these procedures,
large amounts of bone marrow or peripheral blood are required, the collection processes
are very invasive to the patient, and the stem cells are of poor quality because of the
extensive maniplll~tion~ required for pllrifi~tion.
In contrast, peripheral blood transplantations by the ~;ullelll invention have no
such disadvantages. First, only a small sample of peripheral blood is required which is
obtained by a very low-invasive process. Second, no purification steps are necessary.
A concentrated population of autologous stem cells is produced in a single culture.
Moreover, the cells produced can be specifically tailored as required or the entire
WO 94/1671S PCT/US94/01033
4~i'8
hematopoietic system can be reconstituted. This combination of advantages is notpossible using ~ulleuLly available technology.
In accordance with this aspect of the invention, target cell populations of the
hematopoietic system can be used as a therapy and often a cure for certain diseases.
Briefly, a sample of the desired cells may be obtained from the affected patient.
Optionally, the patient may be primed before the sample is taken by ~(1mini~tering a
priming agent such as G-CSF or a chemotherapeutic agent. These cells are treated, if
necessary, to remove any diseased cells and cultured according to the method of the
present invention. The patient is then treated to destroy all affected cells of the body
with, for example, high-dose chemotherapy or radiation treatments, after which cultured
cells are reintroduced. Provided the patient can be cleared of all affected tissue, even
for a short period of time, complete destruction or removal of the affected cell system
or organ becomes a viable option.
Infections and diseases which are localized to a particular cell type or cell
population can also be treated with the methods of the present invention. This inclll~les
the anemias such as red cell membrane disorders, red cell enzyme deficiencies, disorders
of hemoglobin synthesis, isohem~ , erythroblastosis fetalis, malaria, mechanicalor chemical trauma to red blood cells, hypersplenism, sideroblastic ~nemi~, anemia
related to chronic infections, and myelophthisic anemias due to marrow irlfiltrations.
This also includes diseases of the immtlne system such as the non-Hodgkin's lymphomas
including the follicular lymphomas, Burkitt's lymphoma, adult T-cell leukemias and
lymphomas, and acute lellkemi~ litinn~l diseases also treatable by the methods of
this invention are neoplasias such as breast cell car~inom~, testicular cell carrinom~ the
solid tumors, neuroblastomas, neurofibromas, mel~nomz~, ovarian cancer, pancreatic
cancer, liver cancer, stomach cancer, colon cancer, bone cancer, squamous cell
carcinomas, adenocarcincm~ prostate cancer, and retinoblastoma, and nutritional
diseases such as malabsorption syndromes, vitamin deficiencies and obesity.
As in~liç~ted above, the present invention is equally applicable to non-
hematopoietic cell systems. Target cells produced by the method of the invention can
be used for tissue regeneration, organ replacement or supplement~tion, cell
wo 94/16715 PCT/USg4/0l033
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transplantation, or as a prophylactic or therapeutic against disease and infection. For
example, in tissue regeneration, target cell populations of undiLLerellLiated cells of a
particular tissue can be created. Liver, kidney, nerve, pancreas, skin, and bone cells may
be readily and rapidly produced in large quantities. First, BSFM is cultured with a
population of cells to create ~lillliLiv~ stem cell populations. These plillliLive cells are
then treated with specific factors such as, for example, nerve growth factor, fibroblast-
derived growth factor, GM-CSF, or elyLllLopoietin, and other agents to produce the
desired tissue.
In one embo-liment according to the methods of the present invention, skin can
10 be effectively and safely produced and transplanted to a patient. First, a small sample
of healthy epithelial cells is surgically removed from a patient and placed in tissue
culture. Next, the BSFM is pl~ared from this same sample of cells, from another
epithelial sample or other tissue s~mple taken from the same patient earlier, or from
a cell culture obtained from another source. The patient's epithelial sample is then
15 cultured in the presence of an effective quantity of BSFM. From the resllltin~ emiched
and expanded culture, large numbers of epithelial precursor cells can be made toproliferate and produce natural skin which is grafted onto the patient. When using the
patient's own cells as a sample, the risk of rejection is very low or absent, and new
epithelial cells are very rapidly produced. For patients in need of such therapy, for
20 example burn patients, speed is of the essence as the risk of life-thre~tening secondary
infections is quite severe.
In another embo~liment it is also possible to create entire organ systems from
progenitor cells. For example, a small sample of pancreatic cells is obtained from a
patient or a healthy, immnnologically matched donor. A BSFM composition is prepared
25 using this same sample of pancreatic cells which may be Islets of Langerhans cells,
epithelial pancreatic cells, hematopoietic cells or cells from another source. The
pancreatic cells are then treated with an effective amount of BSFM which expands and
enriches the culture in stem, progenitor, precursor or dirLerenliated pancreatic cells.
From these cells, a pancreas, or at least a portion of a pancreas, (e.g. Langerhans cells
30 inçl~l(ling the A, B, D, and PP cells) can be recreated and surgically reintroduced to the
WO 94/16715 PCT/US94/01033
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-30-
patient. These cells will not be immlmologically rejected and will function in the same
manner as natural cells and produce insulin. Consequently, using the method of the
present invention, in certain instances, type I diabetes (insulin dependent diabetes) could
be cured, and, at the very least, complications attributed to type II diabetes (insulin
resistant diabetes) could be alleviated. This method can also be used to produce islet
cell populations for incorporation into impl~nt~ble devices where they function under
regulation to produce insulin.
