U.S. patent application number 09/946325 was filed with the patent office on 2003-06-05 for multi-lineage directed induction of bone marrow stromal cell differentiation.
Invention is credited to Black, Ira B., Woodbury, Dale.
Application Number | 20030104997 09/946325 |
Document ID | / |
Family ID | 25484316 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104997 |
Kind Code |
A1 |
Black, Ira B. ; et
al. |
June 5, 2003 |
Multi-lineage directed induction of bone marrow stromal cell
differentiation
Abstract
Methods of inducing differentiation of mammalian bone marrow
stromal cells into cells of multiple embryonic lineages by
contacting marrow stromal cells with precursor
differentiation-inducing compounds followed by contacting the
partially differentiated precursor cells with specific cell type
differentiation-inducing compounds. In one embodiment, the MSC
derived precursor cell cultures comprise cells, at least some of
which simultaneously express markers that are characteristic of
endodermal and ectodermal cell types. In another embodiment, the
differentiated cells are insulin-secreting pancreatic islet cells.
Precursor differentiation-inducing compounds of the invention
include anti-oxidants such as, but not limited to,
beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxyanisole,
butylated hydroxytoluene, ascorbic acid, dimethylfumarate, and
n-acetylcysteine. Endodermal cell differentiation-inducing
compounds of the invention include but are not limited to
anti-oxidants and growth factors including basic fibroblast growth
factor. Once induced to differentiate into a particular cell type,
the cells can be used for cell therapy, gene therapy, or both, for
treatment of diseases, disorders, or conditions associated with
tissues of multiple embryonic origins.
Inventors: |
Black, Ira B.; (Skillman,
NY) ; Woodbury, Dale; (Piscataway, NJ) |
Correspondence
Address: |
GIBBONS, DEL DEO, DOLAN, GRIFFINGER & VECCHIONE
1 RIVERFRONT PLAZA
NEWARK
NJ
07102-5497
US
|
Family ID: |
25484316 |
Appl. No.: |
09/946325 |
Filed: |
September 5, 2001 |
Current U.S.
Class: |
424/93.21 ;
435/372; 514/11.7; 514/44R; 514/5.9; 514/7.3; 514/8.2; 514/8.4;
514/9.1 |
Current CPC
Class: |
C12N 2501/999 20130101;
C12N 2506/1353 20130101; A61K 35/12 20130101; C12N 5/0676 20130101;
C12N 5/0619 20130101; C12N 2501/115 20130101; C12N 2501/135
20130101; C12N 5/0622 20130101 |
Class at
Publication: |
514/12 ; 435/372;
514/44 |
International
Class: |
A61K 038/18; A61K
048/00; C12N 005/08 |
Claims
What is claimed is:
1. A method of inducing differentiation of an isolated marrow
stromal cell into an endodermal cell, said method comprising
contacting said isolated marrow stromal cell with at least one
endodermal/neuronal precursor differentiation-inducing compound,
thereby inducing differentiation of said isolated marrow stromal
cell into a endodermal/neuronal precursor cell and contacting said
endodermal/neuronal precursor cell with at least one endodermal
differentiation-inducing compound thereby inducing an endodermal
cell.
2. The method of claim 1, wherein said isolated marrow stromal cell
is a human cell.
3. The method of claim 1, wherein said endodermal/neuronal
precursor differentiation-inducing compound is a trophic
factor.
4. The method of claim 1, wherein said endodermal/neuronal
precursor differentiation-inducing compound is a growth factor.
5. The method of claim 1, wherein said endodermal
differentiation-inducing compound is an antioxidant.
6. The method of claim 1, wherein endodermal
differentiation-inducing compound is a growth factor.
7. The method of claim 4, wherein said growth factor is selected
from the group consisting of platelet-derived growth factor,
fibroblast growth factor 2, and nerve growth factor.
8. The method of claim 6, wherein said growth factor is selected
from the group consisting of basic fibroblast growth factor,
fibroblast growth factor 2
9. The method of claim 1, wherein said endodermal/neuronal
precursor differentiation-inducing compound is an anti-oxidant.
10. The method of claim 5, wherein said anti-oxidant is selected
from the group consisting of beta-mercaptoethanol,
dimethylsulfoxide, butylated hydroxytoluene, butylated
hydroxyanisole, ascorbic acid, dimethylfumarate, and
n-acetylcysteine.
11. The method of claim 5, wherein said anti-oxidant is
beta-mercaptoethanol.
12. The method of claim 5, wherein said anti-oxidant is butylated
hydroxyanisole.
13. The method of claim 9, wherein said anti-oxidant is selected
from the group consisting of beta-mercaptoethanol,
dimethylsulfoxide, butylated hydroxytoluene, butylated
hydroxyanisole, ascorbic acid, dimethylfumarate, and
n-acetylcysteine.
14. The method of claim 9, wherein said anti-oxidant is
beta-mercaptoethanol.
15. The method of claim 9, wherein said anti-oxidant is
dimethylsulfoxide.
16. The method of claim 9, wherein said anti-oxidant is
dimethylsulfoxide and butylated hydroxyanisole.
17. The method of claim 1 wherein said endodermal cell is an
insulin-secreting pancreatic islet cell.
18. The method of claim 17 wherein said endodermal cell
differentiation-inducing compound is basic fibroblast growth factor
and butylated hydroxyanisole.
19. The method of claim 17 wherein said endodermal cell
differentiation-inducing compound is basic fibroblast growth factor
and beta-mercaptoethanol.
20. A method of producing an isolated endodermal cell, said method
comprising isolating a marrow stromal cell, contacting said
isolated marrow stromal cell with at least one endodermal/neuronal
precursor differentiation-inducing compound, thereby inducing
differentiation of said isolated marrow stromal cell into a
endodermal/neuronal precursor cell and contacting said
endodermal/neuronal precursor cell with an endodermal
differentiation-inducing compound thereby inducing an endodermal
cell.
21. The method of claim 20 wherein said endodermal cell is an
insulin-secreting pancreatic islet cell and said endodermal
differentiation-inducing compound is an insulin-producing
pancreatic islet differentiation-inducing compound.
