Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
TREATMENT USING REPROGRAMMED MATURE ADULT CELLS
INCORPORATION BY REFERENCE
All documents cited or referenced in herein cited documents, together with any
manufacturer's instructions, descriptions, product specifications, and product
sheets for any
products mentioned herein or in any document incorporated by reference herein,
are hereby
incorporated herein by reference, and may be employed in the practice of the
invention.
FIELD OF THE INVENTION
A method of treating various diseases, disorders, or conditions in patient
using
reprogrammed cells such as retrodifferentiated, transdifferentiated, or
redifferentiated cells.
The method comprises obtaining committed cells from the patient,
retrodifferentiating the
committed cells to obtain retrodifferentiated target cells, and administering
the
retrodifferentiated cells to the patient. In certain embodiments, the method
comprises
obtaining committed cells from the patient, transdifferentiating the committed
cells to obtain
transdifferentiated target cells, and administering the transdifferentiated
target cells to the
patient. The retrodifferentiated or transdifferentiated target cells repair or
replenish tissue or
cells in the patient.
BACKGROUND OF THE INVENTION
Stem cells are characterized by their ability to renew themselves through
mitotic cell
division and to differentiate into a diverse range of specialized cell types.
The two broad
types of mammalian stem cells are embryonic stem cells, which are isolated
from the inner
cell mass of blastocysts, and adult stem cells, which are found in adult
tissues. In a
developing embryo, stem cells can differentiate into all of the specialized
embryonic tissues.
In adult organisms, stem cells and progenitor cells replenish specialized
cells and maintain
the normal turnover of regenerative organs such as blood, skin or intestinal
tissues.
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Stem cells are abundant in a developing embryo, although, the quantity of stem
cells
decreases as development progresses. By contrast, an adult organism contains a
limited
number of stem cells which are confined to certain body compartments.
The therapeutic applications of stein cells have the potential to alter
treatments for
multiple diseases or disorders. While some adult stem cell therapies, such as
bone marrow
transplants, already exist, medical researchers anticipate using stem cells to
treat a wider
variety of diseases including cancer, Parkinson's disease, spinal cord
injuries, amyotrophic
lateral sclerosis, multiple sclerosis, and muscle damage, among others. Such
therapies may
take advantage of the stem cells' capability of differentiating into cell
types that are
necessary to treat the disease.
However there is uncertainty as to the success in treating such ailments using
stem
cells, as well as concerns as to the ease by which stem cells may be obtained.
For example,
haematopoietic stem cells are traditionally extracted by isolation from bone
marrow, growth
factor mobilized peripheral blood, or cord blood (placenta). Haematopoietic
stem cells may
also be prepared from embryonic stem (ES) cells, which are extracted from
embryos
obtained using in vitro fertilization techniques. However, extraction from
these sources is
cumbersome and sometimes hazardous, and may be challenged by ethical concerns.
Further, the number of stein cells that may be obtained from these sources are
limited.
Moreover, the stem cells may experience difficulty in differentiating into the
cells necessary
to treat the ailment.
SUMMARY OF THE INVENTION
The present invention relates to the use of reprogrammed cells to repair
tissue or
replenish tissue or cells in a patient. For example, the present invention
relates to the use of
retrodifferentiated cells, obtained from retrodifferentiation of
differentiated or committed
cells, to repair tissue or replenish tissue or cells in a patient. The present
invention also
relates to the use of transdifferentiated cells, obtained from
transdifferentiation of
differentiated or committed cells, to repair tissue or replenish tissue or
cells in a patient.
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The application is based, in part, on Applicant's discovery that committed or
somatic
cells obtained from a patient may be reprogrammed to result in cells of
different lineages,
and these reprogrammed cells can be administered to the patient to repair or
replenish
tissues or cells. Examples of the process of reprogramming include
retrodifferentiation and
transdifferentiation.
Therefore, Applicants discovered that committed cells may undergo
reprogramming
to result in cells of a different lineage. For instance, committed cells may
undergo
retrodifferentiation to result in retrodifferentiated cells, e.g., cells that
are less differentiated,
such as pluripotent stem cells, and that these retrodifferentiated cells can
be administered to
the patient to repair or replenish tissues or cells. As another example,
committed cells
obtained from a patient may undergo transdifferentiation to result in
transdifferentiated cells,
e.g., cells of a different lineage than the committed cells, and that these
transdifferentiated
cells may be administered to the patient to repair or replenish tissues or
cells.
The invention encompasses a method of repairing or replenishing tissue or
cells of a
cell lineage in a patient by administering reprogrammed cells to the patient.
In particular,
the invention encompasses a method of repairing or replenishing tissue or
cells of a cell
lineage in a patient, comprising (i) obtaining committed cells, (ii)
retrodifferentiating the
committed cells to obtain retrodifferentiated target cells, and (iii)
administering the
retrodifferentiated target cells to the patient, wherein the
retrodifferentiated target cells
redifferentiate into cells of the cell lineage. These redifferentiated cells
may be of the same
cell lineage or of a different cell lineage as the committed cells.
The invention also encompasses a method of repairing or replenishing tissue or
cells
of a cell lineage in a patient, comprising (i) obtaining committed cells, (ii)
transdifferentiating the committed cells to obtain transdifferentiated target
cells, and (iii)
administering the transdifferentiated target cells to the patient.
In some embodiments, the patient may be suffering from a disease, disorder, or
condition including, but not limited to, bone marrow failure, haematological
conditions,
aplastic anemia, beta-thalassemia, diabetes, motor neuron disease, Parkinson's
disease,
spinal cord injury, muscular dystrophy, kidney disease, multiple sclerosis,
congestive heart
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failure, hepatitis C virus, human immunodeficiency virus, head trauma, spinal
cord injuries,
lung disease, depression, non-obstructive azoospermia, andropause, menopause
and
infertility, rejuvenation, scleroderma ulcers, psoriasis, wrinkles, liver
cirrhosis, autoiimnune
disease, alopecia, retinitis pigmentosa, crystalline dystrophy/blindness,
diabetes, and
infertility. Hence, in some embodiments, the reprogrammed cells may be bone
marrow cells
that treat aplastic anemia, leukemia, lymphoma, or human immunodeficiency
virus in the
patient.
In some embodiments, the reprogrammed target cells, such as
retrodifferentiated
cells, transdifferentiated target cells, or redifferentiated cells, may
include, but are not
limited to, pluripotent stem cells, pluripotent genn cells, haernatopoietic
stem cells, neuronal
stem cells, epithelial stem cells, mesenchymal stein cells, endodermal and
neuroectodermal
stem cells, germ cells, extraembryonic, embryonic stem cells, kidney cells,
alveolar
epithelium cells, endoderm cells, neurons, ectoderm cells, islet cells, acinar
cells, oocytes,
sperm, haematopoietic cells, hepatocytes, skin/keratinocytes, melanocytes,
bone/osteocytes,
hair/dermal papilla cells, cartilage/chondrocytes, fats cells/adipocytes,
skeletal muscular
cells, endothelium cells, cardiac muscle/cardiomyocytes, and tropoblasts.
In certain embodiments, the committed cells are obtained from blood or related
tissues including bone marrow. The committed cells may be obtained from whole
blood,
and/or may be obtained through apheresis. The blood may be mobilized or
unmobilized
blood. Such committed cells include, but are not limited to, T cells, B cells,
eosinophils,
basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes,
mast cells,
lymphocytes, leukocytes, platelets, and red blood cells. Alternatively, the
committed cells
may be obtained from neuronal tissue from the central nervous system or
peripheral nervous
system, muscle tissue, or epidermis and/or dermis tissue from skin.
In certain embodiments, the committed cells are obtained from the blood or
tissue of
a patient. In some embodiments, the patient from which the committed cells are
obtained,
and to which the reprogrammed target cells such as retrodifferentiated target
cells or the
transdifferentiated target cells are administered, is the same patient.
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In some embodiments, the committed cells are retrodifferentiated by contacting
the
committed cells with an agent. For example, the committed cells may be
incubated with the
agent. In certain embodiments, the agent engages a receptor that mediates
capture,
recognition or presentation of an antigen at the surface of the committed
cells. The receptor
may be an MHC class I antigen or an MHC class II antigen. In some embodiments,
the
class I antigen is an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an
HLA-E
receptor, an HLA-F receptor or an HLA-G receptor and said class II antigen is
an HLA-DM
receptor, an HLA-DP receptor, an HLA-DQ receptor or an HLA-DR receptor.
In certain embodiments, the agent may be an antibody to the receptor, such as
a
monoclonal antibody to the receptor. In some embodiments, the antibody is
monoclonal
antibody CR3/43 or monoclonal antibody TAL 1135. In further embodiments, the
agent
modulates MHC gene expression, such as MHC class I+ and/or M-IC class II+
expression.
In some embodiments, the retrodifferentiated cells may undergo
redifferentiation in a
separate step. For example, the retrodifferentiated cells may be
redifferentiated by
contacting the retrodifferentiated cells with growth factors including, but
not limited to,
basic fibroblast growth factor, epidermal growth factor, granulocyte
macrophage colony-
stimulating factor, stem cell factor, interleukins-1, -3, -6, and -7, basic
fibroblast growth
factor, epidermal growth factor, granulocyte-macrophage colony stimulating
factor,
granulocyte-colony stimulating factor, erythropoietin, stem cell factor, and
bone
morphogenetic proteins. The resulting redifferentiated target cells may then
be administered
to a patient.
In embodiments of the invention, the committed cells can be
transdifferentiated by
culturing the committed cells in particular culture conditions. For example,
the committed
cells may be cultured in particular types of culture media in conjunction with
the
retrodifferentiation agents. Examples of these culture media may include
Dulbecco's
Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), etc.
The tissue culture media may also comprise differentiation promoting agent
such as vitamin
and/or mineral supplements, hydrocortisone, dexamethasone, (3-mercaptoethanol,
etc.
Furthermore, additional culturing conditions include using chelating agents or
antibiotics,
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culturing at certain temperatures or carbon dioxide or oxygen levels, and
culturing in certain
vessels. The culture conditions can determine the type of transdifferentiated
target cell that
results.
One aspect of the invention is thereby a method of obtaining target cells. The
method may comprise obtaining committed cells and then reprogramming the
committed
cells. These processes are as described in this application. In some
embodiments, the
method may comprise retrodifferentiating committed cells. In other
embodiments, the
method may comprise transdifferentiating committed cells. In yet other
embodiments, the
method may comprise retrodifferentiating committed cells, and then
redifferentiating the
retrodifferentiated cells.
Another aspect of the invention is use of one or more reprogrammed target
cells in
preparation of a medicament for repairing or replenishing tissue or cells of a
cell lineage in a
patient, or for treating a disease or tissue injury.
Yet, another aspect of the invention is a method of treating a disease or
tissue injury
in a patient in need thereof. In certain embodiments, the method comprises
obtaining
committed cells, reprogramming the committed cells to obtain reprogrammed
target cells,
and administering reprogrammed target cells to the patient. In some
embodiments, the
target cells may be reprogrammed via retrodifferentiation,
transdifferentiation, and/or
redifferentiation. In particular embodiments, the target cells are
retrodifferentiated target
cells, transdifferentiated target cells, and/or redifferentiated target cells.
The proceses of
obtaining the committed cells and reprogramming the committed cells are as
described in
this application.
In some embodiments, the reprogrammed target cells, such as
retrodifferentiated
target cells, transdifferentiated target cells, or redifferentiated target
cells, may be
administered via injection, implantation, or infusion. These cells may be
administered by
parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral,
transdermal injection,
or injection into spinal fluid. In certain embodiments, the
retrodifferentiated target cells or
transdifferentiated target cells are administered in a pharmaceutical
composition. The
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pharmaceutical composition may comprise the retrodifferentiated target cells
or
transdifferentiated target cells, and at least one pharmaceutically acceptable
excipient.
One aspect of the invention is the pharmaceutical composition for
administering the
reprogrammed target cells, such as retrodifferentiated target cells or
transdifferentiated
target cells. The pharmaceutical composition may comprise one or more types of
target
cells, and at least one pharmaceutically acceptable carrier. Optionally, the
pharmaceutical
composition may include adjuvants and/or other excipients suitable for
administration into a
patient.
Another aspect of the invention is a method of preparing a pharmaceutical
composition or medicament comprising (i) obtaining committed cells, (ii)
reprogramming
the committed cells to obtain reprogrammed target cells, and (iii) optionally,
combining the
reprogrammed target cells with one or more pharmaceutical excipients. In some
embodiments, the reprogrammed target cells are combined with one or more
pharmaceutical
excipients. In other embodiments, the reprogrammed target cells are not
combined with one
or more pharmaceutical excipients. The processes of obtaining committed cells
and
reprogramming the committed cells are as described in this application.
It is noted that in this disclosure and particularly in the claims and/or
paragraphs,
terms such as "comprises", "comprised", "comprising" and the like can have the
meaning
attributed to it in U.S. Patent law; e.g., they can mean "includes",
"included", "including",
and the like; and that terms such as "consisting essentially of' and "consists
essentially of'
have the meaning ascribed to them in U.S. Patent law, e.g., they allow for
elements not
explicitly recited, but exclude elements that are found in the prior art or
that affect a basic or
novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed
by, the following Detailed Description.
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BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description, given by way of example, but not intended
to
limit the invention solely to the specific embodiments described, may best be
understood in
conjunction with the accompanying drawing, in which:
FIG. 1 shows immunophenotyping of aphaeresed mononuclear cells before (top
panel) and after (lower panel) induction of reprogramming. Cells were labeled
with
monoclonal antibodies conjugated to R-phycoerythrin (RPE) Cy-5 or
phycoerythrins (PE)
(vertical legends) for immunoglobulin G1 (IgGi) isotype control and CD34 or
CD19,
respectively. Cells were also stained for CD45, CD38, CD61, and IgGI isotype
controls
monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC)
(horizontal legends).
Lower panel show increase in haematopoietic stem cells as depicted by in
crease in the
relative number of CD34 and CD34CD38- cells accompanied by decrease in
leukocytes
mature markers such as CD45 and CD 19.
FIG. 2a shows sequential immunophenotyping of peripheral blood samples of a
patient with severe aplastic anemia following infusion of autologous HRSC
(post Day 1,
Day 2, Day 3, Day 6, and Day 14). Cells were labeled with monoclonal
antibodies against
CD34 and CD45 (2"d horizontal panel), and CD34 and CD38 (3rd horizontal
panel). The top
horizontal panel shows forward and side scatter is flow cytometry, bone marrow
smear, and
tehphin section of autologous HRSC. Dayl to Day14 shows an increase in cells
having
large forward and side scatter that is indicative of granulocytes, and Dayl to
Day3 shows an
increase in the relative number of circulating CD34 haernatopoietic stem
cells. FIG. 2b
shows sequential immunophenotyping of peripheral blood samples of a patient
with severe
aplastic anemia following infusion of autologous human reprogrammed stein cell
(HRSC)
(post Day 1, Day 2, Day 3, Day 6, and Day 14). Cells were labeled with
monoclonal
antibodies against CD34 and CD61 (1st horizontal panel), CD19 and CD3 (2" d
panel), and
CD33&13 and CD7 (3rd horizontal panel). The FACScan plots show increase in the
number
of myeloid cells, as depicted by a gradual increase in cells expressing
CD33&13 including
progenitor cells, which is depicted by an increase in the relative number of
cell co-
expressing CD33&13 with CD7. Also, there was a gradual increase in the
relative number
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of lymphocytes, as shown by an increase in the relative number of CD 19 and
CD3
lymphocytes.
FIG. 3 shows bone marrow analysis of a severe aplastic anaemia patient before
and
after infusion of autologous HRSC. Bone marrow smear before and after therapy
(a and b)
shows increase in red blood cells; terphine section before and after therapy
(c and d) shows
increase in bone marrow cellularity; clonal analysis of bone marrow following
infusion of
HRSC showing increased growth of colony forming unit megakarocyte (e); colony
forming
monocyte (f); colony forming granulocyte and macrophage (g); and colony
forming
myelocyte and erythroid (h); and burst forming erythroid (i).