Cell populations produced by the method of the present invention also can be
used to supplement, repopulate, or totally recreate cell systems such as the kidney, liver~
gall bladder, colon, lung, selected muscle tissues, nerves, selected veins, arteries and
capillaries, tendons and li~3mentc and the cells of the gastrointestinal system. 'It is one
aspect of this invention that cells and cell populations created can be utilized for whole
organ reconstruction, ntili7ing methods currently available and known to one of ordinary
skill in the art such as those disclosed in U.S. Patent No. 5,032,508, which is hereby
specifically incorporated by reference.
Cell populations according to the present invention can also be used as a
therapeutic agent against disease or infection. Cell pop~ si- n.c to be transplanted can
be screened and treated for infections such as viruses and bacteria, which can be
effect*ely and completely elimin~ted using procedures which cannot be lltili7e~1 in_o.
For ex~mple, a small sample of a patient's fluid or tissue co~ g the infected cells
can be purged of infection and the cell numbers amplified by cllltllring with BSFM. In
vitro procedures for the selective destruction of diseased cells in transpl~nt~tion therapy
are known. Briefly, the cell sample is placed in culture and treated with, for example,
an alllivilal agent such as a reverse transcriptase inhibitor (e.g. 3'-azido-3'-deoxy-
thymidine (AZT)). BSFM treatment may be performed before, after or concurrently
with dn~ivildl treatment. Alternatively, the cells may be cloned and individually
screened to select for uninfected cells and the reslllting population expanded. At the
same time, the patient is treated with high doses of chemical agents, drugs, or radiation
to elimin~te all infected cells in the body. Once the disease is elimin~ted from the
WO 94/16715 PCT/US94/01033
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expanded cell sample and the patient, the expanded cell sample is then reinfused into
the patient, recreating a total or partial cell system.
Infections caused by viruses such as the human immllnodeficiency virus (HIV)
which causes the acquired immlme deficiency syndrome (AIDS) can be treated in this
m~nner. A small sample of blood is removed and its T-cell populations amplified
according to the method of this invention. All traces of virus are then elimin~ted from
the sample using procedures which are well known in the art and the patient is treated
with high doses of alllivi,al agents (e.g. AZT) to destroy all virus particles and, if
necessary, other agents to destroy virus-infected cells. These steps are not possible using
~ullell~ transplantation technology because secondary infections would set in and kill the
patient before transplanted cells were established and functioning. Accordillg to the
method of this invention, the patient can be infused with a sufficient amount and variety
of cells to reco~LiLuLe an active immlme system. Alternatively, an AlDS-infectedpatient can be colll;IIllously treated with a fresh supply of uninfected T-cells which had
been cleansed of virus and PYI~n(led from a sample of the patient's blood accordiug to
the method of the present invention. Although this may not elimin~te the virus, the
patient may never develop AIDS-related complications, remain symptom-free, and be
able to lead a relatively normal life. Additionally, having the patient in a stronger
physical condition may allow for more aggressive therapeutic measures which may clear
the infection.
In another embodiment, cell populations prepared by the method of the invention
could be used prophylacticly against disease or infection. For example, a sample of
blood could be taken from a patient and treated to expand a population of
stem/precursor cells. These cells are then treated in a manor to select for the
expansion of pre-B cell populations. These pre-B cells are treated with antigens to a
particular disease which could be a viral infection from influenza, herpes simplex virus
I or II, cytomegalo virus, the human T-cell leukemia viruses (e.g., HIV), polio virus,
rhinovirus, respiratory syncytial virus, hepatitis A, B, and C viruses, measles virus, and
JC virus, a bacterial or fungal infection from an organism such as Staphylococcus,
Streptococcus, Mycobacterium, Clostridium, Neisseria, Enterobacter, Pseudomonas,
WO 94116715 ~ PCT/US94/01033
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S~lmonell~, Treponema, C~n~ e, and Aspergillas or a parasitic infection such as
amebiasis, pneumocystis, malaria, trypanosomiasis, the holminthic diseases and
sarcoidosis.
The target cells are treated with antigen in a manner to stim~ te antigen specific
antibody production and the target cells are injected back into the same or a different
patient. That patient would have an antibody resistance to the particular disease chosen
without ever having been exposed to the infection or a vaccination. In a similar fashion,
any cell type of the immllne system could be treated to produce a cellular or humoral
resistance to infection or disease. Alternatively, using this method it may also be
possible to treat these same diseases and infections may be treatable.
Another embodiment of the present invention is directed to a procedure for
providing a effect*e means for genetic therapy. In view of the relative ease by which
cell populations can be removed and cultured, one of ordillaly skill in the art can also
introduce a genetic element into the cells prior to their reintroduction into the patient.
This can cure many of the deficiency disorders which can be attributed to a single
miSsing or defective genetic element. In ~flrlitit~n, hematopoietic stem cells lend
themselves very well as a vehicle for dissemin~ting the mi~ing product to various parts
of the body. Hematopoietic stem cells as well as other cells produced by the method
of the invention can also be used for lalge~ g a specific recombinant product to a
specific area of the patient's body, for example, by targeting an anticancer agent to
hepatic cells in patients with primary carcinnm~ of the liver, by targeting cancer
suppressor gene products to specific cells in patients with retinoblastoma, or by targeting
anti-plaque forming enzymes to hematopoietic cells in patients with atherosderosis.