22. A method of treating a human patient having a disease, disorder
or condition of an endodermal tissue, said method comprising
obtaining a bone marrow sample from a human donor, isolating
stromal cells from said bone marrow sample, inducing said stromal
cells to differentiate into selected isolated endodermal cells, and
administering said selected isolated endodermal cells to the body
of said human patient, wherein the presence of said selected
isolated neuronal cells in said body of said human patient effects
treatment of said disease, disorder or condition.
23. The method of claim 22, wherein said disease, disorder or
condition of an endodermal tissue is selected from the group
consisting of Type I diabetes, Type II diabetes, pancreatitis,
inflammatory bowel disease, stomach cancer, colon cancer,
colo-rectal cancer and liver disease.
24. The method of claim 22, wherein prior to administering said
selected isolated endodermal cells, said selected isolated
endodermal cells are transfected with an isolated nucleic acid
encoding a therapeutic protein or peptide, wherein when said
protein or peptide is expressed in said cells said protein or
peptide serves to effect treatment of said disease, disorder or
condition.
25. The method of claim 24, wherein said isolated nucleic acid
encodes a therapeutic protein or peptide selected from the group
consisting of a cytokine, a chemokine, insulin, glucagon, another
endocrine hormone, a trophic protein, a growth factor, an antibody,
and a tumor toxic protein.
26. A method of treating a human patient in need of endodermal
cells, said method comprising obtaining marrow stromal cells from a
human patient, propagating said marrow stromal cells in culture
under conditions that induce their differentiation into selected
endodermal cells, transplanting said selected endodermal cells into
said human patient in need of said selected endodermal cells,
thereby treating said human patient in need of endodermal
cells.
27. The method of claim 26 wherein said endodermal cells are
insulin-secreting pancreatic islet cells and said endodermal
differentiation-inducing compound is an insulin-producing
pancreatic islet differentiation-inducing compound.
28. An isolated endodermal cell made by a method of inducing
differentiation of an isolated marrow stromal cell, said method
comprising contacting said isolated marrow stromal cell with at
least one endodermal/neuronal precursor differentiation-inducing
compound, thereby inducing differentiation of said isolated marrow
stromal cell into a endodermal/neuronal precursor cell and
contacting said endodermal/neuronal precursor cell with an
endodermal differentiation-inducing compound thereby inducing an
endodermal cell.
29. The cell of claim 28, wherein said cell is a human cell.
30. The cell of claim 28 wherein said endodermal cell is an
insulin-secreting pancreatic islet cell and said endodermal
differentiation-inducing compound is an insulin-producing
pancreatic islet differentiation-inducing compound.
31. An isolated endodermal cell made by a method of inducing
differentiation of an isolated marrow stromal cell, said method
comprising contacting said isolated marrow stromal cell with at
least one endodermal/neuronal precursor differentiation-inducing
compound, thereby inducing differentiation of said isolated marrow
stromal cell into a endodermal/neuronal precursor cell and
contacting said endodermal/neuronal precursor cell with an
endodermal differentiation-inducing compound thereby inducing an
endodermal cell wherein said endodermal cell is further transfected
with an isolated nucleic acid encoding a therapeutic protein or
peptide, and further wherein when said protein or peptide is
expressed in said cell said protein or peptide serves to effect
treatment of a disease, disorder, or condition associated with a
tissue of endodermal origin.
32. The cell of claim 31, wherein said isolated nucleic acid
encodes a protein or peptide selected from the group consisting of
a cytokine, a chemokine, insulin, glucagon, another endocrine
hormone, a trophic protein, a growth factor, an antibody, and a
tumor toxic protein.
33. The cell of claim 31, wherein said cell is a human cell.
34. An isolated endodermal cell made by a method of producing an
isolated endodermal cell, said method comprising isolating a marrow
stromal cell, contacting said isolated marrow stromal cell with at
least one endodermal/neuronal precursor differentiation-inducing
compound, thereby inducing differentiation of said isolated marrow
stromal cell into a endodermal/neuronal precursor cell and
contacting said endodermal/neuronal precursor cell with an
endodermal differentiation-inducing compound thereby inducing said
endodermal cell
35. The cell of claim 34, wherein said cell is a human cell.
36. The isolated endodermal cell of claim 34 wherein said isolated
endodermal cell is an insulin-secreting pancreatic islet cell
endodermal differentiation-inducing compound is an
insulin-producing pancreatic islet differentiation-inducing
compound.
37. An isolated endodermal cell made by a method of producing an
isolated endodermal cell, said method comprising isolating a marrow
stromal cell, contacting said isolated marrow stromal cell with at
least one endodermal/neuronal precursor differentiation-inducing
compound, thereby inducing differentiation of said isolated marrow
stromal cell into a endodermal/neuronal precursor cell and
contacting said endodermal/neuronal precursor cell with an
endodermal differentiation-inducing compound thereby inducing said
endodermal cell wherein said endodermal cell is further transfected
with an isolated nucleic acid encoding a therapeutic protein or
peptide, and further wherein when said protein or peptide is
expressed in said cell, said protein or peptide serves to effect
treatment of a disease, disorder, or condition associated with a
tissue of endodermal origin.
38. The cell of claim 37, wherein said isolated nucleic acid
encodes a protein or peptide selected from the group consisting of
a cytokine, a chemokine, insulin, glucagon, another endocrine
hormone, a trophic protein, a growth factor, an antibody, and a
tumor toxic protein.
39. The cell of claim 37, wherein said cell is a human cell.
40. The isolated endodermal cell of claim 37 wherein said isolated
endodermal cell is an insulin producing pancreatic islet cell and
said endodermal differentiation-inducing compound is an
insulin-producing pancreatic islet differentiation-inducing
compound.
41. An MSC derived cell culture that comprises cells, at least some
of which simultaneously express polypeptide or mRNA markers that
are characteristic of at least endodermal and ectodermal cell
types.
42. The culture of claim 41 wherein said cells simultaneously
express nestin and a polypeptide or mRNA marker characteristic of
an endodermal cell type.