FIG. 4 shows karyotyping and g-banding of peripheral blood sample of a patient
with severe aplastic anemia following 4 years of infusion with autologous HRSC
showing
no genetic abnormalities.
FIG. 5 shows increase in absolute mean fetal hemoglobin levels in patients
with
thalassemia, post-treatment with autologous reprogrammed cells.
FIG. 6 shows increase in mean corpuscular volume, which represents the average
size of red blood cells, expressed in femtoliters, in patients with
thalassemia, post-treatment
with autologous reprogrammed cells.
FIG. 7 shows increase in mean cell hemoglobin, which is the weight of the
hemoglobin per cell, expressed in picograms, in patients with thalassemia,
post-treatment
with autologous reprogrammed cells.
FIG. 8 shows decrease in serum ferritin levels, expressed in nanograms per
milliliter,
in patients with thalassemia, post-treatment with autologous reprogrammed
cells.
FIG. 9 shows increase in C-peptide levels fasting and post mixed meal diet
intake in
patients with diabetes, post-treatment with autologous reprogrammed cells.
FIG. 10 shows decrease in glycosylated hemoglobin (HbA1C) levels in patients
with
diabetes, post-treatment with autologous reprogrammed cells.
FIG 11 shows decrease in creatine phosphokinase (CPK) and lactate
dehydrogenase
(LDH) levels in patients with muscular dystrophy, post-treatment with
autologous
reprogrammed cells.
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FIG. 12 shows decrease in liver enzymes alanine aminotransferase (ALT) and
aspartate aminotransferase (AST) levels in patients with muscular dystrophy,
post-treatment
with autologous reprogrammed cells.
FIG. 13 shows decrease in microalbumin urea levels in 12 patients with kidney
disease, post-treatment with autologous reprogrammed cells.
FIG 14 shows decrease in glycosylated hemoglobin (HbA1C) levels in 12 patients
with kidney disease due to diabetes, post-treatment with autologous
reprogrammed cells.
FIG 15a shows magnetic resonance imaging (MRI) scans of the brain of a patient
with multiple sclerosis before (left scan) and three months after (right scan)
treatment with
autologous reprogrammed cells depicting decrease in lesion enhancement post
stem cell
therapy. FIG. 15b shows MRI scans of a different part of the brain of the
patient before (left
scan) and three months after (right scan) treatment with autologous
reprogrammed cells. In
the scans depicting before treatment, the arrows point to a lesion in the
brain, while in the
scans depicting after treatment, the arrows point to the improvement in the
lesion.
FIG. 16a shows magnetic resonance imaging (MRI) scans of the brain of a
patient
with multiple sclerosis before (top scans) and six months after (bottom scans)
treatment with
autologous reprogrammed cells. FIG l6b shows additional MRI scans of the brain
of the
patient before (top scans) and six months after (bottom scans) treatment with
autologous
reprogrammed cells. In the scans depicting before treatment, the arrows point
to a lesion in
the brain, while in the scans depicting after treatment, the arrows point to
the improvement
in the lesion with reduction in brain atrophy as depicted by reduction in
ventricle and sulci
dilatation.
FIG. 17a shows sagittal magnetic resonance imaging (MRI) scans of a patient
with
multiple sclerosis before (left scan) and six months after (right scan)
treatment with
autologous reprogrammed cells. FIG 17b shows transverse MRI scans of the
spinal cord of
the patient before (left scan) and six months after (right scan) treatment
with autologous
reprogrammed cells. In the scans depicting before treatment, the arrows point
to a lesion on
the spinal cord, while in the scans depicting after treatment, the arrows
point to the
improvement in the lesion.
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FIG. 18a shows decrease liver enzymes alanine aminotransferase (ALT) levels
and
FIG. 18b shows decrease in aspartate aminotransferase (AST) levels in patients
infected with
hepatitis C, post-treatment with autologous reprogrammed cells
FIG. 19a shows magnetic resonance imaging (MRI) scans of the brain of a
patient
with head trauma due to a motor accident. Before treatment (top scans), the
ventricles show
dilatation and displacement with a wide speared haematoma. After treatment
with
autologous reprogrammed cells (bottom scans), the ventricles show decrease in
brain
atrophy parameters such as reduction in ventricle and sulci dilatation with
amelioration of
haematoma. FIG. 19b shows additional MRI scans of the brain of the patient
before (top
scans) and after (bottom scans) treatment.
FIG 20 shows chest x-rays of a patient with lung disease before (left x-ray)
and after
(right x-ray) treatment with autologous reprogrammed cells. After treatment,
the patient
shows improvement in lung volume and reduction in lesion size as depicted by
decrease in
hypo-dense areas.
FIG. 21 shows sex hormone levels for follicle-stimulating hormone (fsh),
luteinizing
hormone (lh), progesterone (pro), and testosterone (test) in patients with non-
obstructive
azoospermia and treated with autologous reprogrammed cells. The levels show
significant
increase in free testosterone with an increase in testis size (data not shown)
determined by
ultrasound.
FIG. 22 shows retinal sensitivity and visual impairment in a patient suffering
from
impaired vision before (top panel) and after (bottom panel) treatment with
autologous
reprogrammed cells. In the Retinal Sensitivity results, the orange area
indicates decreased
retinal sensitivity. In the Visual Impairment results, the white areas
indicate normal vision
and the pink, orange, and black areas indicate increased visual impairment.
After treatment,
the patient experienced improvement in his visual field, as orange areas in
the retinal
sensitivity results before treatment turned green and white after treatment,
and black areas in
the visual impairment results before treatment turned white after treatment.
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DETAILED DESCRIPTION
Definitions
As used herein, "committed cells" are cells that display a differentiated
character.
These cells are often considered mature and specialized. Examples include
white blood
cells, red blood cells, epithelial cells, neurons, and chondrocytes.
As used herein, "uncommitted cells" are cells that do not display a mature
differentiated character. These cells are often considered immature and are
not specialized.
An example of an uncommitted cell is a stem cell, which is an immature cell
that is capable
of self-renewal (division without limit) and differentiation (specialization).
As used herein, "reprogramming" refers to a process by which a committed cell
of a
first cell lineage is changed into a cell of a different cell type. This
different cell type may
be of a different cell lineage. Reprogramming may occur through such processes
as
retrodifferentiation, transdifferentiation, or redifferentiation.
As used herein, a "reprogrammed cell" is a cell that underwent reprogramming
of a
committed cell. A reprogrammed cell may include a retrodifferentiated cell,
transdifferentiated cell, and/or a redifferentiated cell.
As used herein, "retrodifferentiation" is the process by which a committed
cell, i.e.,
mature, specialized cell, reverts back to a more primitive cell stage.
"Retrodifferentiated
cell" is a cell that results from retrodifferentiation of a committed cell.
As used herein, "transdifferentiation" is the process by which a committed
cell of a
first cell lineage is changed into another cell of a different cell type. In
some embodiments,
transdifferentiation may be a combination of retrodifferentiation and
redifferentiation.
"Transdifferentiated cell" is a cell that results from transdifferentiation of
a committed cell.
For example, a committed cell such as a whole blood cell may be
transdifferentiated into a
neuron.
As used herein, "redifferentiation" refers to the process by which an
uncommitted
cell or a retrodifferentiated cell differentiates into a more mature,
specialized cell.
"Redifferentiated cell" refers to a cell that results from redifferentiation
of an uncommitted
cell or a retrodifferentiated cell. If a redifferentiated cell is obtained
through
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redifferentiation of a retrodifferentiated cell, the redifferentiated cell may
be of the same or
different lineage as the committed cell that had undergone
retrodifferentiation. For example,
a committed cell such as a white blood cell may be retrodifferentiated to form
a
retrodifferentiated cell such as a pluripotent stem cell, and then the
retrodifferentiated cell
may be redifferentiated to form a lymphocyte, which is of the same lineage as
the white
blood cell (committed cell), or redifferentiated to form a neuron, which is of
a different
lineage than the white blood cell (committed cell).
As used herein, "target cell" is a cell that is obtained for administration
into a patient
to repair or replenish tissue or cells. For instance, a target cell may be a
reprogrammed
target cell, such as a retrodifferentiated target cell or a
transdifferentiated target cell,
whereby the retrodifferentiated or transdifferentiated target cell is
administered to the
patient.
Committed Cells
As described above, committed cells of the invention are cells that display a
differentiated character. The committed cell may comprise any components that
are
concerned with antigen presentation, capture or recognition. For example, the
committed
cell may be an MHC Class I+ and/or an MHC Class II+ cell.
The committed cell may also be any cell derived or derivable from an
undifferentiated cell. Thus, in one embodiment, the committed cell is also an
undifferentiated cell. By way of example, therefore, the committed cell can be
a lymphoid
stem cell or a myeloid stein cell, which is differentiated relative to a
pluripotent stem cell.
Committed cells may be derived from biological material, such as blood or
related
tissues including bone marrow or cord blood, neuronal tissue from the central
nervous
system or peripheral nervous system, muscle tissue, or epidermis and/or
der7nis tissue from
skin (i.e. by way of oral scraping for instance). The biological material may
be of post-natal
origin.
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The biological material may be obtained using methods known in the art that
are
suitable for the tissue type. Examples include, but are not limited to,
excision, needle
withdrawal, swabbing, and apheresis.
In particular embodiments, the committed cells are derived from whole blood or
processed products thereof, such as plasma or the buffy coat, since their
removal from
subjects can be carried out with the minimum of medical supervision. Blood
samples are
typically treated with anticoagulants such as heparin or citrate. Cells in the
biological
sample may be treated to enrich certain cell types, remove certain cell types
or dissociate
cells from a tissue mass. Useful methods for purifying and separating cells
include
centrifugation (such as density gradient centrifugation), flow cytometry and
affinity
chromatography (such as the use of magnetic beads comprising monoclonal
antibodies to
cell surface markers or panning) (see Vettese-Dadey, The Scientist 1999, 13:
21). By way
of example, Ficoll-Hypaque separation is useful for removing erythrocytes and
granulocytes
to leave mononuclear cells such as lymphocytes and monocytes.
Examples of committed cells that can be derived from blood include, but are
not
limited to, CFC-T cells, CFC-B cells, CFC-Eosin cells, CFC-Bas cells, CFC-Bas
cells, CFC-
GM cells, CFC-M, CFC-MEG cells, BFC-E cells, CFC-E cells, T cells, B cells,
eosinophils,
basophils, neutrophils, monocytes, megakaryocytes, and erythrocytes.
Blood derived committed cells may be identified by their expression of
particular
antigens. For instance, B cells are CD19+, CD21+, CD22+ and DR+ cells. T cells
are CD2+,
CD3+, and either CD4+ or CD8+ cells. Immature lymphocytes are CD4+ and CD8+
cells.
Activated T cells are DR+ cells. Natural killer cells (NKs) are CD56+ and CD
16+ cells. T
lymphocytes are CD7+ cells. Leukocytes are CD45+ cells. Granulocytes are CD
13+ and
CD33+ cells. Monocyte macrophage cells are CD14+ and DR+ cells.
In certain embodiments, the committed cell may be a B lymphocyte (activated or
non-activated), a T lymphocyte (activated or non-activated), a cell from the
macrophage
monocyte lineage, a nucleated cell capable of expressing class I or class II
antigens, a cell
that can be induced to express class I or class II antigens or an enucleated
cell (i.e. a cell that
does not contain a nucleus--such as a red blood cell).
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In alternative embodiments, the committed cell may be selected from any one of
a
group of cells comprising large granular lymphocytes, null lymphocytes and
natural killer
cells, each expressing the CD56 and/or CD 16 cell surface receptors.
Since the committed cells are essentially primary cultures, it may necessary
to
supplement populations of cells with suitable nutrients to maintain viability.
Suitable
culture conditions are known by the skilled person in the art. Nonetheless,
treatment of cell
populations is preferably initiated as soon as possible after removal of
biological samples
from patients, typically within 12 hours, preferably within 2 to 4 hours. Cell
viability can be
checked using well known techniques such as trypan blue exclusion or propidium
iodide.
Retrodifferentiated Cells
Retrodifferentiation is a type of a reprogramming process whereby structures
and
functions of cells are progressively changed to give rise to less specialized
cells.
Retrodifferentiation can occur naturally, wherein cells may undergo limited
reverse
differentiation in vivo in response to tissue damage. Alternatively,
retrodifferentiation may
be induced using the methods described in U.S. application Serial No.
08/594,164, now U.S.
Patent No. 6,090,625; U.S. application Serial No. 09/742,520, now U.S. Patent
No.
7,112,440; U.S. application Serial No. 10/140,978, now U.S. Patent No.
7,220,412; U.S.
application Serial No. 10/150,789, now U.S. Patent No. 7,410,773; and U.S.
application
Serial No.09/853,188, which are all incorporated herein by reference.
Retrodifferentiated cells of the invention may include, but are not limited to
pluripotent stem cells, lymphoid stem cells, myeloid stem cells, neural stem
cells, skeletal
muscle satellite cells, epithelial stem cells, endodermal stem cells,
mesenchymal stem cells,
and embryonic stern cells.
In particular embodiments, the committed cells are derived from blood and are
retrodifferentiated to form retrodifferentiated cells of the haematopoietic
cell lineage.
Examples of these retrodifferentiated cells include, but are not limited to,
pluripotent stem
cells, lymphoid stem cells, and myeloid stem cells.
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Committed cells may be retrodifferentiated by contacting the cells with an
agent that
operably engages the cells. The cells are then incubated so as to allow those
cells that have
been operably engaged by the agent to progress through the
retrodifferentiation process and
ultimately become undifferentiated.
The contacting step may comprise the agent engaging with surface antigens on
the
committed cell. The agent may act in direct engagement or in indirect
engagement with the
committed cell. An example of direct engagement is when the committed cell has
at least
one cell surface receptor on its cell surface, such as a (3-chain having
homologous regions
(regions that are commonly found having the same or a similar sequence) such
as those that
may be found on B cells, and wherein the agent directly engages the cell
surface receptor.
Another example, is when the committed cell has a cell surface receptor on its
cell surface
such as an a-chain having homologous regions such as those that may be found
on T cells,
and wherein the agent directly engages the cell surface receptor.
An example of indirect engagement is when the committed cell has at least two
cell
surface receptors on its cell surface and engagement of the agent with one of
the receptors
affects the other receptor which then induces retrodifferentiation of the
committed cell.
The agent for the retrodifferentiatiog the committed cell may be a chemical
compound or composition. For instance, the agent may be capable of engaging a
cell
surface receptor on the surface of the committed cell. In certain embodiments,
the agent
operably engages a receptor present on the surface of the committed cell -
which receptor
may be expressed by the committed cell, such as a receptor that is capable of
being
expressed by the committed cell.
For example, agents may include, but are not limited to, any one or more of
cyclic
adenosine monophosphate (cAMP), a CD4 molecule, a CD8 molecule, a part or all
of a T-
cell receptor, a ligand (fixed or free), a peptide, a T-cell receptor (TCR),
an antibody, a
cross-reactive antibody, a monoclonal antibody, or a polyclonal antibody.
Growth factors
may also be used, such as haematopoietic growth factors, for example
erythropoietin and
granulocyte-monocyte colony stimulating factor (GM-CSF).
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If the agent is an antibody, a cross-reactive antibody, a monoclonal antibody,
or a
polyclonal antibody, then the agent may be any one or more of an antibody, a
cross-reactive
antibody, a monoclonal antibody, or a polyclonal antibody to any one or more
of: the beta.
chain of a MHC class II antigen, the (3-chain of a MHC HLA-DR antigen, the a-
chain of a
MHC class I or class II antigen, the a-chain of HLA-DR antigen, the a- and the
(3-chain of
MHC class II antigen or of a MHC class I antigen. An example of an antibody is
CR3/43
(supplied by Dako).