Examples of other c~n(lid~te diseases for gene therapy according to the method
of the present invention inchltle hemophilia A (glucose 6-phosphate hydrogenase
deficiency), f~mili~l hypercholesterolemia (e.g., cholesterol 7-alpha-hydro~ylase
deficiency), thalassemia, sickle-cell ~nemi~, cystic fibrous, Tay-Sachs disease (GM2-
gangliosidosis), glycogen and lysosomal storage diseases (e.g., sphingolipidoses,
mucolipidoses, Wolman's disease, Pompe's disease, Gaucher's disease), and SCID, to
name just a few.
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Procedures for genetic therapy are known in the art. Briefly, a sample of cells
is removed from the patient and placed in culture accordillg to the method of the
invention. The sample is m~int~ined in culture treated in a manner in accordance with
the present invention to favor the expansion of a fraction of the cell population which
includes the stem cells. These cells are then subjected to techniques for the introduction
and stable incorporation of genetic elementc. Introduction of genetic elements may be
accomplished before, during, or after cell expansion according to the method of the
present invention. Incorporation of genetic elements during expansion may be pl eLell ed
in some cases. Examples of these techniques inclllde transfection, such as calcium-
mediated or microsome-mediated transfection, cell fusion, electroporation,
microinjection, or infection using recombinant vaccinia virus or lell~vuus vectors.
These vectors would conlail- functional genetic elements which express products, such
as, adenosine de~min~ce for the treatment of SCID. Cells which acquire the genetic
element are selected, further expanded and reintroduced to the p~tient These cells
circulate throughout the body and supply adenosine ~le~min~ce, or some other product,
where needed.
Another lJlerelled example for this approach is in the treatment and possible
cure of diabetes. A sample of pancreas tissue from an insulin-dependent diabeticpatient is biopsied and placed in tissue culture. The sample is cultured with BSFM to
selectively proliferate stem cells, progenitor cells, precursor cells or differentiated
pancreatic cells. These cells are then transfected with the gene which codes for the
expression of in.~lllin Preferably, the gene is under the control of a promoter which is
transcriptionally and translationally regulated in a manner similar or identical to the
natural insulin gene, which may be the natural insulin promoter. These recombinant
cells are then injected or surgically transplanted back into the pancreas of the diabetic
patient. The recombinant cells incorporate into the pancreas and perform the function
of pancreatic B cells, which is to supply insulin to the bloodstream as required. This
treatment can be repeated as necessary, however, once biopsied, pancreatic cells do not
have to be contiml~lly ta~en from the patient, but can be m~int~ined in culture or
WO 94/16715 PCTIUS94/01033
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stored (e.g. cryopreserved) for later use. These are significant advantages not envisioned
by currently available techniques.
Another embodiment of the present invention is directed toward a genetic
therapy for treating an infection with celhll~r transplantation. For example, target cells~
preferably hematopoietic cells, could be prepared according to the method of thepresent invention. These cells are transfected or infected with a recombinant DNA
sequence which expresses a therapeutic product, and inoculated back into the host to
effect the therapy. Therapeutic products which are useful include antibiotics, anticancer
agents including chemotherapeutic drugs, peptides and cytotoxic compounds, and
expression products such as antisense RNA and ribozymes. A particularly useful
product is the expression product of the mlllticlrug resistance (MDR) gene which, when
incorporated into a patient's hematopoietic cells, allows for the use of higher, and
consequently more effective, doses of chemotherapeutie agents in the treatment of
certain cancers. In view of the fact that the methods of the present invention can
selectively expand a desired population of cells, drugs and other agents can be
preferentially targeted to certain tissues and organs for a m~xi,,,ll,,, therapeutic effect
with a Illil~illllllll of side effects. In ~-l(lition, these same procedures could be utilized
to introduce genetic elements into a cell to provide resi~t~nce to disease or infection as
a prophylactic or prec~ntion~ry measure. These forms of treatment are not possible
with current technologies.
Alternatively, target cells could be integrated with a genetic element which
expresses an antigenic or immnnc)genic product. Once the cells are placed back into the
patient, an immnne response would occur creating circnl~ting antigen specific antibodies
and cells to any substance which expresses the antigen such as an infecting organi~m
In effect, such recombinant cells would collsLilule a vaccine against the infecting
org~ni~m
In another embodiment, a bank of cell cultures is provided comprising multiple
populations of stem cells, progenitor cells, or precursor cells produced according to the
method of this invention which are able to differentiate into a variety of specific cell
lineages. This cell bank can be created for a single individual and placed in long-term
WO 94/16715 PCTIUS94/01033
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storage by, for example, cryopreservation. Briefly, a sample of cells from various organs
and cell systems of an individual are obtained, HLA typed, and cultured accordhlg to
the method of the present invention with a selected BSFM. Target cell populations
which develop are expanded, pelleted by ce~ irllg~tion~ suspended in a ~;lyop~eservation
medium such as dimethylsulfoxide (DMSO), divided into samples, and deep-frozen.