43. The culture of claim 41 wherein said cells simultaneously
express nestin and a polypeptide or mRNA marker characteristic of a
pancreatic cell.
44. The culture of claim 41 wherein said cells simultaneously
express nestin and ceruloplasmin.
45. The culture of claim 41 that proliferates under conditions that
are non permissive for the proliferation of bone marrow stromal
cells.
46. A method of producing an isolated endodermal/neuronal precursor
cell, said method comprising 1) isolating a marrow stromal cell 2)
culturing the marrow stromal cell under conditions suitable to
produce an endodermal/neural precursor culture that comprises cells
at least some of which simultaneously express polypeptide or mRNA
markers that are characteristic of at least endodermal and
ectodermal cell types.
47. The method of claim 46 comprising selecting a single
endodermal/neuronal precursor cell from the endodermal/neuronal
precursor culture and culturing the endodermal/neuronal precursor
cell to produce a clonal a endodermal/neuronal precursor
culture.
48. The method of claim 46 comprising culturing the
endodermal/neuronal precursor cell in a media comprising an
antioxidant.
49. The method of claim 48 wherein said antioxidant is
beta-mercaptoethanol.
50. The method of claim 48 wherein said antioxidant is butylated
hydroxyanisole.
Description
BACKGROUND OF THE INVENTION
[0001] Pluripotent stem cells have been detected in multiple
tissues in the adult mammal, participating in normal replacement
and repair, while undergoing self-renewal (Hay, 1966, Regeneration,
Holt, Rinehart and Winston, New York; McKay, 1999, Nature Med.
5:261-262; Lemiscka, 1999, Ann. N.Y. Acad. Sci. 872:274-288; Owens
and Friedenstein, 1988, Ciba Foundation Syp. 136, Chichester, U.K.
pp. 42-60; Prockop, 1997, Science 276:71-74; Ferrari et al., 1998,
Science 279:1528-1530; Caplan, 1991, J. Orthop. Res. 9:641-650;
Pereira et al., 1995, Proc. Natl. Acad. Sci. USA 92:4857-4861;
Kuznetsov et al., 1997, Brit. J. Haemotology 97:561-570; Majumdar
et al., 1998, J. Cell Physiol. 176:57-66; Pittenger et al., 1999,
Science 284:143-147). A subclass of bone marrow stem cells is one
prototype, capable of differentiating into osteogenic,
chondrogenic, adipogenic and other mesenchymal lineages in vitro
(Owens and Friedenstein, 20 1988, Ciba Foundation Symp. 136,
Chichester, U.K. pp. 42-60; Prockop, 1997, Science 276; 71-74;
Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991, J.
Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad.
Sci. USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology
97:561-570; Majumdar et al., 1998, J. Cell. Physiol. 176:57-66;
Pittenger et al., 1999, Science 284:143-147). These pluripotent
cells have been termed marrow stromal cells (MSCs), and have been
used clinically to treat osteogenesis imperfecta (Horwitz et al.,
1999, Nature Med. 5:309-313).
[0002] The discovery of stem cell populations in the central
nervous system (CNS) has generated intense interest, since the
brain has long been regarded as incapable of regeneration (Reynolds
and Weiss, 1992, Science 255:1707-1710; Richards et al., 1992,
Proc. Natl. Acad. Sci. USA 89:8591-8595; Morshead et al., 1994,
Neuron 13:1071-1082). Neural stem cells (NSCs) are capable of
undergoing expansion and differentiating into neurons, astrocytes
and oligodendrocytes in vitro (Reynolds and Weiss, 1992, Science
255:1707-1710; Johansson et al., 1999, Cell 96:25-34; Gage et al.,
1995, Annu. Rev. Neurosci. 18:159-192; Vescovi et al., 1993, Neuron
11:951-966). The recent report demonstrating that NSCs can generate
hematopoietic cells in vivo suggests that stem cell populations may
be less restricted than previously thought (Bjornson, 1999, Science
283:534-537).
[0003] Adult MSC cells are both self-renewing and multipotential
(Owens and Friedenstein, 1988, Ciba Foundation Symp. 136,
Chichester, U.K. pp. 42-60; Prockop, 1997, Science 276; 71-74;
Ferrari et al., 1998, Science 279:1528-1530; Caplan, 1991, J.
Orthop. Res. 9:641-650; Pereira et al., 1995, Proc. Natl. Acad.
Sci. USA 92:4857-4861; Kuznetsov et al., 1997, Brit. J. Haemotology
97:561-570; Majumdar et al., 1998, J. Cell. Physiol. 176:57-66;
Pittenger et al., 1999, Science 284:143-147; Sanchez Ramos et al.
Exp. Neurol. 2000 164(2) 247-56), thereby fulfilling many of the
criteria of a stem cell population.
[0004] Recent studies show that neural stem cells have broad
developmental potential, contributing to the development of blood
as well as to all germ layers in chimeric embryos (Clarke, D. L. et
al., Science (2000) 288: 1660-1663). During early embryonic
development of mammalian embryos, developing neural cells share
features with developing islet cells. A specific characteristic of
neural precursor cells is their expression of the protein nestin
(U. Lendhal, L. D. Zimmerman, R. D. McKay Cell: 23, 585 (1990) as
described in U.S. Pat. No. 5,338,839. Recently, researchers have
found the stem cell marker nestin within developing islet cells (H.
Zulewski et al., 2001, Diabetes 50: 521) and others report the
differentiation of nestin-positive embryonic stem cells to
insulin-secreting structures similar to pancreatic islets in mice
(N. Lumelsky et al. supra) and humans (Assady et al., Diabetes 50,
August 2001). However, differentiation of insulin-secreting
islet-type cells from adult stem cells has not previously been
demonstrated. Common mechanisms of control and shared nestin
expression point to a close relationship between pancreatic and
neural progenitors which give rise to tissues of endodermal and
ectodermal embryonic origin respectively.
[0005] We have recently demonstrated that MSCs can be induced to
differentiate into neuronal cells (D. Woodbury et al., 2000, J.