The term "antibody" may include the various fragments (whether derived by
proteolytic cleavage or recombinant technology) and derivatives that retain
binding activity,
such as Fab, F(ab')2 and scFv antibodies, as well as mimetics or bioisosteres
thereof. Also
included as antibodies are genetically engineered variants where some of the
amino acid
sequences have been modified, for example by replacement of amino acid
residues to
enhance binding or, where the antibodies have been made in a different species
to the
organism whose cells it is desired to treat according to the methods of the
invention, to
decrease the possibility of adverse immune reactions (an example of this is
'humanized'
mouse monoclonal antibodies).
Agents used to effect the conversion of a committed cell to a
retrodifferentiated cell
preferably may act extracellularly of the committed cell. For example, the
committed cell
may comprise a receptor that is operably engageable by the agent and the agent
operably
engages the receptor.
For example the receptor may be a cell surface receptor. Specific examples of
cell
surface receptors include, but are not limited to, MHC class I and class II
receptors. The
receptor may comprise an a-component and/or a (3-component, as is the case for
MHC class
I and class II receptors.
The receptor may comprises a (3-chain having homologous regions, for example
at
least the homologous regions of the (3-chain of HLA-DR.
Alternatively, or in addition, the receptor may comprise an a-chain having
homologous regions, for example at least the homologous regions of the a-chain
of HLA-
DR. The receptor may be a Class I or a Class II antigen of the major
histocompatibility
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complex (MHC). In certain embodiments the cell surface receptor may include,
but are not
limited to, an HLA-DR receptor, a DM receptor, a DP receptor, a DQ receptor,
an HLA-A
receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor, an HLA-F
receptor,
or an HLA-G receptor. In some embodiments, the cell surface receptor may be an
HLA-DR
receptor.
The agent may be an antibody to the receptor, such as a monoclonal antibody to
the
receptor.
An example of an agent may be one that modulates MHC gene expression such as
MHC Class I+ and/or MHC Class II+ expression.
In certain embodiments, the agent may be used in conjunction with a biological
response modifier. Examples of biological response modifiers include, but are
not limited
to, an alkylating agent, an inununomodulator, a growth factor, a cytokine, a
cell surface
receptor, a hormone, a nucleic acid, a nucleotide sequence, an antigen or a
peptide. For
instance, an alkylating agent may be or may comprise cyclophosphoamide.
Other biological response modifiers may include compounds capable of
upregulating
MHC class I and/or class II antigen expression, which, in some embodiments,
may allow an
agent that binds to an MHC receptor to work more effectively.
As any cell type can be made to express MHC class I and/or class II antigens,
this
may provide a method for retrodifferentiating a wide variety of cell types
whether they
constitutively express class I and/or class II MHC antigens or not.
Committed cells are generally incubated with an agent for at least two hours,
typically between 2 and 24 hours, preferably between 2 and 12 hours.
Incubations are
typically performed at from about room temperature or for example about 22 C,
up to about
37 C, including 33 C. The progress of the retrodifferentiating procedure can
be checked
periodically by removing a small aliquot of the sample and examining cells
using
microscopy and/or flow cytometry. Alternatively, the device can comprise
tracking means
for on-line monitoring the progress of the retrodifferentiating procedure.
In addition to the use of retrodifferentiating agents, the committed cells may
be
cultured in autologous plasma or serum, or in fetal blood serum or horse
serum. Optionally,
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the committed cells may be cultured with anticoagulants, chelating agents, or
antibiotics.
The temperature range for incubating the cells may be extended to 18-40 C,
and may also
include 4-10 % CO2 and/or 10-35%02. Furthermore, incubation may occur in blood
bags,
tissue culture bags, or plastic vessels, which are coated or uncoated.
Certain types of retrodifferentiated cells may be obtained by
retrodifferentiating
using particular culture conditions. For example, committed cells may be
retrodifferentiated
into pluripotent cells by culturing the committed cells in Dulbecco's Modified
Eagle
Medium (DMEM), non-essential amino acids (NEAA), L-glutamine (L-glu), and 13-
mercaptoethanol (2 (3ME), in conjunction with the retrodifferentiating agents.
The
committed cells may also be initially exposed to chelating agents.
As another example, to obtain mesenchymal cells, committed cells may be
cultured -
in conjunction with retrodifferentiating agent(s) - using DMEM (low glucose)
and L-glu, or
DMEM (low glucose), L-glu, 2 (3ME, and NEAA. Further, the antibiotic
gentamycin may
also be used in the cell culture.
Trans differentiated Cells
Transdifferentiated cells are obtained by culturing committed cells with a
tissue
culture media in conjunction with retrodifferentiating agents. The committed
cells thereby
undergo transdifferentiation, wherein the committed cells are transformed to
cells of another
cell type; in some embodiments, the committed cells are transformed to cells
of a different
lineage.
The type of target cell obtained through transdifferentiation is dependent on
the
culturing conditions. These conditions vary according to the type of tissue
culture media,
the presence/absence of various differentiation promoting agents, the
presence/absence of
different serums, the incubation temperature, the presence/absence of oxygen
or carbon
dioxide, and the type of container or vessel used for incubation.
Examples of tissue culture media used for transdifferentiation include, but
are not
limited to, Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified
Eagle
Medium (DMEM), Eagle's minimum essential (EME) medium, alpha-minimum essential
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medium (a-MEM), Roswell Park Memorial Institute (RPMI; site where medium was
developed) 1640, Ham-F-12, E199, MCDB, Leibovitz L-15, Williams Medium E, or
any
commercially formulated tissue culture medium.
Differentiation promoting agents include anticoagulants, chelating agents, and
antibiotics. Examples of such agents may be one or more of the following:
vitamins and
minerals or derivatives thereof, such as A (retinol), B3, C (ascorbate),
ascorbate 2-
phosphate, D2, D3, K, retinoic acid, nicotinamide, zinc or zinc compound, and
calcium or
calcium compounds; natural or synthetic hormones such as hydrocortisone, and
dexamethasone; amino acids or derivatives thereof, such as L-glutamine (L-
glu), ethylene
glycol tetracetic acid (EGTA), proline, and non-essential amino acids (NEAA);
compounds
or derivatives thereof, such as (3-mercaptoethal, dibutyl cyclic adenosine
monophosphate
(db-cAMP), monothioglycerol (MTG), putrescine, dimethyl sulfoxide (DMSO),
hypoxanthine, adenine, forskolin, cilostamide, and 3-isobutyl-l-
methylxanthine; nucleosides
and analogues thereof, such as 5-azacytidine; acids or salts thereof, such as
ascorbic acid,
pyruvate, okadic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA),
anticoagulant
citrate dextrose formula A (ACDA), disodium EDTA, sodium butyrate, and
glycerophosphate; antibiotics or drugs, such as G418, gentamycine,
Pentoxifylline (1-(5-
oxohexyl)-3, 7-dimethylxanthine), and indomethacin; and proteins such as
tissue
plasminogen activator (TPA).
These differentiation promoting agents may be used to obtain particular types
of
target cells. For instance, vitamin B3 may be used to yield acinar cells such
as islet cells or
hydrocortisone; dexamethasone may be used to yield cells of mesenchymal origin
or
epithelial origins (e.g. kidney epithelial cells, skin and associated
structures such as dermal
papilla cells); and (3-mercaptoethal may be used to yield ectodermal cells
such as neuronal
cells, including accessory cells of the CNS.
The culture medium may contain autologous plasma; platelets; serum such as
fetal
blood sampling; or sera of mammalian origin such as horse serum. Furthermore,
the
conversion process can occur inside blood bags, scaffolds, tissue culture
bags, or plastic
tissue culture vessels. The tissue culture vessels may be adherent or non-
adherent tissue
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culture vessels, or may be coated or uncoated with agents such as gelatin,
collagen,
matrigels or extracellular matrices that either promote adherence or
floatation depending on
the required type of tissue or specialized cells to be prepared.
Additional culturing conditions include the temperature, which may be between
about 10 and about 60 C, or between about 18 and about 40 C; levels of
carbon dioxide
(CO2), which may be between about 0 and about 20 %, or about 4 and about 10 %;
and
oxygen (02), which may be between about 0 and about 50 %, or about 10 and
about 35 %.
Examples of methods to obtain the target cells and used in conjunction with
retrodifferentiating agents are discussed in Table 1.
Table 1. Methods obtain various types of target cells in conjunction with
retrodifferentiating
agents.
Target Cell Culture Conditions Used in Additional Culture Conditions
Type Conjunction With
Retrodifferentiating Agents
Pluripotent Stem = DMEM, NEAA, L-glu, 2(3ME = Prior exposure of committed
cells or cells to chelating or
heterogenous anticoagulants agents.
population of = For progenitors, withdrawal of
pluripotent retrodifferentiating agent by
progenitor cells diluting with culture media only
Mesenchymal = DMEM (low glucose), L-glu; or = Gentamycin
stem cells = DMEM (low glucose), L-glu,
2(3ME, NEAR
= Pluripotent = RPMI 1640, optionally with GAIC
germ cells NEAA, L- glu, 2(3ME for = incubation can be at about 30-32
= Oocytes oocytes; or C for sperm and about 38-39 C
= Sperms = EME medium, retinol, L-glu, for oocytes.
sodium pyruvate, sodium lactate, = cells can be exposed to chelating
NEAA for oocytes; or agents such as EDTA and EGT
= DMEM, Hams F12, vitamin C, prior to retrodifferentiation and
vitamin E, Retinoic acid, retinol, redifferentiation.
pyruvate, optionally with = For sperm, can include addition
Pentoxifylline for sperm; or of okadic acid, DMSO and zinc
o Culture condition for pluripotent or zinc compound; Gamete 100
stem cells listed above and with can be used as a basal medium
additional culture condition instead
listed in the next column for = For oocytes, can include addition
either sperm or oocytes. of dp-cAMP, disodium EDTA,
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forskolin, cilostamide and
hypoxanthine
= For oocytes, Medium 199 can be
used as a basal medium instead
Kidney = DMEM, Hams F 12, and
hydrocortisone, optionally with
vitamin K; or
= Culture conditions for
pluripotent stem cells listed
above, with hydrocortisone
= Alveolar = DMEM, NEAA, L-glu, = With antibiotic G418 and
epithelium optionally with 2 (3ME, matrigel coated tissue culture
= Endoderm nicotinamide; or vessels
= Culture conditions for
pluripotent stein cells listed
above, with nicotinamide,
optionally with dexamethasone,
retinoic acid, db-cAMP; or
= IMDM, L-glu, ascorbic acid,
MTG
= Neuron o DMEM with Hams F 12, NEAA,
= Ectoderm 2(3ME, optionally with
putrescine, retinoic acid, L-glu,
hydrocortisone, ascorbate; or
= Culture conditions for
pluripotent stem cells listed
above, optionally with
putrescine, retinoic acid,
hydrocortisone, ascorbate
Islet cells = DMEM, Hams F12, vitamin 133;
Acinar or
= RPMI 1640 with vitamin B3; or
= Culture conditions for
pluripotent stein cells listed
above, with Vitamin B3
(nicotinamide), optionally with
dexamethasone
Haematopoietic = IMDM, optionally with = Incubation can be at 33 C
cells hydrocortisone; or = Incubation can be at room
= IMDM, L glutamine and MTG; temperature for amplifying
or megakaryocytes in culture
= Culture conditions for = differentiated cells can be
pluripotent stem cells listed exposed to chelating agents prior
above, with MTG substituted for to conversion in order to amplify
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2(3ME, optionally with vitamins for erythroid progenitors in
culture
= RPMI 1640 can be used as a
basal medium for enrichment of
lymphoid progenitors
= Sodium butyrate and/or 5-
azacytidine can be added to
culture to promote primitive
erythroid differentiation
Hepatocytes of = DMEM or IMDM or a-minimum
the liver essential medium, L-glu,
optionally with dexamethasone,
L ascorbic acid-2-phosphate and
nicotinamide; or
= Williams Medium E, sodium
pyruvate, dexamethasone; or
= Culture conditions for
pluripotent stem cells listed
above, with dexamethasone
Skin/ = Hams F12, DMEM (media ratio = Can be with gentamycine as
keratinocytes 3:1), hydrocortisone, L-glu, antibiotic
optionally with adenine; or = Can incubate at 36.4 C
= E199 or DMEM with L-glu,
optionally with hydrocortisone
and adenine; or
= Culture conditions for
pluripotent stem cells listed
above, with hydrocortisone,
optionally with calcium or
calcium compounds or ascorbate
Melanocytes = MCDB and Leibovitz L- 15,
TPA, optionally with 3-isobutyl-
1-methylxanthine; or
= medium 199 and hydrocortisone
Osteocytes/bone = DMEM,13-glycerophosphate,
dexamethasone, ascorbate and L-
glu, optionally with vitamin 133;
or
= Culture conditions for
pluripotent stem cells listed
above, with glycerophosphate,
dexamethasone and ascorbate,
optionally with vitamin D3
Dermal Papila = William E medium, L-glu,
cells/hair hydrocortisone and/or vitamin
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D2, adenine and linoleic acid; or
= DMEM, Hams F12 as basal
medium, L-glu, hydrocortisone
and/or vitamin D2, adenine and
linoleic acid; or
= Culture conditions for
pluripotent stem cells listed
above, hydrocortisone, vitamin
D2, adenine and linoleic acid
Chondrocytes/ = DMEM, pyruvate, ascorbate 2-
cartilage phosphate, dexamethasone and
proline; or
= Culture conditions for
pluripotent stem cells listed
above, ascorbate 2-phosphate,
dexamethasone and proline
Adipocytes/fat = DMEM, dexamethasone, and
cells indomethacin; or
= dexamethasone and
indomethacin
Skeletal muscles = DMEM, low glucose, optionally = Can be with gentamycin as
with hydrocortisone, antibiotic
dexamethasone, L-glutamine and = Can be with low serum
sodium pyruvate; or concentration.
= DMEM and Ham F12 or F10 as o Can be with culture vessels with
basal medium, or DMEM and gelatin
medium 199; or o Can be at a temperature raised to
= Culture conditions for 39 C
pluripotent stem cells listed = Can include addition of 5-
above, with glucose, azacytidine
gentamycin and low serum
Blood vessels = DMEM, NEAA and 2 (3ME; or
(endothelium) o IMDM and dexamethasone
Cardiac muscle/ = 4:1 DMEM and M199 media or = Gelatin coated culture vessels
for
cardiomyocytes DMEM (low glucose), L-glu, full differentiation and
NEAA; or DMEM (low glucose) autologous serum or platelets
with ascorbic acid and/ or depleted plasma
DMSO; or e Full differentiation can be
= Culture conditions for observed on cover slip slide
pluripotent stem cells listed
above with anti-gravity culturing
or in a vibrating environment
Tropoblast = DMEM, L-glu, 2 I3ME, NEAA = Can be used to produce
with continuous dilution with the autologous growth factors and
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same culture medium minus the hormones necessary for
retrodifferentiating agent; or differentiating cells into
= RPMI 1640, 2 (3ME, sodium mesoderm and ectoderm,
pyruvate and L-glutamine including germ stem cells and
progenitors
Notably, for the culture conditions described for each cell type in Table 1,
withdrawal of the retrodifferentiation agent by subsequent dilution of the
culture condition
with the corresponding culture medium results in more enhanced
transdifferentiation.
During culturing, the culture medium optionally containing the various
differentiation promoting agents may be diluted by the addition of more medium
but without
the retrodifferentiating agent. Not to be bound by theory, dilution seems to
increase
differentiation because cells become less dense and proliferation stimulating
factors are less
concentrated. Thus, the addition of medium may further enhance
transdifferentiation and
will affect what type of cell within the cell lineage is obtained. For
instance, if the target
cell is a neuron, the addition of culture medium may result in a shift in
development towards
a more mature neuron rather than a neuron progenitor (both are of the same
lineage). As
another example, forward differentiation skeletal muscle progenitors will only
differentiate
by consecutive dilution of culture medium which is achieved by gradually
decreasing the
serum concentration.