Samples can be thawed and expanded in culture when needed as, for example, when the
patient is in need of cell transplantation therapy. Alternatively, the samples could be
frozen to create a cell bank, and later thawed and expanded accordillg to the method
of the invention only when needed. These approaches are not possible using current
technology because large numbers of undifferentiated cells cannot be m~int~ined or
expanded in culture.
In a similar fashion, target cell populations can be stockpiled from different
individuals creating banks of stem, progenitor, and precursor cells representative of a
complete or partial spectrum of, for example, the human leukocyte antigens (HLA) for
use in autotologous or allogeneic transplantation therapy. A bank of these cells can be
m~int~ined or clyopreserved for each organ, tissue, and tissue system. ~nlli(l~te
patients for establishing such cell banks other than hilm~nc include economically
valuable m~mm~lc such as cattle, sheep, pigs, and horses, domesticated ~nim~lc such as
dogs and cats, zoo and wild ~nim~lc such as various monkeys and other primates. It is
also possible to establish HLA-typed blood and/or tissue sample banks in which the
typed samples can be frozen until needed and then expanded using the process of this
invention.
In another embodiment, fetal genetic testing can be performed according to the
method of the invention. First, a sample of peripheral blood is taken from a pregnant
woman which cu~ c a small number of fetal cells. Fetal cells are expanded by themethod of the present invention. Separation of fetal cells from maternal cells, either
before or after exp~n.cion, can be employed if desired, by FACS sorting or similar
processes. The reslllting expanded population of fetal cells can be easily and reliably
genetically tested by procedures which are known and ~;ullelllly available such as by
amniocentesis.
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The following examples are offered to illustrate embodiments of the present
invention, but should not be used as limiting the scope of the invention.
EXAMPLES
Example 1
FxI)~n~ n of CD34+ Cells
A. Preparation of BSFM
Peripheral blood cells obtained by leukaphoresis were diluted 1.10 in culture
medium CCM-2 (Verax Corporation) co~ g 20 units of heparin/ml. An equal
volume of the diluted blood sample was mixed with 2% acetic acid and the total number
of mononucleated cells estimated using a hemocytometer. In general, the number of
mononucleated cells from a leukaphoresis unit was about 30 to 50 x 106 ce!ls/ml.To induce BSFM, the peripheral blood cells were diluted to a final concentrationof about 4 x 106 mononucleated cells/ml in serum-free CCM-2 medium col~ lg 20
units/m1 of heparin. A total volume of 80 ml of cell suspension in a T-150 flask was
pre-incubated with 10 ng/ml of mezerein in a hnmi(1ified 5% CO2 incubator at 37C for
about 2 hours. Concanavalin-A was added to a final concentration of 20 ~ug/ml and the
cells were incubated at 37C for about 4 days. The supern~t~nt was harvested and the
cell debris was removed by ce-ll-iLugation. The clarified supernatant was stored at 4C
or frozen at -20C before use.
B. Op~ F BFSM Addition Level
The ~pLilllulll potential of BSFM level for expanding a peripheral blood cell(s)population was PY~mined in this example using the BSFM as prepared in example lA.
Unfractionated and ficoll-fractionated peripheral blood cells were used. The
ficoll-fractionated blood cells were prepared by ~ lting 10 ml of peripheral blood from
a leukaphoresis unit with 20 ml of CCM-2 medium col~ lil-g 20 units/ml of heparin
and 10% total calf serum. 15 ml of the diluted blood samples were loaded onto 10 ml
of Ficoll-Hypaque in a sterile 50 ml culture tube. The cells were centrifuged in a
table-top centrifuge at 400 x g for 30 mimltes at room temperature (25C). Cells were
harvested from the aqueous ficoll interphase and washed twice in CCM-2 medium
WO 94/16715 PCT/US94/01033
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cont~inin~ 10% fetal calf serum. The cells were then resuspended before use in
serum-free CCM-2 medium or CCM-2 medium cont~inin~ 10% FCS.
Unfractionated cells were diluted to a final cell density of about 2 x 105/ml inCCM-2 medium co~ 1% to 5% BSFM as indicated and in the presence or
absence of fetal calf serum. One and one-half mls of cell suspension per well were
suspended in a 24-well plate and incubated at 37C in a 5% CO2 incubator. The cell
count and viability were determined daily for 5 days. As in~ic~ted in Table 1, the
highest rate of proliferation was observed in CCM-2 medium co~ 5% BSFM.
Further studies confirm that the rate of cell proliferation peaks in CCM-2 medium
co"t~i~-i"~ 5% BSFM. There was no difference between the rate of cell proliferation
in medium CO"~ ,;"~ 5% or 10% BSFM. In ~ltlition~ the cells have been observed to
proliferate equally well in both serum-free and serum-c~ medium. Tbe ability
of the CCM-2 medium coll~;"ill~ 5% BSFM to support the proliferation of
ficoll-fractionated PBMNC was comparable to that for unfr~cti-)n~te-l PBMNC.
TABLE 1
Proliferation of Unfractionated PBMNC
TOTAL CELL COUNT (105/ml)
DAY 0% BSFM* 1% BSFM 2% BSFM 5% BSFM
0 3.00 3.00 3.00 3.00
5.75 5.55 4.75 5.65
2 4.36 4.96 5.96 6.88
3 4.20 5.45 6.00 6.16
4 6.35 5.55 7.40 8.60
6.80 7.00 5.40 7.00
* The basal medium is CCM-2 co"l~;"i"~ 10% FCS.