Neur. Sci. Res. 61, 364). Differentiation of MSCs into astrocytes
and glial cells (WO 99/43286) has also been demonstrated. These
recent studies indicate that rat and human MSCs are capable of
differentiating into non-mesenchymal derivatives, suggesting that
intrinsic genomic mechanisms of commitment, lineage restriction and
cell fate are mutable. Environmental signals apparently can elicit
the expression of pluripotentiality that extends well beyond the
accepted fate restrictions of cells originating in classical
embryonic germ layers.
[0006] To define the process of stem cell differentiation and
elucidate underlying mechanisms, we have characterized MSCs and
developing neurons more extensively, defining expression patterns
for representative genes of different lineages and correlating
expression with morphologic maturation. Our observations indicate
that the "undifferentiated" MSCs express germline, endodermal and
ectodermal genes, as well as the expected mesodermal genes.
Neuronal differentiation of the MSCs involves complex modulation of
these different gene sets, rather than simple on-off switching of
neural and non-neural genes. We now describe, for the first time,
conditions which permit the growth and expansion of endodermal
cells, particularly insulin-producing pancreatic islet cells,
differentiated from adult bone marrow stromal cells (MSCs). MSCs
constitute a novel source of pancreatic islet cells and represent
the only adult cells used for this purpose.
[0007] A number of disease states are associated with organs of
endodermal lineage which include the liver, stomach, intestine,
pancreas, and other endocrine glands. Type 1 and Type 2 diabetes
and chronic pancreatitis result from the anatomical and functional
loss of insulin-producing beta cells and the ductal and acinar
cells, respectively, while uncontrolled proliferation of the ductal
cells leads to pancreatic carcinogenesis. The replacement of these
cells through regeneration or transplantation could offer lifelong
treatment for diabetics and for patients with chronic pancreatitis.
However, a major problem in implementing treatment is the lack of
sufficient pancreatic/islet cell tissue for transplantation. The
present invention officers the potential of generating sizable
quantities of insulin-producing cells from adult bone marrow
stromal cells.
[0008] Gut malignancies and inflammatory bowel diseases are major
causes of morbidity and mortality. The cell differentiation
techniques disclosed herein may be utilized to gain new insights
about initiation, progression and treatment of tumorigenesis and
offer new strategies to increase the absorptive function of the
intestine.
[0009] Liver transplantation is the treatment of choice for many
liver diseases. Unfortunately, the supply of donor organs is
limiting and therefore many patients cannot benefit from this
therapy. Therapeutic liver re-population with bone marrow derived
cells holds the hope of overcoming the shortage in donor
livers.
[0010] Despite the crucial need for obtaining endodermal cells for
treatment a number of diseases, disorders, and conditions,
associated with tissues of endodermal lineage, no method has
previously been available for obtaining large numbers of endodermal
cells without encountering the technical and ethical hurdles
involved in obtaining adult human or fetal tissue. The present
invention overcomes that need, offering the potential of generating
sizable quantities of endodermal tissue from adult bone marrow
stromal cells.
[0011] While previous studies have demonstrated that intrinsic
genomic mechanisms of commitment, lineage restriction and
mesenchymal cell fate of MSCs are mutable, it was unexpected that
these adult cells could be induced to differentiate to cells
associated with tissues of endodermal lineage including the liver,
stomach, intestine, pancreas, and other endocrine glands.
SUMMARY OF THE INVENTION
[0012] In one aspect of the invention, a cell culture is provided
that comprises cells, at least some of which simultaneously express
polypeptide or mRNA markers that are characteristic of at least
ectodermal and endodermal cells. Useful ectodermal markers include
but are not limited to nestin, tau, neuron specific enolase, glial
fibrillary acidic protein, especially nestin. Useful endodermal
markers include but are not limited to ceruloplasmin. According to
another aspect of the invention, at least some cells of the culture
simultaneously express the neuronal marker nestin and an endodermal
marker, such as ceruloplasmin.
[0013] According to another aspect of the invention there is
provided a method of producing a nestin-positive
endodermal/neuronal precursor cell capable of differentiation into
cells of at least endodermal or ectodermal lineage.
[0014] The present invention includes a method of inducing
differentiation of an isolated marrow stromal cell into an
endodermal cell, particularly a pancreatic islet cell. The method
comprises contacting the isolated marrow stromal cell with at least
one endodermal/neuronal precursor differentiation-inducing compound
thereby producing a nestin-positive endodermal/neuronal precursor
cell. The endodermal/neuronal precursor cell is contacted with at
least one endodermal differentiating inducing compound,
particularly an insulin-secreting pancreatic islet
differentiation-inducing compound. This induces differentiation of
the isolated marrow stromal cell into an endodermal cell,
particularly an insulin-secreting pancreatic islet cell. In one
embodiment, the at least one insulin-secreting pancreatic islet
differentiation-inducing compound comprises a defined insulin
secreting pancreatic islet differentiation-inducing medium.
[0015] In one aspect, the isolated marrow stromal cell is a rat
cell. Preferably, the isolated marrow stromal cell is a human
cell.
[0016] In one aspect, the nestin-positive pancreatic/neuronal
precursor differentiation-inducing compound is an anti-oxidant. In
another aspect, the anti-oxidant is selected from the group
consisting of beta-mercaptoethanol, dimethylsulfoxide, butylated
hydroxytoluene, butylated hydroxyanisole, ascorbic acid,
dimethylfumarate, and n-acetylcysteine. In one aspect, the
pancreatic islet differentiation-inducing compound is an
anti-oxidant. In another aspect, the anti-oxidant is selected from
the group consisting of beta-mercaptoethanol and butylated
hydroxyanisole. In yet another aspect, the pancreatic
differentiation-inducing anti-oxidant is beta-mercaptoethanol. In
another aspect, the pancreatic islet differentiation-inducing
anti-oxidant is butylated hydroxyanisole.
[0017] The pancreatic islet cell differentiation-inducing compound
is also a growth factor in another aspect. In a preferred aspect,
the growth factor is basic fibroblast growth factor.