Redifferentiated Cells
The retrodifferentiated cells may be used to obtain target cells by
recommitting or
redifferentiating the retrodifferentiated cells to a target cell type. This
may be performed by
contacting the retrodifferentiated cells with growth factors. For example,
retinoic acid has
been used to differentiate stem cells into neuronal cells. Methylcellulose
followed by co-
culture with a bone marrow stromal line and IL-7 has been used to
differentiate stem cells
into lymphocyte precursors (Nisitani et al., Int Immuno 1994, 6: 909-916). Le
Page (New
Scientist Dec. 16, 2000) teaches that stein cells can be differentiated into
lung epithelial
cells. Bischoff (Dev Biol 1986, 115: 129-39) teaches how to differentiate
muscle satellite
cells into mature muscle fibres. Neural precursor cells can be expanded with
basic fibroblast
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growth factor and epidermal growth factor (Nakafuku and Nakamura, J Neurosci
Res 1995,
41: 153-168). Haematopoietic stem cells can be expanded using a number of
growth factors
including GM-CSF, erythropoietin, stem cell factor and interleukins (IL-1, IL-
3, IL-6)--see
Metcalf (Nature 1989, 339: 27-30) for a review of these various factors.
Potocnik et al. (EMBO J 1994, 13: 5274-83) even demonstrated the
differentiation of
stem cells to haematopoietic cells using low oxygen (5%) conditions.
The redifferentiated cell may be of the same lineage as the committed cell
from
which the retrodifferentiated cell was derived. Alternatively, the
redifferentiated cell may
be of a different lineage than the committed cell from which the
retrodifferentiated cell was
derived. For example, a B lymphocyte may be retrodifferentiated to a CD34+CD38-
HLA-
DR" stem cell. This stem cell may be subsequently redifferentiated or
recommitted along a
B cell lineage (the same lineage) or a lymphoid lineage (different lineage).
Target Cells
The target cells of the invention are reprogrammed cells that can be obtained
by
retrodifferentiation, transdifferentiation, or redifferentiation as described
above. According
to the invention, target cells may include, but are not limited to,
pluripotent stem cells,
lymphoid stem cells, myeloid stem cells, neural stem cells, skeletal muscle
satellite cells,
epithelial stem cells, endodermal and neuroectodermal stem cells, germ cells,
extraembryonic and embryonic stem cells, mesenchymal stem cells, , kidney
cells, alveolar
epithelium cells, endoderm cells, neurons, ectoderm cells, islet cells, acinar
cells, oocytes,
sperm, haematopoietic cells, hepatocytes, skin/keratinocytes, melanocytes,
bone/osteocytes,
hair/dermal papilla cells, cartilage/chondrocytes, fats cells/adipocytes,
skeletal muscular
cells, endothelium cells, cardiac muscle/cardiomyocytes, and tropoblasts.
As described above, committed cells and/or retrodifferentiated cells are
cultured
under particular conditions to induce retrodifferentiation and/or
transdifferentiation and/or
redifferentiation and obtain the target cells. The duration for which the
committed cells
and/or retrodifferentiated cells are cultured is not controlled by a
particular length of time,
but rather by a determination that the target cells have been produced.
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The determination of the production of, or the changes in the number of,
retrodifferentiated, transdifferentiated, or redifferentiated target cells may
be performed by
monitoring changes in the relative number of committed cells that
downregulates expression
of lineage-associated markers or transcription factors, and/or changes in the
relative number
of cells having cell surface markers characteristic of the target cells.
Alternatively, or in
addition, decreases in the numbers of cells having cell surface markers
typical of the
committed cells and not the target cells may be monitored. For example, the
target cell may
be an embryonic stem cell, which are characterized by many stage-specific
markers such as
POU5F1 (OCT-4), TERT, KLF4, UTFI, SOX2, Nanog or stage-specific embryonic
markers
3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoproteins TRA-1-60 and
TRA-1-
81 and alkaline phosphatase (Andrews et al., Hybridoma 1984, 3: 347-361;
Kannagi et al.,
EMBO J 1983, 2: 2355-2361; Fox et al., Dev Biol 1984, 103: 263-266; Ozawa et
al., Cell
Differ 1985, 16: 169-173). They also do not express SSEA-1, the presence of
which is an
indicator of differentiation. Other markers are known for other types of stem
cells, such as
Nestein for neuroepithelial stem cells (J Neurosci 985, 5: 3310). Mesenchymal
stem cells
are positive for SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a and CD124,
for
example, and negative for CD34, CD45 and CD 14. Pluripotent stem cells are
CD34+ DR-
TdT- cells (other useful markers being CD38- and CD36+). Lymphoid stem cells
are DR+,
CD34+ and TdT+ cells (also CD38+). Myeloid stem cells are CD34+, DR+, CD 13+,
CD33+,
CD7+ and TdT+ cells.
Additional cell markers for target cells may be discovered through microarray
analysis. The analysis may involve isolating RNA from target cells that were
retrodifferentiated and/or transdifferentiated and/or redifferentiated,
labeling the isolated
RNA with a dye, and hybridizing the isolated RNA to a microarray. The
microarray may
comprise genes or oligonucleotides representing a whole genome, or may
comprise genes or
oligonucleotides directed to a particular organ system, tissue system,
disease, pathology, etc.
Cell markers may be identified by which genes/oligonucleotides exhibit high
signal
intensity, and are thereby upregulated or downregulated in the target cells.
This information
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can then be applied to determining target cells based on the presence of
markers, or even
groups or a pattern of markers, that were identified by the microarray
analysis.
Confirmation of target cells can also be performed using a number of in vitro
assays
such as CFC assays (see also, the examples). Very primitive haematopoietic
stem cells are
often measured using the long-term culture initiating cell (LTC-IC) assay
(Eaves et al, J Tiss
Cult Meth 1991, 13: 55-62). LTC-ICs sustain haemopoiesis for 5 to 12 weeks.
For other cell types such as cells of the central nervous system, pancreas,
liver,
kidney, skin, etc., cell culturing may continue until the target cells emerge
as characterized
by immunohistochemistry, flow cytometry, microarrays, or reverse-transcription
polymerase
chain reaction (RT-PCR), which are techniques known in the art. This may also
include
functional assays, for example, engrafting an immunodeficient host or
correcting or
ameliorating an underlying clinical condition, as observed herein.
The target cells can be identified by microarray or RT-PCR that show
acquisition of
new lineage-specific transcription factors, proteins, and signals in the
target cells. For
example, retrodifferentiated stem cells converted to target cells of the
ectodermal lineage
may express genes such as Nestin, Criptol, isll, LHX1, and/or EN1, and if
further
differentiated into neurons, will be expressing neurofilaments (NF). On the
other hand, cells
converted into target cells of the endodermal lineage may express genes such
as sox7,
sox 17, Nodal, PDX1, and/or FOXA2; but target cells further differentiated
towards
pancreatic islet cells may express genes such as insulin (INS) and neurog3
(NGN3).
Notably, the conversion towards the desired target cells may be accompanied by
down
regulation of mature transcription factors associated with the original
starting population that
has undergone conversion.
In addition, the determination of the production of target cells may occur by
recognizing particular structural and/or morphological characteristics of the
target cells, e.g.,
cell shape, size, etc. These characteristics are known in the art for the
target cells of the
invention.
Once the relative numbers of the desired cell type have increased to a
suitable level,
which may for example be as low as 0.1% or as high as 5%, the resulting
altered cell
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populations may be used in a number of ways. With respect to the numbers of
target cells,
e.g., pluripotent stem cells, formed, it is important to appreciate the
proliferative ability of
stem cells. Although under some circumstance, the numbers of stem cells or
other
retrodifferentiated cells formed may appear to be low, studies have shown that
only 50
pluripotent haematopoietic stem cells can reconstitute an entire
haematopoietic system in a
donor mouse. Thus therapeutic utility does not require the formation of a
large number of
cells.
Conversion of committed cells to retrodifferentiated, transdifferentiated, or
redifferentiated target cells may also be carried out in vivo by
administration of the agent,
admixed with a pharmaceutically carrier or diluent, to a patient. However it
is preferred in
many cases that retrodifferentiating, transdifferentiating, or
redifferentiating is performed in
vitrolex vivo.
Treated populations of cells obtained in vitro may be used subsequently with
minimal processing. For example they may be simply combined with a
pharmaceutically
acceptable carrier or diluent and administered to a patient in need of stem
cells.
It may however be desirable to enrich the cell population for the
retrodifferentiated,
transdifferentiated, or redifferentiated target cells or purify the cells from
the cell population.
This can conveniently be performed using a number of methods (see Vattese-
Dadey--The
Scientist 1999, 13). For example cells may be purified on the basis of cell
surface markers
using chromatography and/or flow cytometry. Nonetheless, it will often be
neither
necessary nor desirable to extensively purify retrodifferentiated,
transdifferentiated, or
redifferentiated target cells from the cell population since other cells
present in the
population (for example stromal cells) may maintain stem cell viability and
function.
Flow cytometry is a well-established, reliable and powerful technique for
characterizing cells within mixed populations as well as for sorting cells.
Thus, the
purification or isolation means may comprise a flow cytometer. Flow cytometry
operates on
the basis of physical characteristics of particles in liquid suspension, which
can be
distinguished when interrogated with a beam of light. Such particles may of
course be cells.
Physical characteristics include cell size and structure or, as has become
very popular in
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recent years, cell surface markers bound by monoclonal antibodies conjugated
to fluorescent
molecules.
Kreisseg et al. (J Hematother 1994, 3: 263-89) state, "[b]ecause of the
availability of
anti-CD34 monoclonal antibodies, multiparameter flow cytometry has become the
tool of
choice for determination of haematopoietic stem and progenitor cells."
Kreisseg further
describes general techniques for quantization and characterization of CD34-
expressing cells
by flow cytometry. Further, Korbling et al., (Bone Marrow Transplant 1994, 13:
649-54)
teaches purification of CD34+ cells by immunoadsoiption followed by flow
cytometry based
on HLA-DR expression. As discussed above, CD34+ is a useful marker in
connection with
stem cells/progenitor cells. Flow cytometry techniques for sorting stem cells
based on other
physical characteristics are also available. For example, Visser et al. (Blood
Cells 1980,
6:391-407) teach that stem cells may be isolated on the basis of their size
and degree of
structuredness. Grogan et al. (Blood Cells 1980, 6: 625-44) also teach that
"viable stem
cells may be sorted from simple haematopoietic tissues in high and verifiable
purity".
As well as selecting for cells on the basis of the presence of a cell surface
marker or
other physical property (positive selection), cell populations may be
enriched, purified using
negative criteria. For example, cells that possess lineage specific markers
such as CD4,
CD8, CD42 and CD3 may be removed from the cell population by flow cytometry or
affinity chromatography.
A very useful technique for purifying cells involves the use of antibodies or
other
affinity ligands linked to magnetic beads. The beads are incubated with the
cell population
and cells that have a cell surface marker, such as CD34, to which the affinity
ligand binds
are captured. The sample tube containing the cells is placed in a magnetic
sample
concentrator where the beads are attracted to the sides of the tube. After one
or more wash
stages, the cells of interest have been partially or substantially completely
purified from
other cells. When used in a negative selection format, instead of washing
cells bound to the
beads by discarding the liquid phase, the liquid phase is kept and
consequently, the cells
bound to the beads are effectively removed from the cell population.
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These affinity ligand-based purification methods can be used with any cell
type for
which suitable markers have been characterized or may be characterized.
Urbankova et al.
(J Chromatogr B Biomed Appl 1996, 687: 449-52) teaches the micropreparation of
haematopoietic stem cells from a mouse bone marrow suspension by gravitational
field-flow
fractionation. Urbankova et al. further comments that the method was used for
the
characterization of stem cells from mouse bone marrow because these cells are
bigger than
the other cells in bone marrow and it is therefore possible to separate them
from the mixture.
Thus physical parameters other than cell surface markers may be used to
purify/enrich for
stem cells.
Cell populations comprising reprogrammed target cells such as
retrodifferentiated,
transdifferentiated, or redifferentiated target cells, and/or purified
reprogrammed target cells,
such as retrodifferentiated, transdifferentiated, or redifferentiated target
cells produced by
the methods of the invention, may be maintained in vitro using known
techniques.
Typically, minimal growth media such as Hanks, RPMI 1640, Dulbecco's Minimal
Essential
Media (DMEM) or Iscove's Modified Dulbecco Medium, are used, supplemented with
mammalian serum such as FBS, and optionally autologous plasma, to provide a
suitable
growth environment for the cells. Stem cells may be cultured on feeder layers
such as layers
of stromal cells (see Deryugina et al. Crit Rev Immunology 1993, 13: 115-150).
Stromal
cells are believed to secrete factors that maintain progenitor cells in an
undifferentiated state.
A long term culture system for stem cells is described by Dexter et al. (J
Cell Physiol 1977,
91: 335) and Dexter et al. (Acta Haematol 1979, 62: 299).
For instance, Lebkowski et al. (Transplantation 1992, 53: 1011-9) teaches that
human CD34+ haematopoietic cells can be purified using a technology based on
the use of
monoclonal antibodies that are covalently immobilized on polystyrene surfaces
and that the
CD34+ cells purified by this process can be maintained with greater than 85%
viability.
Lebkowski et al., (J Hematother 1993, 2: 339-42) also teaches how to isolate
and culture
human CD34+ cells. See also Haylock et al. (Immunomethods 1994, 5: 217-25) for
a review
of various methods.
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Cell populations comprising stem cells and purified preparations comprising
stem
cells may be frozen/cryopreserved for future use. Suitable techniques for
freezing cells and
subsequently reviving them are known in the art.
In one aspect, the retrodifferentiating, transdifferentiating, or
redifferentiating occurs
to cells from or in buffy coat blood samples. The term "buffy coat" means the
layer of white
cells that forms between the layer of red cells and the plasma when unclotted
blood is
centrifuged or allowed to stand.
Methods of Treatment
Reprogrammed target cells of the present invention, such as
retrodifferentiated target
cells, transdifferentiated target cells, and redifferentiated target cells,
may be combined with
various components to produce compositions of the invention. The compositions
may be
combined with one or more pharmaceutically acceptable carriers or diluents to
produce a
pharmaceutical composition (which may be for human or animal use). Suitable
carriers and
diluents include, but are not limited to, isotonic saline solutions, for
example phosphate-
buffered saline. The composition of the invention may be administered by
direct injection.
The composition may be formulated for parenteral, intramuscular, intravenous,
subcutaneous, intraocular, oral, transdermal administration, or injection into
the spinal fluid.
Compositions comprising target cells may be delivered by injection or
implantation.
Cells may be delivered in suspension or embedded in a support matrix such as
natural and/or
synthetic biodegradable matrices. Natural matrices include, but are not
limited to, collagen
matrices. Synthetic biodegradable matrices include, but are not limited to,
polyanhydrides
and polylactic acid. These matrices may provide support for fragile cells in
vivo.
The compositions may also comprise the retrodifferentiated or
transdifferentiated or
redifferentiated target cells of the present invention, and at least one
pharmaceutically
acceptable excipient, carrier, or vehicle.
Delivery may also be by controlled delivery, i.e., delivered over a period of
time
which may be from several minutes to several hours or days. Delivery may be
systemic (for
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example by intravenous injection) or directed to a particular site of
interest. Cells may be
introduced in vivo using liposomal transfer.
Target cells may be administered in doses of from 1 x 105 to 1 x 107 cells per
kg. For
example a 70 kg patient may be administered 14 x 106 CD34+ cells for
reconstitution of
tissues. The dosages may be any combination of the target cells listed in this
application.