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C. Expansion of Peripheral Blood Mononucleated Cells
The expansion of the PBMNC cells was studied by culturing both unfractionated
and fractionated cells in CCM-2 medium co,~ i"g 5% BSFM, 10 ng/ml of
recombinant human stem cell factor (SCF) and 1 unit/ml of recombinant human
erythropoietin (EPO) in a 24-well plate. Each well was seeded with 1l/~ ml of cell
suspension with a cell density of 1 to 2 x 105 mononucleated cells/ml. The cell count
and viability were estimated after 5-6 days of incubation. At the end of the incubation,
the cells were subcultured in the same culture condition with a seeding density of 1 to
2 x 105 cells/ml for another 5 to 6 days. ~rom the initial cell culture, the cells were
subcultured twice. As indicated in Table 2, the fold increase of cell numbers exceeded
600 in both unfractionated and ficoll-fr~ction~ted cell cultures for a period of 16 days
with two passages.
TABLE 2
TOTAL PBMNC EXPANSION
TOTAL FOI;D OF INCR~E
CULTURE* Fractionated Unfractionated
Initial 7.6 4.8
1st Passage 62.2 67.7
2nd Passage 613.1 602.7
* Total of 16 days.
D. Analvsis of the Cell Population Durin~ Expansion
The composition of the hem~topoietic cells in the total cell population was
analyzed by three methods: FACS analysis to determine the expressed surface markers,
e.g. CD34; colony-forming assay to estimate the total number of progenitor cells,
specifically BFU-E and CFU-GM in the cell population; and morphological analysis by
light microscopy.
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The list of surface markers used for FACS analysis is sllmm~rized in Table 3.
In most inct~nces, three color st~ining~c were used to assign and determine the
subpopulation of CD34+ cells in the culture (Table 4). For example, three separate
antibodies vs. CD34, CD38, HLA-DR respectively were used to determine the cell type
which expressed the listed, if any, markers as a means to determine the CD34+ cell
subpopulations. Other combin~tion~ inchl-led CD34, CD33 and CD3 to monitor
differentiation into lymphoid and myeloid lineage cells.
TABLE 3
CD MARKERS USEFUL FOR CHARA( l~RIZING
~T T~C!T'T~ ~T' ~T'T T ~
MARKER DESCRIPTION
CD3 Pan T-Cell
CD4 Helper T-Cell
CD8 Cytotoxic/Suppressor T-Cell
CDl9 Early B-Cell, B-Cell Specific
CD33 Early Myeloid Cells
CD34 Stem Cells, Progenitor Cells
CD38 Activated T-Cells, Early Progenitor Cells
HLA-DR ¦ Activated T-Cell, Monocytes, B-Cell
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TABLE 4
CELL MARKER COMBINATIONS
s
CD34 / CD33 / CD19
CD3 / CD4 / CD8
CD34 / CD3 / CD33
CD34 / CD38 / DR
To determine the total number of progenitor CFU-GM cells obtained in the
culture, 1 x 105 monoml~leated cells were suspended in 1 rnl of CCM-2 medium
co~ i"it~g 20% FCS, 0.9% methyl cellulose, and 10 ng/ml of recombinant lluman
GM-CSF and plated in a 35 mm tissue culture dish with grid. The colonies formed were
scored after 14 days of inçubation in a 5% CO2 incubator at 37C. To determine the
number of el~Lhloid progenitor cells, BFU-E, 1 x 105 mont)nllçleated cells were placed
in a medium as described in the CFU-GM assay except that recombinant GM-CSF was
replaced with 1 unit/rnl of recombinant human elyL}~o~oietin. Again, the total colony
forming units were determined on Day 14 of incubation by light microscopy.
To study the cell morphology, cytospin st~ining technique was used. Briefly, thecells were concentrated by cenllirugation and the cell smear was stained with Wright's
st~ining Cell morphology was analyzed by light microscopy.
E. Expansion of Myeloid and Erythroid Pro~enitor Cell
In a 24-well plate culture, ficoll-fractionated PBMNCs were seeded at a cell
density of about 2 x 105/ml and 1.5 rnls/well. The cells were cultured in CCM-2
medium co~ g 5% BSFM, 10 ng/rnl recombinant human SCF and 1 unit/ml of
recombinant human elyLlllo~oietin for S days. The total CFU-GM and BFU-E
progenitor cells in the Day 0 culture as well as Day S culture were estimated, using
colony-forming assay as described previously. Representative CFU-GM and lBFU-E
colonies are shown in Figures 3 and 4 respectively. As indicated in Table 5, there was
a 32.3-fold increase in CFU-GM and a 237.5-fold increase in BFU-E progenitor cells
WO 94/16715 ~S~?,7t PCT/US94/01033
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after 5 days of incubation. For this example, the total cells in the culture increased by
about 5-fold after 5 days of incubation, indicating a preferential expansion of CFU-GM
and BFU-E cells or CD34+ precursor cells thereof.