[0018] The invention further includes a method of producing an
isolated insulin-secreting pancreatic islet cell. The method
comprises isolating a marrow stromal cell, contacting the marrow
stromal cell with at least one endodermal/neuronal precursor
differentiation-inducing compound that differentiates the marrow
stromal cell into a nestin-positive endodermal/neuronal precursor
cell and contacting the nestin-positive endodermal/neuronal
precursor cell with at least one differentiation-inducing compound
that induces the nestin-positive precursor cell to differentiate
into an isolated pancreatic islet cell, thereby producing an
isolated pancreatic islet cell.
[0019] According to another aspect of the invention there is
provided a method of producing an isolated nestin-positive
endodermal/neuronal precursor cell capable of differentiating into
an insulin producing pancreatic islet cell. The method comprises
isolating a marrow stromal cell and contacting a isolated marrow
stromal cell with at least one endodermal/neuronal precursor
differentiation-inducing compound thereby producing an isolated
nestin-positive endodermal/neuronal precursor cell capable of
differentiating into an isolated insulin producing pancreatic islet
cell To induce insulin producing pancreatic islet cell
differentiation, the isolated endodermal/neuronal precursor cell is
contacted with at least one insulin-secreting pancreatic islet
differentiation-inducing compound. This induces differentiation of
the isolated endodermal/neuronal precursor cell into an isolated
insulin-secreting pancreatic islet cell.
[0020] The invention further includes a method of producing an
isolated endodermal cell. The method comprises isolating a marrow
stromal cell, contacting the marrow stromal cell with at least one
endodermal/neuronal differentiation-inducing compound that
partially differentiates the marrow stromal cell into a
nestin-positive precursor cell and contacting the nestin-positive
endodermal/neuronal precursor cell with at least one endodermal
differentiation-inducing compound that induces the
endodermal/neuronal precursor cell to differentiate into an
isolated endodermal cell, thereby producing an isolated endodermal
cell.
[0021] In addition, the invention includes a method of treating a
human patient having a disease, disorder or condition associated
with tissues of endodermal origin, particularly of pancreatic
origin. The method comprises obtaining a bone marrow sample from a
human donor, isolating stromal cells from the bone marrow sample,
inducing the stromal cells to differentiate into nestin-positive
endodennal/neuronal precursor cells and inducing the
nestin-positive endodermal/neuronal precursor cells to
differentiate into isolated endodermal cells, particularly
insulin-secreting pancreatic islet cells, and administering the
isolated endodermal cells to the body of the human patient. The
presence of the isolated endodermal cells, particularly
insulin-secreting pancreatic islet cells, in the body of the human
patient effects treatment of the disease, disorder or condition of
endodermal origin, particularly pancreatic origin.
[0022] In one aspect, the disease, disorder or condition associated
with a tissue of endodermal origin is selected from the group
consisting of Type I diabetes, Type II diabetes, pancreatitis,
inflammatory bowel disease, stomach cancer, colon cancer,
colo-rectal cancer and liver disease.
[0023] In another aspect, prior to administering the isolated
endodermal cells, particularly insulin-secreting pancreatic islet
cells, the isolated endodermal cells are transfected with an
isolated nucleic acid encoding a therapeutic protein or peptide,
particularly insulin wherein, when the protein or peptide is
expressed in the cells, the protein or peptide serves to effect
treatment of the disease, disorder or condition.
[0024] In an alternative aspect, the endodermal cells, particularly
pancreatic islet cells are transfected with an isolated nucleic
acid encoding a cytokine, a chemokine, insulin, glucagon, another
endocrine hormone, a trophic protein, a growth factor, an antibody,
and a tumor toxic protein.
[0025] The present invention further includes a method of treating
a human patient in need of endodermal cells, particularly
insulin-secreting pancreatic islet cells. The method comprises
obtaining marrow stromal cells from a human patient, propagating
the marrow stromal cells in culture under conditions that induce
their differentiation, inducing the stromal cells to differentiate
into nestin-positive endodermal/neuronal precursor cells, inducing
the nestin-positive endodermal/neuronal precursor cells to
differentiate into isolated endodermal cells, particularly
insulin-secreting pancreatic islet cells, transplanting the
endodermal cells into the human patient in need of the endodermal
cells, thereby treating the human patient in need of endodermal
cells.
[0026] A preferred embodiment of the invention includes an isolated
endodermal cell, particularly an insulin-secreting pancreatic islet
cell, transfected with a therapeutic protein or peptide. The
endodermal cell is isolated by the method of inducing
differentiation of an isolated marrow cell into an endodermal cell,
particularly an insulin-secreting pancreatic islet cell, as recited
above. The endodermal cell is then transfected with an isolated
nucleic acid encoding a therapeutic protein or peptide that, when
expressed, will effect treatment of a disease, disorder, or
condition associated with a tissue of endodermal origin,
particularly pancreatic origin. In an aspect of the invention, the
therapeutic protein or peptide encoded by the isolated nucleic acid
is a cytokine, a chemokine, insulin, glucagon, another endocrine
hormone, a trophic protein, a growth factor, an antibody, or a
tumor toxic protein. The invention encompasses diseases, disorders,
or conditions of organ systems of endodermal lineage including, but
not limited to, Type I diabetes, Type II diabetes, pancreatitis,
inflammatory bowel disease, stomach cancer, colon cancer,
colo-rectal cancer and liver disease.
[0027] In one aspect, the transfected endodermal cell, particularly
an insulin-secreting pancreatic islet cell, made by this method is
a rodent cell. In another aspect the transfected endodermal cell is
a rat cell. In a preferred aspect, the transfected cell made by
this method is a human cell.
[0028] The invention also includes endodermal cells, particularly
insulin-secreting pancreatic islet cells, made by the method of
inducing differentiation of isolated marrow cells into
nestin-positive endodermal/neuronal precursor cells and inducing
the nestin-positive endodermal/neuronal precursor cells into
endodermal cells, particularly insulin-secreting pancreatic islet
cells. The method comprises contacting the isolated marrow stromal
cells with at least one differentiation-inducing compound which
induces the isolated marrow cells to differentiate to
nestin-positive endodermal/neuronal precursor cells. Contact
between the nestin-positive endodermal/neuronal precursor cells and
at least one endodermal differentiation-inducing compound, in
particular an insulin-secreting pancreatic islet cell inducing
compound, induces differentiation of the nestin-positive
endodermal/neuronal precursor cells into the endodermal cells of
the invention, particularly insulin-secreting pancreatic islet
cells.