The methods of the invention can be used to treat a variety of diseases,
conditions, or
disorders. Such conditions include, but are not limited to, bone marrow
failure,
haematological conditions, aplastic anemia, beta-thalassemia, diabetes, motor
neuron
disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney
disease, liver
disease, multiple sclerosis, congestive heart failure, hepatitis C virus,
human
immunodeficiency virus, head trauma, lung disease, depression, non-obstructive
azoospermia, andropause, menopause and infertility, rejuvenation, scleroderma
ulcers,
psoriasis, wrinkles, liver cirrhosis, autoimmune disease, alopecia, retinitis
pigmentosa, and
crystalline dystrophy/blindness or any disorder associated with tissue
degeneration.
Aplastic anemia is a rare but fatal bone marrow disorder, marked by
pancytopaenia
and hypocellular bone marrow (Young et al. Blood 2006, 108: 2509-2519). The
disorder
may be caused by an immune-mediated pathophysiology with activated type I
cytotoxic T
cells expressing Thl cytokine, especially y-interferon targeted towards the
haematopoietic
stem cell compartment, leading to bone marrow failure and hence anhaematoposis
(Bacigalupo et al. Hematology 2007, 23-28). The majority of aplastic anaemia
patients can
be treated with stem cell transplantation obtained from HLA-matched siblings
(Locasciulli
et al. Haematologica. 2007; 92:11-18.), though, extending this approach to
older patients or
those that lack family donors remain a great challenge. Despite the reasonable
survival rate
after HLA-matched allogenic stem cell transplantation, the procedure carries
some potential
risks due to the immunosuppressive regime used to prevent graft versus host
disease
(GVDH). For example high dose cyclophosphoamide with or without antithymocyte
globulin (ATG) leads to prolonged period of immunosuppression and predisposes
the patient
to opportunistic infection. Other potential risk is graft failure which may
ensue weeks or
months after stem cell transplantation (Gottdiener et al., Arch Intern Med
1981, 141: 758-
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763; Sanders et al. Semin Hematol 1991, 28: 244-249). Moreover the risk of
graft failure
increases with the number of blood transfusion received prior stem cell
transplantation.
Thalassaernia is an inherited autosomal recessive blood disease marked by a
reduced
synthesis rate of one of the globin chains that make up hemoglobin. Thus,
there is an
underproduction of normal globin proteins, often due to mutations in
regulatory genes,
which results in formation of abnormal hemoglobin molecules, causing anemia.
Different
types of thalassernia include alpha thalassemia, beta thalassemia, and delta
thalassernia,
which affect production of the alpha globin, beta globin, and delta globin,
respectively.
Treatments include chronic blood transfusion, iron chelation, splenectomy, and
allogeneic
haematopoietic transplantation. However, chronic blood transfusion is not
available to most
patients due to the lack of an HLA-matched bone marrow donor, while allogeneic
hematopoietic transplantation is associated with many possible complications
such as
infections and graft-versus-host disease.
Diabetes is a syndrome resulting in abnormally high blood sugar levels
(hyperglycemia). Diabetes refers to a group of diseases that lead to high
blood glucose
levels due to defects in either insulin secretion or insulin action in the
body. Diabetes is
typically separated into two types: type 1 diabetes, marked by a diminished
production of
insulin, or type 2 diabetes, marked by a resistance to the effects of insulin.
Both types lead
to hyperglycemia, which largely causes the symptoms generally associated with
diabetes,
e.g., excessive urine production, resulting compensatory thirst and increased
fluid intake,
blurred vision, unexplained weight loss, lethargy, and changes in energy
metabolism.
Diabetes is considered to be a chronic disease, without a cure. Treatment
options are limited
to insulin injections, exercise, proper diet, or, for patients who have type 2
diabetes, some
medications, e.g., those that promote insulin secretion by the pancreas,
decrease glucose
produced by the liver, increase sensitivity of cells to insulin, etc.
Motor neuron diseases refer to a group of neurological disorders that affect
motor
neurons. Such diseases include arnyotrophic lateral sclerosis (ALS), primary
lateral
sclerosis (PLS), and progressive muscular atrophy (PMA). ALS is marked by
degeneration
of both the upper and lower motor neurons, which ceases messages to the
muscles and
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results in their weakening and eventual atrophy. PLS is a rare motor neuron
disease
affecting upper motor neurons only, which causes difficulties with balance,
weakness and
stiffness in legs, spasticity, and speech problems. PMA is a subtype of ALS
that affects only
the lower motor neurons, which can cause muscular atrophy, fasciculations, and
weakness.
There are no known cures for motor neuron diseases. Riluzole, which is
believed to reduce
damage to motor neurons, has been has approved as a medication for ALS,
although it slows
down the progression of ALS rather than improves its effects. For PLS,
treatments only
address symptoms, such as baclofen which can reduce spasticity or quinine
which may
decrease cramps.
Parkinson's disease (PD) is a neurodegenerative disorder marked by the loss of
the
nigrostriatal pathway, resulting from degeneration of dopaminergic neurons
within the
substantia nigra. The cause of PD is not known, but is associated with the
progressive death
of dopaminergic (tyrosine hydroxylase (TH) positive) mesencephalic neurons,
inducing
motor impairment. Hence, PD is characterized by muscle rigidity, tremor,
bradykinesia, and
potentially akinesia. Thus, there is currently no satisfactory cure for
Parkinson's disease or
treatments for preventing or treating Parkinson's disease or its symptoms.
Symptomatic
treatment of the disease-associated motor impairments involves oral
administration of
dihydroxyphenylalanine (L-DOPA), which can lead to a substantial improvement
of motor
function, but its effects are reduced as the degeneration of dopaminergic
neurons progresses.
Alternative strategies include neural grafting, which is based on the idea
that dopamine
supplied from cells implanted into the striatum can substitute for lost
nigrostriatal cells, and
gene therapy, which can be used to replace dopamine in the affected striatum
by introducing
the enzymes responsible for L-DOPA or dopamine synthesis such as by
introducing
potential neuroprotective molecules that may either prevent the TH-positive
neurons from
dying or stimulate regeneration and functional recovery in the damaged
nigrostriatal system.
Spinal cord injury is characterized by damage to the spinal cord and, in
particular, the
nerve fibers, resulting in impairment of part or all muscles or nerves below
the injury site.
Such damage may occur through trauma to the spine that fractures, dislocates,
crushes, or
compresses one or more of the vertebrae, or through nontraumatic injuries
caused by
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arthritis, cancer, inflammation, or disk degeneration. While treatments
following spinal cord
injury may involve medications such as methylprednisolone, which is a
corticosteroid that
reduce damage to nerve sells and decreases inflammation in the injured area,
or medications
that control pain and muscle spasticity, as well as immobilization of the
spine or surgery to
remove herniated disks or any objects that may be damaging the spine, there is
no known
means to reverse the damage to the spinal cord.
Muscular dystrophy (MD) refers to a set of hereditary muscle diseases that
weaken
skeletal muscles. MD may be characterized by progressive muscle weakness,
defects in
muscle proteins, muscle cell apoptosis, and tissue atrophy. There are over 100
diseases
which exhibit MD characteristics, although nine diseases in particular -
Duchene, Becker,
limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal,
distal, and
Emery-Dreifuss - are classified as MD. There are no known cures for MD, nor
are there
any specific treatments. Physical therapy may maintain muscle tone and surgery
may be
used to improve quality of life. Further, symptoms such as myotonia may be
treated with
medications, but there are no long term treatments.
Kidney disease refers to conditions that damage the kidneys and decrease their
ability
to function, which includes removal of wastes and excess water fro the blood,
regulation of
electrolytes, blood pressure, acid-base balance, and reabsorption of glucose
and amino acids.
The two main causes of kidney disease are diabetes and high blood pressure,
although other
causes include glomerulonephritis, lupus, and malformations and obstructions
in the kidney.
There is no cure for kidney disease, and thereby therapy focuses on slowing
the progression
of the disease and treating the causes of the disease, such as through
controlling blood
glucose and high blood pressure and monitoring diet; treating complications of
the disease,
for example, by addressing fluid retention, anemia, bone disease; and
replacing lost kidney
function, such as through dialysis or transplantation.
Multiple sclerosis is an autoimmune condition in which the immune system
attacks
the central nervous system, leading to demyelination. MS affects the ability
of nerve cells in
the brain and spinal cord to communicate with each other, as the body's own
immune system
attacks and damages the myelin which enwraps the neuron axons. When myelin is
lost, the
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axons can no longer effectively conduct signals. This can lead to various
neurological
symptoms which usually progresses into physical and cognitive disability.
There is no
known cure for MS; treatments attempt to return function after an attack
(sudden onset or
worsening of MS symptoms), prevent new attacks, and prevent disability. For
example,
treatment with corticosteroids may help end the attack, while treatment with
interferon
during an initial attack has been shown to decrease the chance that clinical
MS will develop.
Human immunodeficiency virus (HIV) is a lentivirus that can lead to acquired
immunodeficiency syndrome (AIDS), a condition wherein the immune system begins
to fail.
HIV primarily infects vital cells in the human immune system such as helper T
cells,
macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T
cells by
direct viral killing of infected cells, by increased rates of apoptosis in
infected cells, or by
killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize
infected cells.
Currently, there is no vaccine or cure for HIV or AIDS. Treatment for HIV
infection
consists of highly active antiretroviral therapy, or HAART. Current HAART
options are
combinations (or "cocktails") consisting of at least three drugs of at least
two types
antiretroviral agents. Typically, these classes are two nucleoside analogue
reverse
transcriptase inhibitors (NARTIs or NRTIs) plus either a protease inhibitor or
a non-
nucleoside reverse transcriptase inhibitor (NNRTI).
Congestive heart failure refers to a condition in which the heart cannot pump
enough
blood to the body's other organs. This condition can result from coronary
artery disease,
scar tissue on the heart cause by myocardial infarction, high blood pressure,
heart valve
disease, heart defects, and heart valve infection. Treatment programs
typically consist of
rest, proper diet, modified daily activities, and drugs such as angiotensin-
converting enzyme
(ACE) inhibitors, beta blockers, digitalis, diuretics, vasodilators. However,
the treatment
program will not reverse the damage or condition of the heart.
Hepatitis C is an infectious disease in the liver, caused by hepatitis C
virus. Hepatitis
C can progress to scarring (fibrosis) and advanced scarring (cirrhosis).
Cirrhosis can lead to
liver failure and other complications such as liver cancer. Current treatments
include use of
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a combination of pegylated interferon alpha and the antiviral drug ribavirin.
Success rates
can vary between 50-80% depending on the virus genotype.
Head trauma refers to an injury of the head that may or may not cause injury
to the
brain. Common causes of head trauma include traffic accidents, home and
occupational
accidents, falls, and assaults. Various types of problems may result from head
trauma,
including skull fracture, lacerations of the scalp, subdural hematoma
(bleeding below the
dura mater), epidural hematoma (bleeding between the dura mater and the
skull), cerebral
contusion (brain bruise), concussion (temporary loss of function due to
trauma), coma, or
even death. Treatment for head trauma will vary with the type of injury. If
the brain is
damaged, there is no quick means to fix it, and often the damage may be
irreversible by
available treatment means.
Lung disease is a broad term for diseases of the respiratory system, which
includes
the lung, pleural cavity, bronchial tubes, trachea, upper respiratory tract,
and nerves and
muscles for breathing. Examples of lung diseases include obstructive lung
diseases, in
which the bronchial tubes become narrowed; restrictive or fibrotic lung
diseases, in which
the lung loses compliance and causes incomplete lung expansion and increased
lung
stiffness; respiratory tract infections, which can be caused by the common
cold or
pneumonia; respiratory tumors, such as those caused by cancer; pleural cavity
diseases; and
pulmonary vascular diseases, which affect pulmonary circulation. Treatment for
lung
disease varies according to the type of disease, but can include medication
such as
corticosteroids and antibiotics, oxygen, mechanical ventilation, radiotherapy,
and surgery.
Depression is a mental disorder characterized by a low mood accompanied by low
self-esteem and loss of interest or pleasure in normally enjoyable activities.
Biologically,
depression is accompanied by altered activity in multiple parts of the brain,
including raphe
nuclei, which are a group of small nuclei in the upper brain stem that is a
source of
serotonin; suprachiasmatic nucleus, which controls biological rhythms such as
the
sleep/wake cycle; hypothalamic-pituitary-adrenal axis, which is a chain of
structures that are
activated during the body's response to various stressors; ventral tegmental
area, which is
considered to be responsible for the "reward" circuitry of the brain; nucleus
accumbens,
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which us thought to play a role in reward, laughter, pleasure, addiction, and
fear; and the
anterior cingulated cortex, which is activated by negative experiences.
Treatments for
depression include antidepressants that increase the amount of extracellular
serotonin in the
brain, exercise, and psychotherapy. However, the efficacy of these treatments
continues to
be questioned.
Non-obstructive azoospermia is a medical condition of a male not having any
measureable level of sperm in his semen due to a problem with spermatogenesis.
This is
often caused by hormonal imbalance and can be treated using medications which
restore the
imbalance.
Andropause is a menopause-like condition experienced in middle-aged men, which
involves a reduction in the production of the hormones testosterone and
dehydroepiandrosterone. Treatments include hormone replacement therapy and
exercise.
Scleroderma is a chronic autoimmune disease that affects connective tissue.
Hardening of the skin is the most visible manifestation of the disease,
although it can affect
connective tissue throughout the body. There is no known direct cure for
scleroderma.
Psoriasis is a chronic autoimmune disease that causes red, scaly patches to
appear on
the skin. The cause is psoriasis is linked to the excessive growth of skin
cells. One
hypothesis suggests that it is linked to T-cells which migrate to the dermis
and trigger the
release of cytokines that induce the rapid production of skin cells.
Treatments for psoriasis
include drugs that target the T-cells.
Retinitis pigmentosa is type of progressive retinal dystrophy in which the
photoreceptors or the retinal pigment epithelium is abnormal and leads to
visual loss.
Therapies for treating retinitis pigmentosa are limited
The conditions described herein may be treated with a particular type, or a
combination of types, of target cells. In preferred embodiments, the condition
described
herein may be treated by infusion of the cell types outlined in Table 2.
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Table 2. Treatment regimes of various conditions using reprograrmned, i.e.,
retrodifferentiated or transdifferentiated or redifferentiated, target cells
Condition Treatment Cell Type
(single or a combination thereof)
Aplastic Anemia haematopoietic cells
Beta-thalassemia haematopoietic cells
Diabetes mesenchymal stem cells, pluripotent stem cells
and/or islet cells
Motor Neuron Disease pluripotent stem cells, alveolar epithelium cells,
ectoderm cells and/or neurons
Parkinson's Disease pluripotent stem cells and/or neurons
Spinal Cord Injury pluripotent stem cells and/or neurons
Muscular Dystrophy pluripotent stem cells, mesenchymal stem cells
and/or skeletal muscle cells
Kidney Disease Kidney cells, mesenchymal stem cells and/or
pluripotent stem cells
Multiple Sclerosis pluripotent stem cells, mesenchymal stem cells
and/or neurons
Congestive Heart Failure cardiomyocytes, mesenchymal stein cells,
pluripotent stem cells and/or endothelium cells
Hepatitis C Virus haematopoietic cells, pluripotent stem cells,
mesenchymal stem cells, and/or hepatocytes of
the liver
Human Immunodeficiency Virus haematopoietic cells
Head Trauma pluripotent stem cells and/or neurons
Lung Disease pluripotent stem cells, mesenchymal stem cells,
alveolar epithelium cells and/or endothelium cells
Depression pluripotent stem cells and/or neurons
Non-obstructive Azoospermia or pluripotent stein cells, pluripotent germ cell,
Andropause and/or sperm
Menopause and infertility pluripotent stem cells, oocytes, pluripotent germ
cells
Rejuvenation pluripotent germ cells, pluripotent stem cells,
keratinocytes, dermal papilla cells, neurons,
sperm, osteocytes, chondrocytes, cardiomyocytes,
skeletal muscular cells, neurons, endothelium
cells, and/or melanocytes
Scleroderma Ulcers pluripotent stem cells, endothelium cells,
keratinocytes and/or mesenchymal stem cells
Psoriasis mesenchymal stem cells and or pluripotent stein
cells
Wrinkles mesenchymal stem cells, keratinocytes, and/or
pluripotent stein cells
Liver cirrhosis hepatocytes of the liver, mesenchymal stem cells,
endoderm cells, pluripotent stem cells
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Autoimmune Disease mesenchymal stem cells and/or pluripotent stem
cells
Alopecia dermal papilla cells and/or melanocytes
Retinitis pigmentosa or crystalline neurons and/or pluripotent stem cells
dystrophy/blindness
By way an example, a patient may be treated for a condition as described above
through the following steps:
1) Fistula-cannula is inserted in a patient's arm;
2) White blood cells are harvested through aphaeresis using an automated
system,
such as the COBE Spectra Device (Gambro PCT);
3) Autologous retrodifferentiated stem cells are produced from the patient's
white
blood cells;
4) The autologous retrodifferentiated stem cells are washed and then infused
intravenously into the patient;
5) Patient's progress is monitored, including taking blood tests and assessing
the
injured areas.