TABLE 5
EXPANSION OF PROGENITOR CF.T TA~
PROGENITOR CELLS / 105 (~FT TAS
DAY TOTAL CELL~ FOLD OF INCREASE
CFU-GM BFU-E CFU-FM BFU-E
3 2
97 475 32.3 327.5
Total CFU-GM and BFU-E were 950 cells/ml and 4,655 cells/ml, respectively.
F. Expansion of CD34+ Cells
The selected BSFM prepared as described in Example lA was intended for the
expansion of CD34+ cells which favor lymphoid lineage differenti~tinn. Accordingly,
both unfractionated and ficoll-fr~ tion~ted PBMNC were cultured in CCM-2 medium
con~ining 5% BSFM, 10 ng!ml recombinant human SCF and 1 unit/ml recombinant
human ely~ o~oietin in a 24-well plate as described previously. The cells were
subcultured on Day 5 and Day 11 and the surface marker, CD34 was detçnnined by
FACS analysis on cells harvested on Day 0, Day 5, Day 11 and Day 16. As in~1ir~ted
in Table 6, CD34+ cells increased from less than 0.5% to greater than 70% of the cell
population. Figures 5 and 6 show the 1 and 3 day, respectively, cultures for
unfractionated cells. Figures 7 and 8 show the 1 and 3 day, respectively, cultures for
fractionated cells.
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TABLE 6
CD34+ CELLANALYSIS
~ of Total Cells
DAY Fractionated Unfractionated
0 <0.5 <0.5
55-63
11 50-52 73-75
16 35-36 74-75
Not Done
G. Analysis of CD34+ and CD3+ Cell Subpopulations.
In a separate test to analyze the CD34+ and CD3+ cells subpop~ tion~, both
unfractionated and fractionated cells were cultured as described previously in CCM-2
medium cont~ining 5% BSFM, 10 ng/rnl SCF and 1 u/ml elyLl~opoietin in 24-well plate
for S and 6 days respectively. The rate of PBMNC proliferation is sllmm~Tized inTable 7.
TABLE 7
PBMNC Proliferation
UNFRACTIONATED CELL FRACTIONATED CELL
DAYTOTAL COUNTI FOLD OF TOTAL COUNT' FOLD OF
INCREASE INCREASE
0 0.33 --- 0.17 ---
1.47 4.4
6 l.so2 8.7
l x106/ml
2 Viability is 87.2%
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To determine both the CD34+ and CD3+ cell subpopulations, both the unfractionated
and fractionated PBMNC were subjected to FACS analysis based on the following surface
markers: CD34, CD38, HLA-DR, CD3, CD33, CD4, CD8. The results of the analysis are
sllmm~rized in Table 8. The total CD34+ cells in the unfractionated and fractionated cell
5 culture are 28.2% and 36.5% of the total cell population respectively. In addition, based on
CD38, DR and CD3 surface markers, an array of CD34 + cell subpopulations, ranging from more
ive to more co"",lil~ed progenitor cells were obtained during culture. Some representative
FACS analyses are shown in Figures 9 and 10.
TABLE 8
FACS ANALYSISl
~o OF TOTAL CELL POPULATION
SURFACE MARKER UNFRACTIONATED FRACTIONATED
DAY 0 DAY 5 DAY 0 DAY 6
CD34+CD38+DR+ 0 1.6 0 0.6
CD34 + CD38 + DR- 0 3.5 0 0.8
CD34+CD38-DR+ 0 7.8 0 13.5
CD34 + CD38-DR- 0 15.3 0 21.6
Peripheral blood cells from Subject 6.
Example 2
Preparation of Modified BSFM
A modified BSFM was obtained by the protocol described in Example 1.A. Five
mls of modified BSFM were passed through an anti-IL-2 afflnity column co~
ml of matrix. The flow-through fraction (the modified BSFM) was used for the
preparation of culture medium for myeloid lineage cell expansion. The culture medium
contains CCM-2 medium, 5% modified BSFM, 10 ng/ml of recombinant human SCF
and 1 u/ml of recombinant human elyLhlopoietin.
Two x 105 PBMNC/ml were seeded in a 24-well plate with 1.5 mls of cell culture
per well. The cells were subcultured every 5 to 6 days for 2 passages. At the end of
WO 94/16715 ~ PCT/US94/01033
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each of 5 to 6 days of incubation, total cell count and viability were determined. The
composition of the cell population related to the expression of CD34, CD33, CD19, CD3
markers were analyzed by FACS. In addition, colony-forming assays were performedon the cells obtained from cultures. The results indicate expansion of CD34 + cells, the
population of which consists mainly of myeloid lineage cells (CD33+) with various
degrees of differentiation.
Example 3
Expansion of Lvmphoid Linea~e Cells
The expansion of lymphoid cells was studied by culturing unfractionated and
fr~ction~ted PBMNCs in a 24-well plate. Both the culture conditions and medium, are
described in Example 1. The cultures were passed twice for a total of 16 days ofincubation. On day 0, day 5, 11, and 16, the CD3 +, CD4+ and CD8 + cell populations
were analyzed by FACS. As indicated in Table 9, the CD3+, CD4+ and CD8+ cell
populations were expanded and m~int~ined. After 16 days of incubation, the total cell
pop~ tion~ in both unfractionated and fr~cti-)n~ted cell cultures expanded in excess of
600-fold.