[0029] In an aspect, the endodermal cells, particularly
insulin-secreting pancreatic islet cells, made by this method are
rodent cells. In another aspect, the endodermal cells, particularly
insulin-secreting pancreatic islet cells, are rat cells. In a
preferred aspect, the endodermal cells, particularly
insulin-secreting pancreatic islet cells, made by this method are
human cells.
[0030] The invention also includes an isolated endodermal cell,
particularly an insulin-secreting pancreatic islet cell, produced
by a method comprising isolating a marrow stromal cell and
contacting the isolated marrow stromal cell with at least one
endodermal/neuronal precursor differentiation-inducing compound
that induces the isolated marrow stromal cell to differentiate into
an isolated nestin-positive endodermal/neuronal precursor cell. The
nestin-positive endodermal/neuronal precursor cell is contacted
with at least one endodermal differentiation-inducing compound.
This induces the isolated nestin-positive endodermal/neuronal
precursor cell to differentiate into an isolated endodermal cell,
particularly an insulin-secreting pancreatic islet cell
[0031] The invention also includes an isolated transfected
endodermal cell, particularly an insulin-secreting pancreatic islet
cell, produced by a method comprising inducing differentiation of
an isolated marrow cell into an endodermal cell, particularly an
insulin-secreting pancreatic islet cell, as recited above. The
endodermal cell is then transfected with an isolated nucleic acid
encoding a therapeutic protein or peptide that, when expressed,
will effect treatment of a disease, disorder, or condition
associated with a tissue of endodermal origin, particularly
pancreatic origin. In an aspect of the invention, the therapeutic
protein or peptide encoded by the isolated nucleic acid is a
cytokine, a chemokine, insulin, glucagon, another endocrine
hormone, a trophic protein, a growth factor, an antibody, or a
tumor toxic protein.
[0032] In an aspect, the transfected endodermal cell is rodent
cell. In another aspect, the transfected endodermal cell is a rat
cell. In a preferred aspect, the transfected endodermal cell is a
human cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiment(s) which are presently preferred. It should be
understood, however, that the invention is not limited to the
precise arrangements and instrumentalities shown.
[0034] FIG. 1. Undifferentiated MSCs express genes representative
of all germ layers. Messenger RNA was harvested from
undifferentiated MSCs, converted to cDNA, and target genes were
amplified using specific primer pairs (see Table 1 for symbol and
primer sequences). Amplification resulted in a single band in each
reaction where cDNA target was provided (RT+). Control lanes, where
no RTase was present during the cDNA synthesis reaction, did not
yield product. Kilobase ladder (Kb) and X/Hind III DNA (M) were
employed as markers.
[0035] FIG. 2. Neuronal differentiation of MSCs alters the pattern
of gene expression. RNA was harvested from undifferentiated MSCs
(S) or from MSC-derived neurons (N) 48 hours after neuronal
induction. RT-PCR was performed to amplify gene products
representing specific germ layers. Neuronal induction decreased
expression of ceruloplasmin, NMDA receptor glutamate binding
subunit, and protamine2 significantly, while expression of aldolase
C, SM22.alpha. and syntaxin was less affected. The level of
expression of APP was not changed by neuronal induction.
.lambda./Hind III DNA was used as M. W. marker (M).
[0036] FIGS. 3A-B. MSC-derived neurons express the neuronal marker
NF-M. A. RNA was harvested from undifferentiated MSCs maintained in
SFM (SFM) and from MSCderived neurons maintained in NIM (NIM) for
24 hours. RT-PCR performed with primers specific for NF-M yielded a
single band from MSC-derived neurons. A very faint band
corresponding to the NF-M product was also detected in cells
maintained in SFM. RT-PCR of RNA derived from the cerebellum of
adult rats yielded a NF-M product of the same size, demonstrating
specificity of the reaction. Equal amplification of noggin (inset)
indicates equivalence of cDNA target. .lambda./Hind III DNA was
used as M.W. marker (M). B. NF-M message was undetectable in MSCs
maintained in SFM, but is just above the level of detection in
cells exposed to NIM at 5 hours. The level of expression of NF-M
increased dramatically after a 24-hour exposure to NIM.
.lambda./Hind III DNA was used as M. W. marker (M).
[0037] FIGS. 4A-B. Neuronal differentiation alters expression of
neural genes. A. RT-PCR analysis was utilized to assess expression
of neuroglial genes during the initial 48 hours of neuronal
differentiation. APP expression in undifferentiated MSCs (S),
MSC-derived neurons at 24 hours (N-24) and 48 hours (N-48)
post-induction remained constant. GFAP and NeuroD (NeD) mRNA levels
were elevated in MSCs and decreased as neuronal differentiation
proceeded. Message for the neuronal markers NF-M and tau were
undetectable in MSCs but increased with ongoing neuronal
differentiation. tau message was present in multiple isoforms
(bracketed bands). .lambda./Hind III DNA was used as M. W. marker
(M). B. RT-PCR followed by high resolution electrophoresis revealed
three distinct bands corresponding to known splice variants of tau
message that were present in MSC-derived neurons at 48 hours
post-induction (<). No signal was discernible in
undifferentiated MSCs. Sizes (bp) of low M. W. fragments of
Kilobase ladder (Kb) are indicated.
[0038] FIGS. 5A-B. MSC-derived neurons express tau 10 days after
induction. MSC-derived neurons were fixed and processed
immunocytochemically for tau expression. A. Intensely tau-positive
neuron (<) displays contracted cell body and long processes
studded with varicosity-like swellings (arrows) while neighboring
cells lack both neuronal morphologies and strong tau staining. B.
tau-positive MSC-derived neuron (>) exhibits an extremely long
process which terminates in a growth conelike structure (arrow).