The invention will now be further described by way of the following non-
limiting
examples which further illustrate the invention, and are not intended, nor
should they be
interpreted to, limit the scope of the invention.
EXAMPLES
Example 1
Materials and Methods
This clinical study assessed the safety of infusing a single dose of
autologous 3 hr
reprogrammed cells following exposure to haematopoietic inductive culture
condition into
four patients with aplastic anemia.
This clinical study was approved by the ethical committee of the King Edward
Memorial (KEM) Hospital and was performed in joint collaboration with the
Institute of
Immunohematology (IIH). Patients were required to fulfill the criteria
outlined in Table 3.
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As a result, four patients with severe (3 males) and hypo-plastic (1 female)
anaemia were
enrolled into the study. These 4 patients were selected and monitored by
IIH/KEM staff.
The patients' clinical and treatment history are described in Table 4, while
their CD34+ cells
infusion dosage are shown in Table 5.
Table 3: Inclusion Criteria
Each 1- Absolute Neutrophil count < 0.5X109/L
criterion
is 2- Platelet count <20X109/L
Required
3- Anemia with corrected Reticulocyte <1%
Only One 4- Bone marrow cellularity <25%
of the
Criteria is 5- Bone marrow cellularity <50% with fewer than 30% hematopoietic
cells
Required
6- Subject evaluated within first 3 months of diagnosis
7- Subject did not receive prior immunosuppressive therapy
The patients were transfused with 2 units of irradiated packed red blood cells
and 4
units of platelets to maintain their hemoglobin level above 8 g/dl and
platelets counts above
50,000. Patient were apheresed by processing 2-3 times their total blood
volume using the
Cobe Spectra apheresis machine and the white blood cells separation kit (both
from Gambro
BCT). Apheresis involved jugular and anticubetal venous catheterization with
single lumen
catheter for venous access.
An aliquot of cells were collect aseptically for CD34 analysis following
collection of
150-200 ml of buffy coat. Thereafter, the buffy coat was subjected to
reprogramming under
hematopoietic inductive culture condition. Briefly, the reprogramming
procedure involved
the addition of 1000 g of purified CR3/43 (specially prepared by
DakoCytomation for
TriStem Corp.) diluted in 30m1 of Iscove modified media, aseptically into the
white blood
cell bag. The bag was then incubated in a sterile tissue culture incubator
maintained at 37 C
and 5 % CO2 for three hours. Following completion of the reprogramming
process, the
converted cells were analyzed for CD34+ cell content. Thereafter, cells were
washed twice
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with saline solution using the cobe cell processor 2991. Upon agitation and re-
suspension in
saline solution, the cell suspension was infused into the patient via the
jugular vein under
gravity using an infusion set. Vital signs including CBC counts of patients
were
continuously monitored before and following infusion of the autologous
reprogrammed
cells.
Table 4: Clinical and treatment history of aplastic anemia patients up to
Autologous human
reprogrammed stem cell (HRSC) infusion.
Patient Clinical History U to HRSC infusion Treatment tip to RSC infusion
Patient A = Diagnosed with SAA in 2002 = Received a trial of anabolic
25 yrs male = Presented with symptoms of weakness steroid (TabMenabol) for 8
Severe Aplastic and dysponoea months without any
Anaemia = Frequent episodes of rectal and gum significant improvement
bleeding and vomiting
= Receives 4 and 2 units of blood and
platelets respectively, every month
= Appear Jaundiced with severe eye
congestion
Patient B = Diagnosed with Hypoplastic anaemia in = Received Haematics since 3
26 yrs female January 2004 months prior infusion of
Hypoplastic = Presented with symptoms of RSC with no response
anemia polymenorrhagia of 6 months duration
= Only prior RSC infusion patients
received 4 and 6 units, respectively, of
packed red blood cells and platelets,
respectively
Patient C = Diagnosed with very severe aplastic = Received cyclosporine
19 yrs male anaemia in 2003 therapy for 6 months which
Very severe = Presented with anaemia, weakness, ended in march 2003 with no
aplastic anaemia dysponea on exertion effect
= Fever with chills lasting 10-15 days due
to severe neutropaenia
= Multiple episodes of infections,
vomiting and purpuric spots
= Gluteal abscess
= Fainted twice due to brain hemorrhage
= Receives 5 and 2 units of blood and
platelets respectively every 2 weeks
Patient D = Anaemia, weakness, dysponea = Received cyclosporine and
35 yrs male = Fever with chills due to severe anti-tuberculosis therapies.
Very severe neutropenia and multiple episodes of Did not respond to 6 months
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Aplastic infections and bleeding of immunosuppression.
anaemia = Diagnosed 3 years ago
= Receives 4 and 2 units of blood and
platelets, respectively, every month
All clinical monitoring was carried out by the IIHIKEM staff. Transfusion
requirement was determined prior and post infusion from transfusion records
obtained
following receiving any unit of blood product. The transfusion units are only
utilized
following obtaining consent from patients and immediate relatives as witness.
Prior
transfusing the patient all blood products were irradiated at the Tata
Memorial Hospital.
Patients were also asked questions regarding their well-being prior and post
infusion of the
reprogrammed cells. All patients carried a copy or original records of their
conditions and
all laboratory and clinical follow up. The duration of monitoring of these
patients was
originally set for 2 years, but was extended. For the first month post
infusion, patients were
kept in hospital in sterilized positive pressure rooms.
Table 5: Patient, date of infusion, weight and height, mononuclear cells
collect and CD34+
cell infused.
Patient 11) and Weight Height Mononuclear CD34+
date of infusion cell collected Received/
Patient A 48 152 1 x 109 11.7x 106
7/6/2004
Patient B 38 153 3.6X109 20x106
7/14/2004
Patient C 52 166 3.9X 109 25x106
7/27/2004
Patient D 52.5 160 4.3x10 23x106
8/17/2004
One million cells were stained according to the manufacturer instructions with
the
following panels of monoclonal antibodies (all from DakoCytomation):
Panel I consisted of Isotype negative control IgGI-FITC, IgGl-PE-Cy5 and IgGI-
RPE conjugates
Panel 2 consisted of anti-human CD45-FITC and CD34-RPE-Cy5
Panel 3 consisted of anti-human CD38-FITC and CD34-RPE-Cy5
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Panel 4 consisted of CD61-FITC and CD34-RPE-Cy5
Panel 5 consisted of CD33/13 RPE and CD7-FITC
Panel 6 consisted of CD45 and Glycophorin-A-RPE
Panel? consisted of CD3-FITC and CD19-RPE
Cell analysis was performed with a FACSCalibur system (BD bioscience) using
the
BD cell Quest software.
For clonal assay, bone marrow mononuclear cells (MNC) of patient prior and
post
infusion of the reprogrammed cells were seeded into methocult GFH4434
supplemented
with recombinant growth factors according to the manufacturer's instructions
(Stem Cell
Technologies). Differentiation into haematopoietic cell colonies was assessed
and scored
with time using phase contrast inverted microscopy.
Patient CBC, liver enzymes and haemoglobin variants were continuously
monitored
before and post procedure. Following release from hospital patients, CBC,
liver enzymes,
hemoglobin variants, and peripheral blood karyotyping and G banding were
monitored by an
independent laboratory for reconfirmation purposes. These tests were performed
frequently
following infusion of the autologous reprogrammed cells.
Peripheral blood samples and bone marrow cells were analyzed before and
following
infusion of the autologous reprogrammed cells. This test was repeated in six-
month
intervals for the first year and on a yearly basis following 2 years post-
initiation of
autologous reprogrammed stern cell therapy. In addition, reprogrammed cells
were analyzed
prior infusion to look into the stability of the cells, which was also
performed following the
3hr conversion step, as well as post-establishment of a maximum 1 month long
term culture
of the converted cells. Karyotyping and G banding were monitored by a third
independent
laboratory.
Bone marrow smears and trephine section was performed before and post infusion
of
the autologous reprogrammed cells. This test was performed 14-20 days post
infusion of the
autologous reprogrammed cells and thereafter on a yearly basis.
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All smear and trephine sections were scanned using a microscope hooked to a
camcorder before and following infusion of the reprogrammed stem cells to
assess and keep
record of engraftment.
Results
All patients tolerated the aphaeresis and the single reprogrammed stem cells
infusion
procedure with no adverse event. Patient A and Patient D became transfusion-
independent
post single infusion of reprogrammed haematopoietic stem cells (RHSC) (see
Tables 6 and
7). Platelets, neutrophil and red blood cell engraftment ensued 3 and 6 days
post infusion in
Patient A and Patient D, respectively. Fetal hemoglobin switching was noted in
Patient A
and Patient D (see Tables 4 and 5) but not in Patient B and Patient C (data
not shown). Prior
to infusion, liver enzymes were elevated in Patient A and Patient D despite
being negative
for HCV (as measured by ELISA). Liver enzymes started to normalize post
infusion of
RHSC and reached normal levels 4 years post infusion of RHSC. Patient B and
Patient C
died 2 years and six months post infusion, respectively. Fetal Hb switching
was noted in
patients 001 and 004 (see Tables 6 and 7). These two patients exhibited long
term
engraftment post single infusion of the autologous HRSC.
Table 6. Patient A's complete blood counts, hemoglobin variant and liver
enzymes of a
severe aplastic anemia patient before and after in fusion of autologous HRSC.
Nadir prior 07/19/2004 05/26/2005 03/07/2006 01/05/2008
Blood test to infusion Post- infusion Post- infusion Post- infusion Post-
infusion
WBC [103/ L 1.3 3.4 3.1 3.7 5.4
HB [g/dL] 2.6 7.1 9 12 14
RBC 1106/ L] 0.9 2 2.3 2.9 3.86
RETIC % 1 4 1.7
MCV [fL] 101.5 101.5 121.74 100 101.4
MCH P 33.6 35.5 39.13 30.8 36.3
MCHC [g/dL] 33.8 35 32.14 30.8 35.8
PLATELETS
[103 /L] 18 26 37 58 189
NEUTROPHILS
[10 / L] 0.32 1.000 1.054 1.184 1.994
ESO [103/ L] 0.62 0.74 0.65
BASO [103/ L 0 0 0.054
LYMPH [103/ Ll 1.500 1.922 2.220 2.214
MONO 103/ L 0.500 0.062 0.222 0.324
HB A [g/dL] 5.1 6.7 9.2 12
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HBA2 [ /dl] 0.13 0.14 0.24 0.39
HBF [g/dl] ND 1.2 2.4 2.6 1.82
SGOT [U/L] 150 127 74 32 30
SGPT [U/L] 145 181 49 37 44
WBC = white blood cell; HB = hemoglobin; RBC = red blood cell; RETIC =
reticulocyte;
MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean
corpuscular hemoglobin concentration; ESO = eosinophils; BASO = basophils;
LYMPH =
lymphocytes; MONO = monocytes; HB A = hemoglobin A; HBA2 = hemoglobin A2; HBF
= fetal hemoglobin; SGOT = serum glutamic oxaloacetic transaminase; SGPT =
serum
glutamic pyruvic transaminase
Table 7. Patient D's complete blood counts, hemoglobin variant and liver
enzymes of a
severe aplastic anemia patient before and after in fusion of autologous HRSC.
Nadir prior 09/28/2004 05/30/2005 03/07/2006 01/05/2008
Blood Test to infusion post infusion Post infusion Post infusion Post infusion
WBC [103/ L] 1.7 3 2.2 2.7 4
HB 3 11.1 7.7 11.5 13
RBC 3.6 2.4 3.2 3.45
RETIC [%] 2.4 3.4 1.7
MCV 90.3 108.33 113 110.3
MCH 30.9 32 35.9 37.8
MCHC 34.2 29.62 31.9 34.2
PLATELETS
[10 /iL] 5 30 20 25 68
NEUTROPHILS
[103/ L] 0.1 0.72 0.50 1.0 1.24
ESO [103/AL] 0.90 0.22 0.54 0.64
BASO [103/pL] 0 0 0 0
LYMPH [103/gL] 2.01 1.67 1.59 1.88
MONO [103/ L] 0.18 0.44 0.54 0.12
HB A 10.6 7.13 10.33 12.1
HBA2 0.33 0.19 0.28 0.36
HBF 0.11 0.2 0.38 0.9 0.42
SGOT 160 46 46 69 34
SGPT 150 57 32 60 46
WBC = white blood cell; HB = hemoglobin; RBC = red blood cell; RETIC =
reticulocyte;
MCV = mean corpuscular volume; MCH = mean corpuscular hemoglobin; MCHC = mean
corpuscular hemoglobin concentration; ESO = eosinophils; BASO = basophils;
LYMPH =
lymphocytes; MONO = monocytes; HB A = hemoglobin A; HBA2 = hemoglobin A2; HBF
= fetal hemoglobin; SGOT = serum glutamic oxaloacetic transaminase; SGPT =
serum
glutamic pyruvic transaminase
Flow cytometry of aphaeresed mononuclear cells before and 3 hrs post induction
of
haematopoietic reprogramming are shown in FIG. 1. The number of CD34 positive
cell
generated post haematopoietic reprogramming is listed in Table 4. A
representative flow
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cytometry of apheresed mononuclear before and post haematopoietic
reprogramming show
significant increase in the number of CD34 positive cells with and without
expression of
CD45, CD38 and CD7 (FIG. 1). On infusion, the CD34 cells circulated peripheral
blood for
3-6 days and thereafter differentiated at a sustainable level into myelocyte
as depicted by
significant increase in cells expressing CD33&13 with and without CD7 having
high
forward and side scatter (see FIG. 2). This pattern of reprogramming was
observed in all
patients.
Hardly any colonies formed from patients bone marrow aspirate before infusion
of
the autologous HRSC following seeding into methylcellulose cell culture (see
FIG. 3).
Clonal assay only in Patient B was suffering from hypoplastic anaemia showed
depressed
haempoiesis. However, 14 days to 20 days post infusion all bone marrow
aspirates obtained
from patients gave rise to normal range of a variety of haematopoietic
colonies with mild
elevation in the number of Burst forming unit -erythroid (BFU-erythroid). Bone
marrow
smears and trephine sections (see FIG. 3) 14-20 days post infusion of
autologous of HRSC
showed significant increase in the number of myelocytes at various stages of
differentiation
with mature and immature megakaryocytes in all patients when compared to
baseline.
Erythroid hyperplasia at various stages of differentiation was also noted in
all samples.
No changes in Karyotyping and G banding pattern in peripheral blood or bone
marrow samples obtained before and after infusion (in Patient A and Patient D
for up to
more than 4 years) of autologous HRSC in all patients (see FIG. 4).