TABLE 9
Analysis of CD3+, CD4+ and CD8+ Cells
~ of Total Cells
DAY Unfractionated Fractionated
CD3 + CD4 + CD8 + CD3 + CD4 + CD8 +
0 75 -- -- 75 --
-- -- -- 76 54 17
11 74-80 70 77
16 88 65 22 84 37 57
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The CD3+ cells in both unfractionated and fractionated cell cultures after 16
days of incubation were further analyzed by FACS to determine the subpopulations of
CD3+ cells with respect to CD4 and CD8 markers. The results of the analysis are
sllmm~ri7ed in Table 10 and indicate that these cell populations were expanded and
m~int~ined in culture.
TABLE 10
ANALYSIS OF CD3 CD4 CD8 CELLS
% of Total Cells
Cell Markers Unfractionatedl Fractionated
CD3+CD4+CD8+ 3.8 2.1
CD3 + CD4 + CD8- 52.0 28.7
CD3 + CD4-CD8 + 16.7 45.3
CD3 + CD4-CD8- 15.0 7.5
Day 16 culture which has been subcultured twice on Day 5 and Day 11. The
total percentages of CD3+ cells in the total cell population from both unfractionated
and fractionated cell cultures was 84 and 88 respectively.
Example 4
Expansion of Erythroid Linea~e Cells
A cell density of 2 x 10~ cells/ml of both unfractionated and fractionated PBMNCis used to seed a 24-well plate with 1.5 ml of cell culture per well. The culture medium
consists of CCM-2 medium cont~ining the BSFM described in Example 2, 10 ng/ml
recombinant human SCF and 10 u/ml recombinant human erythropoietin and 50
millimolar ferric citrate.
The cell cultures are incubated at 37C and subcultured every 5 to 6 days. At
the end of each of the 5 to 6 days of incubation, the total cell count and viability are
determined. The cell population is analyzed by FACS and morphological observation
using light microscopy. The results indicate expansion of erythroid lineage cells, leading
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to a cell population cont~ining proerythrocytes, erythrocytes, and highly differentiated
erythroid cells.
Example 5
Reconstitution of the Hematopoietic System
To test the ability of the CD34+ cells obtained from expansion in culture to
reco,~LiLule the hematopoietic system, both unfractionated and fractionated PBMNC are
cultured in CCM-2 medium co~ illg 5~o BSFM, 10 ng/ml recombinant human SCF
and 1 u/ml recombinant human elylhlu~oietin for 5 days as described in Example 1.
The cells are harvested and concentrated by ce"l. ;r~g~tinn in a table-top cellL~ifuge at
200 x g for 20 mimltes at room temperature. About 5 x 106 cells in 0.1 to 0.2 ml of
phosphate buffered saline, pH 7.4, from each of the unfr~ction~ted and fractionated cell
culture are injected intraperitoneally (IP) into a SCID mouse. Ten weeks after
injection, cells from under the kidney capsule in the SCID mouse are analyzed by FACS
for evidence of specific cell incorporation. The results indicate that the cultured CD34 +
cells can engraft an intact functinning human hematopoietic system in SCID mice. Example 6
Preparation of BSFM
To demonstrate a potenti~tQr and an inducer other than those described in
Example 1 to induce a BSFM which favors expansion of CD34+ cells toward lymphoidlineage differenti~tion, 4 x 106 mononllcleated cells/ml are pre-treated with 300 IU/ml
of IFN-~B for two hours. The cells are incubated for another 4 days after the addition
of 10 ~ug/ml of PHA. The ability of the BSFM to expand PBMNC is tested as described
in Example 1, using 24-well plate cultures. The results demonstrate that the BSFM
obtained by using a potenti~tQr/inducer comhin~tinn diLrerelll from the one used in
Example 1 supports and expands a comparable CD34+ cell population.
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Example 7
Expansion of Islet Cells
Porcine pancreatic islets are isolated and islets with average diameter of about150~ are obtained by density gradient centri~lg;ltion using Ficoll. About 10,000 islets
are plated on a 60 mm petri dish in the presence or absence of 1 ml of collagen matrix.
The culture medium consists of DMEM, 20% of horse serum, 5~o BSFM as described
in Example 1, and 15~ hypoth~l~mll~ extract. The cells are incubated at 37C on a
rotating platform. On Day 0, Day 7, Day 14, and Day 21, islet cells cultured in the
presence or absence of collagen matrix from duplicate petri dishes are assayed for total
cell count and viability. In addition, the concentrations of insulin and glucagon in the
culture media are determined by radioilllllllll~oassay as a means to demonstrate the
proliferation of A and B cells. The results indicate the proliferation of islet cells and
the intact functionality of both the A and B cells as indicated by the production of
insulin and glucagon.
Example 8
Cell Expansion Using Cells from Dirrerellt Donors
PBMNC's were separately collected from 6 human donors and were specially
Ficoll-fractionated using the protocols described in Example 1.B A BSFM was obtained
by the protocol described in Example l.A.
Cells from each donor were cultured in 24-well plates using one and one-half mlsof cells suspension per well at initial seeding densities of 4 x 105 and 1 x 106 cells per
ml. The cells were incubated at 37C in a 5~o CO2 incubator for 5 to 7 days.