Neighboring cells show varying intensities of tau staining
indicative of heterogeneous level of expression of this neuronal
marker. Magnification 200.times..
[0039] FIGS. 6A-D. MSC-derived neurons express TOAD-64,
.beta.-tubulin III and synaptophysin. MSC-derived neurons were
fixed 10 days after differentiation and processed
immunocytochemically for neuronal markers. MSC-derived neurons
express (A) TOAD-64, (B) .beta.-tubulin III, and (C) synaptophysin,
with intense staining in the varicosity-like swellings (arrows).
Magnification 200.times.. D. RTPCR for synaptophysin was performed
on RNA harvested from undifferentiated MSCs (S) and MSC-derived
neurons 24 (N-24) and 48 (N-48) hours postinduction. A signal
indicating the presence of synaptophysin message is not detected in
MSCs, but becomes increasingly evident with progressing neuronal
differentiation.
[0040] FIGS. 7A-D. MS C-derived neurons express enzymes required
for neurotransmitter biosynthesis. MSC-derived neurons were fixed
at 10 days post-induction and processed immunocytochemically. A, B.
Cells exhibiting neuronal morphologies show intense staining for
CHAT (>), while flat stromal-like cells (arrow) do not. The
perinuclear staining pattern is particularly evident in the bipolar
neuron in A. C, D. A subpopulation of cells express TH (>),
while the majority of MSC-derived neurons (arrow) do not express
this protein. Magnification 200.times..
[0041] FIGS. 8A-C. Neuronal differentiation is reversible
morphologically and transcriptionally. A. MSC-derived neurons at 24
hours post induction display neuronal morphologies with highly
refractile cell bodies and long process like extensions. B. Same
cells as in A. 24 hours after NIM withdrawn. Cell bodies have
flattened and processes have been withdrawn, yielding cells with
stromal morphologies. Magnification 200.times.. C. Morphologic
reversion is accompanied by changes in gene expression, identified
by RT-PCR. Ceruloplasmin, protamine2, GFAP, and NeuroD are
expressed at higher levels in reverted cells (R48) than in
MSC-derived neurons (N-48) at 48 hours post-induction. The level of
expression of APP and SM22 cc is unaltered by the reversion
process.
[0042] FIG. 9A is a graph depicting fluorescent cell sorting of
passage 1 rMSCs using mouse monoclonal antibodies that specifically
bind with cell surface marker CD11b (CD11/integrin alpham/Mac-1
alpha chain; Pharmingen, San Diego, Calif.) (unfilled peaks). The
secondary antibody used was anti-mouse antibody conjugated with
fluoresceine isothiocyanate (FITC). An isotype control is included
in each experiment to identify background fluorescence (filled
peaks). Number of cells analyzed (Events) is plotted on the Y-axis,
while intensity of staining is plotted on the X-axis.
[0043] FIG. 9B is a graph depicting fluorescent cell sorting of
passage 1 rMSCs using mouse monoclonal antibodies that specifically
bind with cell surface marker CD45/leukocyte common antigen
(Pharmingen) (unfilled peaks). The secondary antibody is anti-mouse
antibody conjugated with fluoresceine isothiocyanate (FITC). An
isotype control is included in each experiment to identify
background fluorescence (filled peaks). Number of cells analyzed
(Events) is plotted on the Y-axis, while intensity of staining is
plotted on the X-axis.
[0044] FIG. 9C is a graph depicting fluorescent cell sorting of
passage 1 rMSCs using mouse monoclonal antibodies that specifically
bind with cell surface marker CD90/Thy-1/CD90.1/Thy1.1 (Pharmingen)
(unfilled peaks). The secondary antibody is anti-mouse antibody
conjugated with fluoresceine isothiocyanate (FITC). An isotype
control is included in each experiment to identify background
fluorescence (filled peaks). Number of cells analyzed (Events) is
plotted on the Y-axis, while intensity of staining is plotted on
the X-axis. The data disclosed herein demonstrate that the
fluorescence intensity is greater (shifted to the right) when rMSCs
are incubated with CD90 antibody (unfilled), as compared to control
antibody (filled), indicating that the vast majority of cells in
the rMSC cultures express CD90, consistent with their
undifferentiated state.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The novel methods of the subject invention are based on the
discovery MSC stem cells, which we have previously shown to be
capable of differentiating into neuronal cells, express germline,
ectodermal, endodermal and mesodermal genes prior to neurogenesis.
(Woodbury, et al.--submitted for publication). That is, MSCs are
not undifferentiated but rather "multi-differentiated." The
expression of genes characteristic of the endoderm, e.g.
ceruloplasmin, by MSCs prior to neurogenesis was unexpected and
MSCs represent the first adult stem cells used for the purpose of
generating cells of endodermal lineage.
[0046] According to the present invention, marrow stromal cells are
contacted with at least one endodermal/neuronal
differentiation-inducing agent that mediates partial
differentiation of the cells into precursor cells which express the
specific marker nestin. The nestin-positive precursor cells
prepared according to the methods described herein have the
potential to differentiate into cells of neuronal and/or endodermal
lineage.
[0047] MSC cultures simultaneously express a wide array of mRNA and
protein markers that are normally associated with cells of multiple
distinct developmental lineages including neural (ectodermal),
vascular/hematopoietic (mesodermal), muscle (mesodermal) and
endoderm lineages. Mesodermal cells include, for example,
connective tissue cells, (e.g. fibroblasts), bone, cartilage (e.g.
chondrocytes), muscle (e.g. myocytes), blood and vessels, lymphatic
and lymphoid organ cells, pleura, pericardium, kidney, gonad and
peritoneum. Ectodermal cells include, for example, epidermal cells
such as those of the nail, hair, glands of the skin, nervous
system, external organs (e.g. eyes and ears) and the mucosal
membranes (e.g. mouth, nose, anal, vaginal). Endodermal cells
include, for example, those of the pharynx, respiratory tract,
digestive tracts, bladder, liver, pancreas, and urethra.