Post infusion of the autologous HRSC into aplastic anaemia patients primitive
(CD38
negative) and committed (CD38 Positive) CD34 cells circulated the peripheral
circulation
for three days prior reprogramming into myelocytes (FIG 2). Myeloid
engraftment ensued 3
days post infusion of the HRSC in Patient A, Patient B, and Patient D. On the
other hand
myeloid engraftment ensued at day 20 for patient 003 when analyzed by flow
cytometry.
The single infusion of HRSC, without the use of any pre-conditioning regimen
lead to long
term engraftment of 2 out of 4 patients with aplastic anaemia. Patient A and
Patient B
showed long term engraftment without any transfusion post infusion of the
HRSC.
Engraftment of neutrophils, red blood cells and platelets in such patient was
accompanied by
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switching or increase in Hb F haemoglobin level (Tables 6 and 7). This was not
noted in the
other 2 patients that died. Hb F switching in these two patients confirms the
reprogramming
capabilities of the infused HRSC towards juvenile Hb phenotype and hence
engraftment and
reconstitution as observed with cord blood stem cells transplant (Elhasid et
al., Leukemia
2000, 14: 931-934; Locatelli et al., Bone Marrow Transplant 1996, 18: 1095-
101).
The long term engraftment with preservation of chromosome number and banding
reflect clearly the safety of infusing the HRSC in a haematological condition
where clonal
evolution is not a rare event with conventional therapies
Importantly, the autologous HRSC were capable of long term engraftment and
survival rate in a subset of severe aplastic anaemia patients without use of
any
immunosuppression regiment, just like those seen with syngenic stern cells.
In summary, fourteen days after infusion, the analysis of the bone marrow of
the
infused patients showed an increase in bone marrow cellularity, and myeloid,
erythroid and
megakaryocytic lineages at various stages of differentiation with a drop in
fat cells and
stromal cells, which are the predominant occupant of severe aplastic anaemia
bone marrow.
There was a significant increase in red blood cell mass in the bone marrow
with a
corresponding increase in haemoglobin level as well as reticulocytes count.
There was also
a steady increase in foetal haemoglobin - an important component in
ameliorating sickle cell
anaemia and beta thalassemia - and foetal haemoglobin-expressing red blood
cells following
infusion. Furthermore, there was significant improvement in red blood cell
indices as
determined by red blood cell size, haemoglobin content and concentration. The
reprogrammed cells have exhibited a normal karyotype and genetic stability
after infusion.
Finally, engraftment and long term repopulation was observed in 3 more patient
suffering
from aplastic anemia.
Example 2
Materials and Methods
Autologous reprogrammed haematopoietic cells (target cells) were tested in 21
patients with beta thalassemia. Nineteen patients had beta-thalassemia major
and 2 had
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thalassemia intermedia. One of the beta thalassemia intermedia patient was
thalassemia/Hb
E variant (common in patient of far eastern and Indian origin) and the other
had
Thalassemia/Sickle cell anaemia.
Patients were apheresed by processing 2-3 times their total blood volume. The
autologous reprogrammed cells were generated through reprogramming white blood
cells
until the target cells were obtained, as indicated by their distinguishing
characteristics as
described above. The patients were administered the autologous reprogrammed
cells via
intravenous infusion into the jugular or veins in the arm or thigh.
Results
There was no toxic or adverse side effects were observed following infusion of
the
reprogrammed cells in patients with beta thalassemia as measured by vital sign
monitoring,
echocardiogram, bone densities, liver and kidney enzymes including Karyotyping
and G-
banding when compared to baseline. There was a statistically significant mean
reduction
(50%) in blood transfusion requirement in beta thalassemia major patients now
nearly 9
months following infusion of the reprogrammed cells when compared to base
line. Two
thalassemic patient who are beta thalassemia intermediate (one was
thalassemia/HB E and
the other Thalassaemia /Sickle Cell Anaemia) were transfusion-independent now
nearly 9
months following infusion of the reprogrammed cells.
The mean weight and height following infusion of the reprogrammed cells were
significantly greater when compared to base line, and the organ size in
thalassemic patients
with enlarged spleen and/or liver was normalized. The absolute mean fetal
haemoglobin
concentration significantly increased in patients with thalassemia major and
intermedia
following infusion of the reprogrammed cells when compared to baseline (FIG.
5). Also, the
mean red blood cell indices as reflected by improvement in red blood cell size
haemoglobin
content (FIG 6) and concentration (FIG. 7) was also significantly improved as
compared to
baseline.
Finally, the mean serum ferritin (a biomarker for iron overload) significantly
decreased in thalassemic patients following infusion of the reprogrammed cells
(FIG. 8).
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Iron overload is the major cause of mortality and morbidity in patients with
thalassemia;
sickle cell anaemia and any transfusional iron overload induced disorder.
Example 3
Materials and Methods
Two patients with diabetes were apheresed by processing 2-3 times their total
blood
volume. Autologous reprogrammed mesenchymal stem cells, pluripotent stem
cells, and
islet cells (target cells) were obtained through reprogramming apheresed white
blood cells
until the target cells developed, as indicated by their distinguishing
characteristics as
described above.. The patients were administered the autologous reprogrammed
cells via
intravenous infusion into the jugular or veins in the arm or thigh.
Results
Following infusion of the autologous reprogrammed cells, the patients were
synthesizing normal level of insulin, as measured by fasting and 90 minutes
food intake
stimulated c-peptide. This normal level of c-peptide is being maintained up to
3 months
following infusion of the reprogrammed cells (FIG. 9). In addition, Hb A1C
levels, which
indicate glycemic control, had normalized following infusion of the
reprogrammed cells
(FIG. 10). For example patients with Hb Al C above 10% prior infusion have now
Hb A1C
of 5.8% following receiving the reprogrammed cells. Furthermore blood glucose
levels of
these patients appear to reach normal levels post infusion when compared to
baseline. In
addition a drastic reduction in insulin intake/injection by the diabetic
patient was noted
following infusion of the reprogrammed cell.
Example 4
Materials and Methods
Four patients with amyotrophic lateral sclerosis (ALS) received the autologous
reprogrammed cells. The diagnosis of this disease does not involve a specific
biomarker.
This disease is diagnosed by clinical exclusion of other similar disorders.
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Patients were apheresed by processing 2-3 times their total blood volume.
Autologous reprogrammed pluripotent stem cells, alveolar epithelium cells, and
neurons
(target cells) were obtained through reprogramming apheresed white blood cells
until the
target cells developed, as indicated by their distinguishing characteristics
as described
above. The patients were administered the autologous reprogrammed cells via
intravenous
infusion into the jugular or veins in the arm or thigh.
Results
In patients suffering from ALS and treated with the autologous reprogrammed
cells,
there was a significant improvement in Pulmonary Function test (PFT).
Impairment in this
lung function test is one of the causes that lead to early mortality. Most
patients experienced
less stiffness in limbs and neck, and some reported improvement in their
speech. Others
showed improvement in their walking ability as well as lifting of head.
Example 5
Materials and Methods
Four patients with Parkinson's disease were apheresed by processing 2-3 times
their
total blood volume. Autologous reprogrammed pluripotent stem cells and neurons
(target
cells) were obtained through reprogramming apheresed white blood cells until
the target
cells developed, as indicated by their distinguishing characteristics as
described above. .
The patients were administered the autologous reprogrammed cells via
intravenous infusion
into the jugular or veins in the arm or thigh.
Results
Patients in which the shiverer effect of the disease is pronounced experienced
significant reduction in shaking and conventional medication intake used to
manage this
disorder. The first patient was on 4 tablets of Sinemet (dopamine regulator)
daily and 4-
months post-transfusion is only taking 1 tablet a day.
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Example 6
Materials and Methods
Two patients with spinal cord injury were apheresed by processing 2-3 times
their
total blood volume. Autologous retrodifferentiated pluripotent stem cells and
neurons
(target cells) were obtained through reprogramming apheresed white blood cells
until the
target cells developed, as indicated by their distinguishing characteristics
as described
above. The patients were administered the autologous reprogrammed cells via
intravenous
infusion into the jugular or veins in the arm or thigh.
Results
One patient was lost to follow up. The other patient was a quadriplegic with
C5-C6
spinal cord injury. This patient was unable to sit up or rotate his torso in
bed. Following
treatment, he was able to sit upright for very long periods of time and able
to turn his body
in bed. He was also capable of standing alone following propping his body
against a wall.
Further, he was able to wiggle his toe and is reporting sensation in his
bladder.
An MRI analysis before and following infusion of the reprogrammed cells showed
slight reduction in lesion size following infusion of reprogrammed cells. He
began actively
undergoing physiotherapy and generally felt much better than before.
Example 7
Materials and Methods
Two patients with Muscular Dystrophy (MD) were treated with autologous
reprogrammed pluripotent stem cells, mesenchymal stem cells, and skeletal
muscle cells
(target cells) were obtained through reprogramming apheresed white blood cells
until the
target cells developed, as indicated by their distinguishing characteristics
as described
above. The first patient was inflicted with limb-girdle MD, which refers to a
class of MD
wherein the muscles that are most severely affected generally those of the
hips and
shoulders, and the second patient was inflicted with nemelin MD.
Patients were apheresed by processing 2-3 times their total blood volume. The
autologous reprogrammed cells were generated through reprogramming in
accordance to the
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invention. The patients were administered the autologous reprogrammed cells
via
intravenous infusion into the jugular or veins in the arm or thigh.
Results
The muscle atrophy associated with MD can be measured by monitoring the level
of
the muscle enzyme creatine phosphokinase (CPK). This enzyme decreased in
response to
infusion of the retrodifferentiated stem cells (FIG. 11). Lactate
dehydrogenase, an enzyme
which is elevated during tissue breakdown, also decreased (FIG. 11). Patients
also
experienced a decrease in liver enzymes alanine aminotransferase (ALT) and
aspartate
aminotransferase (AST), which are both associated with inflammation and injury
to liver
cells, as well as skeletal muscle.
Moreover, patients showed an improvement in patient mobility as determined by
cam recording of patients before and following infusion of the reprogrammed
cells, and in
pulmonary function test following infusion of the reprogrammed cells when
compared to
baseline
Example 8
Materials and Methods
Patients with kidney disease were treated with autologous reprogrammed
pluripotent
stem cells, mesenchymal stem cells, and kidney cells (target cells) were
obtained through
reprogramming apheresed white blood cells until the target cells developed, as
indicated by
their distinguishing characteristics as described above. The patients were
apheresed by
processing 2-3 times their total blood volume. The autologous reprogrammed
cells were
generated through reprogramming in accordance to the invention. The patients
were
administered the autologous reprogrammed cells via intravenous infusion into
the jugular or
veins in the arm or thigh.
Results
Patients receiving an infusion of autologous reprogrammed cells experienced an
improvement in various fluid marker levels which were indicative of healthier
kidney
function such as urine output, serum creatinine and BUN or UREA. For example,
a 75-year
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old diabetic male patient showed improved kidney function 24 months after
treatment with
the autologous reprogrammed cells (see Table 8). The patient showed increased
levels in
hemoglobin, which is the protein molecule in red blood cells that carries
oxygen, and
insulin-like growth factor-1 (IGF-1), a growth factor. The patient also showed
decreases in
urea, which is an organic compound, creatinine, which is a breakdown product
of creatine
phosphate in muscle, uric acid, which is an organic compound excreted in
urine, and
phosphorus, which is a mineral found in bone; high amount of these markers are
indicative
of poor kidney function. Further, the patient showed an decrease in
glycosylated
hemoglobin (HbA1C), which is a form of hemoglobin and is commonly used as an
indicator
of plasma glucose concentration of people suffering from diabetes.
Similarly, a 51-year old diabetic male exhibited similar improvement 12 months
after
treatment (see Table 8). This patient showed increased levels of hemoglobin
and decreased
levels of creatinine, HbA1C, and blood urea nitrogen (BUN), which is a
measurement of the
amount of nitrogen in the blood in the form of urea.
Table 8: Kidney function marker levels in two diabetic male patients.
75-Year Old Diabetic Male 51-Year Old Diabetic Male
Baseline 24-coos post- Baseline 12-mos post-
treatment treatment
Hemoglobin [ /dl] 8 12.9 12 14.5
Urea [g/dl] 190 38 n/a n/a
Creatinine [m /dl] 12 1.9 15 4.5
Uric acid [m /dl] 8.5 4 n/a n/a
Phosphorus [mg/dl] 6.1 3.8 n/a n/a
HbA1C [%] 14.4 6.1 12 6
IGF-1 [n /dl] 45 112 n/a n/a
BUN [m /dl] n/a n/a 79 19
HbA1C = glycosylated hemoglobin; IGF-1 = Insulin-like Growth Factor 1; BUN =
blood
urea nitrogen
Analysis of 12 patients having Type II diabetes and treated with autologous
reprogrammed cells revealed decreased levels of microalbumin (FIG. 13), which
is
associated with leakage of albumin into the urine and is an indicator of
kidney disease, as
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well as vascular endothelial dysfunction and cardiovascular disease. The 12
patients also
experienced decreased levels of HbA 1 C (FIG. 14).
In addition, a 45-year old female suffering from autoimmune glomerulonephritis
exhibited a improvement in kidney function within 18-months after treatment
with
autologous reprogrammed cells (see Table 9).
Table 9: Kidney function marker levels in 45-year old female patient suffering
from
autoimmune glomerulonephritis.
Baseline 18-mos post-treatment
Hemoglobin [g/dl] 9 11.5
Creatinine [m /dl] 9.3 2.1
BUN [mg/dl] 89 15
BUN = blood urea nitrogen
Furthermore, a 59-year old female patient suffering from end stage renal
disease
showed improvement in creatinine level, urea, hemoglobin level, and
parathyroid hormone
(PTH) upon treatment with reprogrammed cells (see Table 10). Kidney function
marker
levels in 45-year old female patient suffering from autoimmune. The patient
also decreased
in the number of hemodialysis sessions that she attended per month, from 12
sessions to
about 8 sessions.
Table 10: Kidney function marker levels in 59-year old female patient
suffering from end
stage renal disease before and after therapy with reprogrammed cells.
Kidney Function Pre-flierapy Post therapy
Marker 03/02/2009 04/29/2009 05/23/2009 08/05/2009 08/11/2009
Hemoglobin [g/dl] 9.8 9.2 11.5
TLC/WBC (per l) 3800 6800 5100
PLT (per l) 361000 273000 348000
HbA1C [%] 5.6
AST [U/1] 25 16 14
ALT [U/1] 49 11 12
Urea [mg/dl] 90 70 22.13
Creatinine [mg/dl] 14.5 11.2 11.86 8.86 9.58
S. Albumin [g/dl] 3 3.4 3.3 3
PT [seconds] 31.2
INR 2.71
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Calcium [mg/dl] 8.4 9.5 9.4 8.7 9.5
Phosphorus [mg/dl] 8 9.2 5.9
Potassium 5.9 5.2 6.9 4.1
mmol/dl
Sodium [mmol/dl] 133 136
Uric acid [mg/dl] 7.9 6.1 7.5 4.8
PTH [pg/ml] 195.6 108.2
TLC =total leukocyte count; WBC = white blood cells; PLT = platelet; HbA1C =
glycosylated hemoglobin; AST = aspartate aminotransferase; ALT = alanine
aminotransferase; PT = pro-thrombin time; INR = international normalized
ratio(for blood
coagulation); PTH = parathyroid hormone
A 46-year old patient suffering from chronic renal failure due to diabetes
also
showed improvement in creatinine level, urea, and hemoglobin level upon
treatment with
reprogrammed cells (see Table 11). The patient decreased in the number of
hemodialysis
sessions that he attended per month, from 12 sessions to about 5 sessions, and
decreased in
treatment with epoetin alfa (EPREX ) delivered subcutaneously, from 4000 units
twice a
week to 4000 units once every two weeks.