T_e cells from each donor were suspended in medium form~ tion~ made from
CCM-2 basal medium plus the addition of 2~o BSFM and/or blood pl~m~, and/or 10~ofetal bovine serum (FBS), and/or selected cytokines: 1 unit/ml of recombin~nt human
elyLhl~oietin (EPO), 10 ng/ml of recombinant hllm~n stem cell factor (SCF), 10 ng/ml
of recombinant human interleukin-3 (IL-3), 10 ng/ml of recombinant human interleukin-
6 (IL 6). The medium formulations used are shown in Table 11.
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TABLE 11
Medium Formulations
Medium Formulation Medium Composition*
#1 2~ BSFM, EPO, SCF, Plasma
#2 2% BSFM, Plasma
#3 2~o BSFM, EPO, SCF
#4 2~o BSFM
#5 IL-3, IL-6, EPO, SCF, Plasma
#6 IL3, IL-6, EPO, SCF
#7 10% FBS
* The basal medium is CCM-2
For each donor's cells, the cell count and viability were estimated after 5-7 days
of incubation. The total numbers of CFU-GM cells and BFU-E plus CFU-E cells at the
start and at the end of each culture were determined using the procedures described in
Example lD.
The average total cell expansion (and the st~nd~rd deviation) for the 6 donors'
cells for each of the medium fonmll~tion~ is shown in Table 12.
TABLE 12
Average Total PBMNC Expansion
Medium Total Fold of Increase
25 Form~ tion
(See Table 11) ISDI = 4 x 105 cells/ml ISD~ = 1 x 106 cells/ml
#1 *5.34 (3.18)2 2.26 (1.08)
#2 *5.31 (3.63) 2.36 (1.01)
#3 4.62 (0.92) 2.00 (0.67)
#4 4.60 (1.10) 2.10 (0.80)
~5 *1.40 ((1.50) 1.00 (1.20)
#6 0.40 (0.20) 0.50 (.20)
#7 0.70 (0.40) 0.60 (0.30)
*Standard Deviation value is skewed by the value of a single donor.
l ISD = Initial Seeding Density of cultures.
2 Average Value (Standard Deviation) of the values
for each of the six donor's cells.
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The average expansion of CFU-GM and BFU-E + CFU-E progenitor cells are
shown in Tables 13 and 14, respectively.
In Tables 12, 13, and 14, where the value of the Standard Deviation is large
compared to the Average Value, this Standard Deviation value is skewed by the data
of one donor (which is from two-times to four-times larger than the Average Value of
the donors).
TABLE 13
Average Total CFU-GM FxI-~n~ion
Medium Total fold of Increase
F-)rrmll~tion ISD = 4 X 105 cells/ml ISD = 1 x 106 cells/rnl
#1 *11.2 (12.6) 2.3 (1.4)
#2 5.9 (2.2) 2.5 (2.2)
#3 1.6 (1.1) 2.5 (3.7)
#4 *2.9 (4.0) 1.3 (1.4)
#5 5.1 (3.2) 3.5 (1.1)
#6 1.1 (1.8) 0.7 (0.8)
#7 0.4 (0.3) 0.9 (0.5)
* Standard Deviation value is skewed by the value of a single donor.
TABLE 14
Average Total BFU-E/CFU-E Expansion
Medium Total fold of Increase
(See Table 11)ISD = 4 X 105 cells/ml ISD - 1 x 106 cells/rnl
#1 3.0 (1-8) 2.4 (2.1)
#2 3.7 (1-3) 2.5 (3.4)
#3 0.6 (0.3) 1.1 (1.5)
#4 0.8 (0.1) o.9 (1-0)
#5 *10.0 (6.2) 7.9 (5.6)
#6 1.4 (0.3) 0.8 (1.1)
#7 0.5 (0.4) 1.2 (0.8)
* Standard Deviation value is skewed by the value of a single donor.
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Example 9
Analysis of Cell Size Distribution
PBMNC's were obtained by leukapheresis and a BSFM prepared as described
in Example 1.A. A modified BSFM was prepared by the addition of blood plasma to
S the BSFM (2% plasma). Ficoll-Hypaque fractionated cells were prepared and cultured
as described in Example 1.B. using a CCM-2 basal medium (without FCS) plus 10 ng/ml
of recombinant human stem cell factor (SCF) and 1 unit/ml of recombinant human
elylhlo~oietin (EPO).
Cell count and viability were measured at the end of 5-6 days of incubation and
10 the cells analyzed by FACS to determine cell size and the subpopulation of CD34+ cells.
Representative FACS analysis forward light scatter (FS) and side light scatter
(SS) plots are shown in Figures 11 and 12, respectively, for cells cultured with and
without plasma added to the BSFM. Without plasma added to the BSFM the cell sizedistribution is in two distinct subpopulations as shown in Figure 12. The lower FS
15 subpopulation of cells are smaller size cells and this subpopulation contains the
preponderance of cells that are CD34 positive. With plasma added to the BSFM, cells
in the smaller size subpopulation are negligible and the number of CD34 positive cells
is significantly reduced.
Other embodirnents or uses of the invention will be a~yalellt to those skilled in
20 the art from consideration of the specifications and practice of the invention disclosed
herein. It is intended that the specifications and examples be consideration exemplary
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PCTIUS94/01033
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only, with the true scope and spirit of the invention being indicated by the following
claims.