Table 11: Kidney function marker levels in 46-year old patient suffering from
chronic renal
failure before and after therapy with reprogrammed cells.
Kidney Function Marker Pre-therapy Post-therapy
09/21/2008 02/24/2009 04/20/2009
Hemoglobin /dl 6.5 10.3 9.4
TLC/WBC (per 1 5500 8200 10100
PLT (per 1 159000 246000 300000
FBS [mg/dl] 133 120 121
2 hrs PP [mg/dl] 170 172 169
AST [U/L] 9 34 32
ALT U/L 5 22 15
Urea [mg/dl] 109 51 65
Creatinine m /dl 7.5 4.9 4
S. Albumin /dl 3.5 3.7 4
PT [seconds] 12.7 12 11
INR 1 1 1
Calcium [mg/dl] 10 9.9 11
Phosphorus m /dl 6 5.3 5
Potassium mmol/L 6.2 5.1 4.7
Magnesium m /dl 2.3 2.4 2.8
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TLC = total leukocyte count; WBC = white blood cells; PP = post prandial; PLT
= platelet;
FBS = fasting blood sugar; AST = aspartate aminotransferase; ALT = alanine
aminotransferase; PT = pro-thrombin time; INR = international normalized ratio
(for blood
coagulation)
Example 9
Materials and Methods
Patients with multiple sclerosis were treated with autologous reprogrammed
pluripotent stem cells, mesenchymal stem cells, and neurons (target cells).
The target cells
were through reprogramming apheresed white blood cells until the target cells
developed, as
indicated by their distinguishing characteristics as described above. The
patients were
apheresed by processing 2-3 times their total blood volume. The autologous
reprogrammed
cells were generated through reprogramming in accordance to the invention. The
patients
were administered the autologous reprogrammed cells via intravenous infusion
into the
jugular or veins in the arm or thigh.
Results
Patients treated with autologous reprogrammed cells showed reduction in
lesions in
the brain and in the spinal cord. A reduction in lesions can occur within
three months of
receiving the treatment (FIGS. 15a-b). A reduction in damage to brain tissue
occurred
within six months (FIGS. 16a-b).
Patients treated with autologous reprogrammed cells also showed removal of
spinal
cord lesions (FIGS. 17a-b). Further, these patients exhibited improvement in
the Kurtzke
Expanded Disability Status Scale (EDSS) score, showed remission, and have not
participated in conventional therapy up to four years since receiving a single
infusion.
Example 10
Materials and Methods
A female patient with HIV was treated with autologous reprogrammed
haematopoietic stem cells. The patient was apheresed by processing 2-3 times
her total
blood volume. The target cells were obtained through reprogramming apheresed
white
blood cells until the target cells developed, as indicated by their
distinguishing
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characteristics as described above. The patient was administered the
autologous
reprogrammed cells via intravenous infusion into the jugular or veins in the
arm or thigh.
Results
Before treatment, the screening test for HIV-1 and HIV -2 antibodies showed a
test
value of 3.68. A value of 1.0 or greater is considered positive.
Two months after treatment with autologous reprogrammed cells, the test value
for
the HIV 1 and HIV-2 antibody screening was 0.46, which indicated that the
patient did not
show a positive result for HIV 1 and HIV 2. At six months after treatment, the
test value for
the HIV-1 and HIV-2 screening was 0.48, further demonstrating that the patient
was not
showing a positive result for HIV.
Example 11
The effects of treating with autologous reprogrammed cells are demonstrated in
patients suffering from other conditions and diseases. The target cells listed
herein were
obtained through reprogramming apheresed white blood cells until the target
cells
developed, as indicated by their distinguishing characteristics as described
above
Congestive Heart Failure
A 61-year old male patient suffering from congestive heart failure was infused
with
autologous reprogrammed cardiomyocytes, pluripotent stem cells, mesenchymal
stein cells,
and endothelium cells. The treatment led to a improvement in ejection fraction
(EF); brain
natriuretic peptide precursor levels (Pro BNP), which is a predictor
associated with coronary
artery disease; blood glucose fasting levels; and HbA1C levels (see Table 12).
Furthermore,
the heart, which was previously dilated, returned to normal size as evidenced
in Table 12 by
decrease in end-diastolic left ventricular internal dimension (LVID/D), end-
diastolic left
ventricular internal dimension (LVID/S), and end-diastolic intraventricular
septal thickness
(IVSD) as measured by echocardiogram.
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Table 12. Coronary artery disease markers in a 61-year old patient suffering
from
congestive heart failure before and after therapy with reprogrammed cells.
Coronary 1-mo before 1-mo after 2-coos after 4-coos after 6-mos afterl0-mos
after
Artery Disease treatment treatment treatment treatment treatment treatment
Markers
LVID/D [cm] 6.5 6.3 5.9 6.9 6.5 5.7
LVID/S [cm] 6.1 5.6 5.4 5.9 5.7 4.9
F 15 18 22 29 33 45
IVSD [cm] 1.3 1.3 1.2 1.2 0.6 1.0
VPWD [cm] 0.7 0.6 0.6 0.8 0.6 1.0
V Mass 283 251 209.5 167 151.5
Index [gm/ml 141.5 133.5 111.4 88.8 81
Pro BNP 2969 800 600 600 600 497
In/ml]
Blood Glucose 244 118 145 87 95 80
Fasting [mg/dl]
bA1C [%] 9.2 nd nd 7.2 6.5 6
LVID/D = end-diastolic left ventricular internal dimension; LVID/S = end
systolic left
ventricular internal dimension; EF = ejection fraction; IVSD = end-diastolic
intraventricular
septal thickness; LVPWD = end-diastolic left ventricular posterior wall
thickness; Pro-BNP
= precursor of brain natriuretic peptide; HbA1C = glycosylated hemoglobin
Hepatitis C
Thirteen patients infected with hepatitis C virus (HCV) and having beta-
thalassemia
were infused with autologous reprogrammed haematopoietic cells, pluripotent
stem cells,
mesenchymal stem cells, and hepatocytes. The effects of the treatment revealed
a lower or
clearance of HCV load (see Table 13), as well as improved blood markers (see
Table 14)
such as liver enzymes, bilirubin, albumin, pro-thrombin time, and the
international
normalized ratio (for blood coagulation). In particular, patients experienced
normalization
of liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase
(AST),
which are both associated with inflammation and injury to liver cells (FIGS.
18a-b).
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Table 13. Viral load in twelve patients infected with hepatitis c virus and
treated with
autologous reprogrammed stem cells
Baseline 3 months post-infusion
Patient ID viral load x1000 viral load x1000
1 0___ 0
2 164 82
3 3 0
4 123 16
16 0
6 2 0
6 0 0
8 7.7 million 7.7 million
9 152 106
63 27
11 387 97
12 0 0
13 17 0
Table 14. Blood markers in patients infected with hepatitis C virus and
suffering from liver
5 cirrhosis at baseline and after therapy with reprogrammed cells.
Blood Markers Baseline 3 months post-infusion
Hemoglobin [g/dl] 9.8 12.9
Platelet (per l) 65000 120000
C (per l) 4300 7200
Blood sugar (Fasting) [mg/dl] 210 110
Blood 2 hrs (PP) [mg/dl] 190 134
T [U/L] 50 24
ST [U/L] 32 31
Bilirubin (Total) [mg/dl] 6.9 3.2
Bilirubin (Direct) [Mg/dm] 3.1 1.8
Serum albumin [g/1] 1.9 3.1
Urea [mg/dl] 60 36
Creatinine [mg/dl] 1.6 1.1
NR 1.8 1.0
PT [seconds] 18 12.8
WBC = white blood cells; PP = post prandial; ALT = alanine aminotransferase;
AST =
aspartate aminotransferase; INR = international normalized ratio (for blood
coagulation); PT
= prothrombin time
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For instance, a 44-year old male patient suffering from liver cirrhosis due to
hepatitis
C virus infection also showed improvement in liver enzymes alanine
aminotransferase and
aspartate aminotransferase, the international normalized ratio (for blood
coagulation),
bilirubin, and albumin, as well as random blood sugar (see Table 15). In fact,
this patient
was on albumin treatment before the therapy with reprogrammed cells, but did
not receive
albumin after the therapy.
Table 15: Blood markers in a 44-year old male patient suffering from liver
cirrhosis due to
hepatitis C virus infection at baseline and after therapy with reprogrammed
cells.
Blood Marker Baseline Post-therapy
03/30/2009 04/04/2009 04/23/2009 05/06/2009 06/22/2009
Hemoglobin 10.9 10.4 11.6 13.4 10.2
[ /dl]
TLC (per 1) 2500 2700 6100 4700 2800
PLT (per 1) 20000 17000 43000 50000 27000
AST [U/L] 212 170 131 141 120
ALT [U/L] 62 59 48 51 53
Alkaline
Phosphatase 196 147 58
[U/L]
GGT [U/L] 49 70 63 35
Bilirubin T 12.7 10.9 11.2 8.1 8.8
[mgt]
Bilirubin D 6.8 5.4 4.71 4.9 4
[m /DI]
Urea [m /dl] 13 18 20 16 25
Creatinine 0.7 0.7 0.8 0.7 0.7
[mg/dl]
S. Albumin [g/L] 2.8 2.5 2.9 2.4 1.9
RBS [m /dl] 212 154 158 188 174
INR 1.9 1.8 1.72 1.69 1.72
Potassium 3.2 3.8 4.4
[mmol/L]
Sodium 134 141 137
[mmol/L]
Uric acid 3.5 2.4 3
[M /dl]
Alpha Feto Normal
Protein
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C o lobulins Negative
TLC = total leukocyte count; PLT = platelet; AST = aspartate aminotransferase;
ALT =
alanine aminotransferase; GGT = gamma-glutamyl transferase; RBS = random blood
sugar;
INR = international normalized ratio (for blood coagulation)
Head Trauma
A patient suffering head trauma from a motor accident was treated with an
infusion
of autologous reprogrammed pluripotent stem cells and neurons. The treatment
led to
reparation of damaged brain tissue (FIGS. 19a-b), improvement in ejection
fraction (EF);
brain natriuretic peptide precursor levels (Pro BNP), which is a predictor
associated with
coronary artery disease; blood glucose fasting levels; and HbA1C levels (see
Table 10).
Lung Disease
A patient suffering from restrictive lung disease associated with a motor
neuron
disease was treated with an infusion of autologous reprogrammed pluripotent
stem cells,
mesenchymal stem cells, alveolar epithelium cells, and endothelium cells. At
six months
following treatment, forced vital capacity (FVC), which is the volume of air
that can
forcibly be blown out after full inspiration, increased from 50 % to 71 %,
while forced
expiratory volume in 1 second (FEVI), which is the volume of air that can
forcibly be blown
out in one second, increased from 64% to 68%. Further, the ratio of FEV1 to
FVC, which is
approximately 75-80 % in healthy adults, decreased from 100 % to 82 %. An x-
ray scan of
the lungs shows less opacity of the lung cavity after treatment (FIG. 20).
Menopause
A 51-year old patient in menopause was administered an infusion of autologous
reprogrammed pluripotent stem cells, pluripotent germ cells, and oocytes.
Following the
treatment, the patient experienced an increase in various hormone and protein
levels,
including insulin-like growth factor (IGF-1), esterdiaol, and low-density
lipoprotein (LDL)
(see Table 16).
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Table 16. Effects of administering patient in menopause with autologous
reprogrammed
stem cells.
Blood Markers Pre-therapy Post-therapy
11/23/2006 12/09/2006 01/01/2007 02/17/2009
IGF-1 [ng/ml] 91 53 100 143
GF-1-bind 5.3 4 4.5 5.9
[m /L]
GH [ g/L] 2.4 3.5 3.5 1.5
Progesterone 1.2 1.5 17.5 2.25
[n /ml]
Esterdiaol 26 82 162 168.82
[ /ml]
Cortisol [ g/dl] 17.8 24 4.3 24.5
DHEA-S [Nmol/1] 2.2 3.6 0.4 5.3
drenocort 21.1 10 10
[ mol/1]
Tyroglobulin 47 18 31 1.4
[ng/m1]
Calcitonin 28 40 50
[ng/ml]
FSH [IU/1] 41.2 5.4 3.88
LH [IU/1] 15.1 1.8 6.76
Prolactin [jig/11 16.2 11.1 10.5 0.6
Free test [ mol/1] 8 6.4 7.5
Cholest [mg/dl] 242 258 280 268
HDL [m /dl] 70 76 83 63.22
DL [m /dl] 22 162 173 179.9
Triglycer [mg/dl] 111 102 122 125
Hbalc [%] 6.5 5.3 5.5 5.4
bs [M /dl] 94 90 102 103
Inhibin [U] 40 30
Osteocalcin
6.9
[ng/mL]
TSH [uU/ml] 0.84 0.7 0.5
FT3 [pg/ml] 3.1 2.7 2.7 1.54
Calcitonin [ng/1] 28 40 50
T4 [ /ml] 1.4 0.94 0.71 0.82
IGF-1 = insulin-like growth factor 1; IGF-1-bind = insulin-like growth factor
binding; GH =
growth hormone; DHEA-S = dehydroepiandrosterone sulfate ester; adrenocort =
adrenocortical hormone; FSH = follicle-stimulating hormone; LH = luteinizing
hormone;
HDL = high density lipoprotein; LDL = low density lipoprotein; triglycer =
triglyceride;
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HBA1C = glycosylated hemoglobin; FBS = fetal blood sampling; TSH = thyroid
stimulating
hormone; FT3 = free triiodothryronine; FT4 = free thyroxine
Depression
A patient suffering from depression was treated with an infusion of autologous
reprogrammed pluripotent stem cells and neurons. Following the treatment, the
patient
experienced an increase in various hormone and protein levels, including
insulin-like growth
factor-1 (IGF-1), cortisol, and testosterone (see Table 17).
Table 17. Effects of administering patient in menopause with autologous
reprogrammed
cells.
Pre-therapy Post-therapy
Blood Markers
06/11/2007 07/23/2007
GH [gg/11 <0.05 0.06
GF-1 [ng/ml] 93.4 140
GF-bp [mg/1] 4.4 5.6
Cortisol [ /dl] 4 20.9
drenocort [pg/ml] <10 17.1
Shbg [nmoIIi] 37.9 39.8
FSH [IU/L] 4.94 4.98
LH [IU/L] 10.1 3.72
e2 [ /ml] 29 <28
Testosterone [pmoUl] 28.4 57.8
Prolactin [ /1] 15.7 1.6
DHEA-S [ mol/I] 2.3 2.9
TSH [ U/ml] 1.707 3.318
FT3 [pg/ml] 1.69 1.87
FT4 [ng/dl] 0.81 0.89
GH = growth hormone; IGF-1 = insulin-like growth factor 1; IGF-bp = insulin-
like growth
factor binding protein; Adrenocort = adrenocortical hormone; Sh-bg = sex
hormone-binding
globulin; FSH = follicle-stimulating hormone; LH = luteinizing hormone; e2 =
estradiol;
DHEA-S = dehydroepiandrosterone sulfate ester; TSH = thyroid stimulating
hormone; FT3
= free triiodothryronine; FT4 = free thyroxine
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Non-obstructive Azoospermia
A patient suffering from non-obstructive azoospermia was treated with an
infusion of
autologous reprogrammed pluripotent stem cells, pluripotent germ cells, and
sperm.
Following the treatment, the patient experienced an increase in testosterone
over a period of
eight months (FIG. 21).
Vision Impairment
A patient suffering from vision loss due to a benign tumor that had been
removed
was treated with an infusion of autologous reprogrammed pluripotent stem cells
and
neurons. Following treatment, the patient experienced increased retinal
sensitivity and
improvement in vision (FIG 22).
Having thus described in detail preferred embodiments of the present
invention, it is
to be understood that the invention defined by the above paragraphs is not to
be limited to
particular details set forth in the above description as many apparent
variations thereof are
possible without departing from the spirit or scope of the present invention.
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