[0048] The untreated MSC cultures of the invention simultaneously
express an unexpectedly wide variety of mRNA and polypeptide
lineage markers in a pattern different from naturally occurring
differentiated cells. Thus, the MSC cell cultures of the invention
can be characterized by the presence or absence of the markers.
Such markers include, for example, the endodermal marker
ceruloplasmin, the mesodermal marker SM22.alpha., the germline
marker protamine and ectodermal aldolase C, amyloid precursor
protein, NMDA, glutamate binding subunit and syntaxin. In the mixed
culture, not all markers will be present in all cells, e.g. some
markers may be absent due to differences in developmental state,
culture conditions, etc. All cultures contacted with the
endodermal/neuronal precursor-inducing compound of the invention
will express the ectodermal marker nestin and at least one marker
characteristic of endodermal lineage e.g. ceruloplasmin.
[0049] Expression of markers from different lineages in the
resulting cultures can be due to multiple cell types within the
culture or can result from a multi- or pluri-potent cell capable of
many distinct patterns of expression and physiological roles.
Clonal lines may be isolated and RT-PCR expression profile carried
out on each. Expression of a wide variety of lineages within clonal
lines indicates that differentiated cells are derived from a
multi-potent cell. The unique, multifarious expression
characteristics of MSCs suggest a multifunctional, multipotential
differentiation capability.
[0050] Growth media can be enhanced with a wide variety of
compounds including but not limited to retinoic acid,
dimethylsulfoxide (DMSO), cAMP activators such as forskolin,
isobutylmethylxanthine and dibutyryl cAMP, antioxidants such as
beta-mercaptoethanol, dimethylsulfoxide, butylated hydroxytoluene,
butylated hydroxyanisole, ascorbic acid, dimethylfumarate,
N-acetylcysteine and cytokines such as basic fibroblast growth
factor, epidermal growth factor, and nerve growth factor.
[0051] The nestin-positive precursor cells prepared according to
the methods described herein have the potential to differentiate
into cells of neuronal and/or endodermal lineage, particularly
pancreatic lineage, and may further be genetically modified. Thus,
the cell differentiation methods disclosed herein provide entirely
new strategies for repairing or replacing damaged or diseased
tissues associated with organs of endodermal lineage such as the
liver, stomach, intestine pancreas and other endocrine glands.
[0052] The invention includes a method of inducing isolated marrow
stromal cells to differentiate into endodermal cells, in
particular, pancreatic islet cells. Generally, cells are isolated
from a donor, stromal cells are obtained therefrom, usually using a
cell-sorting method, and the stromal cells are subsequently
cultured in vitro. The donor may be a rat, for example, or the
donor may be a human. The invention is intended to encompass a
mammalian donor and should not be limited to the specific donors
disclosed herein.
[0053] Induction of Endodermal/Precursor Cells
[0054] In one embodiment, to induce partial differentiation of at
least some of the undifferentiated MSCs to endodermal/neuronal
precursor cells, the undifferentiated MSCs are pre-treated with an
effective amount of at least one endodermal/neuronal
precursor-inducing compound which is introduced into the cell
culture for a period of time. The length of time may vary according
to the precise method being contemplated and should not be
construed as limiting the invention in any way. After pretreatment
exposure to the endodermal/neural precursor-inducing compound, the
cells are transferred to a serum-free medium containing an
effective amount of the same endodermal/neuronal precursor-inducing
compound resulting in partial differentiation of the marrow stromal
cells to nestin-positive endodermal/neural precursor cells.
[0055] In preferred embodiments of the invention, antioxidants
serve as the endodermal/neuronal precursor-inducing compounds,
including but not limited to .beta.-mercaptoethanol,
dimethylsulfoxide, butylated hydroxytoluene, butylated
hydroxyanisole, ascorbic acid, dimethylfumarate, and
n-acetylcysteine. Particularly preferred is .beta.-mercaptoethanol.
Antioxidants as used herein should be construed to include all
antioxidants, as well as other compounds which induce the
proliferation of nestin-positive MSCs.
[0056] Cell cultures and clonal lines of the nestin positive
precursor cells of the invention retain a broad pattern of gene
expression including simultaneous expression of markers normally
associated with cells of the neural and endodermal lineages. For
example the simultaneous expression of the neuronal marker nestin
and the endodermal marker ceruloplasmin as detected by reverse
transcriptase polymerase chain reaction (RT-PCR) confirms that
these nestin-positive cells are indeed multi-potent precursor cells
capable of differentiating into cells of ectodermal and endodermal
lineage.
[0057] Induction of Pancreatic Phenotype
[0058] To induce the insulin-secreting pancreatic islet phenotype
from adult stem cells according to one embodiment of the present
invention, MSCs are pre-treated with an effective amount of at
least one endodermal/neuronal precursor-inducing compound that is
introduced into the cell culture for a period of time. The length
of time may vary according to the precise method being contemplated
and should not be construed as limiting the invention in any way.
After pre-treatment exposure to the at least one
endodermal/neuronal precursor differentiation-inducing compound,
the cells are transferred to a serum-free medium containing an
amount of the same endodermal/neuronal precursor-inducing compound
to provide an environment that promotes vigorous proliferation of
nestin-positive endodermal/neuronal precursor cells.
Nestin-positive precursor cells are then transferred to a medium
containing at least one pancreatic islet differentiation-inducing
compound to provide an environment that promotes proliferation of
differentiated pancreatic cell types. Pancreatic morphology in
precursor cells becomes evident after exposure to the pancreatic
islet differentiation-inducing compound and becomes more evident
steadily over time. Pancreatic islet marker expression also becomes
apparent after treatment, in particular, insulin secretion.
Differentiated pancreatic cell types eventually express other
pancreatic markers known to those skilled in the art, such as
glucagon.
[0059] In one aspect of the invention, the pancreatic islet
differentiation-inducing compound for inducing differentiation of
nestin-positive endodermal/neuronal precursor cells to
insulin-secreting pancreatic islet cells is a growth factor. In a
preferred embodiment, the growth factor is basic fibroblast growth
factor (bFGF). The invention contemplates other growth factors
including but not limited to insulin-like growth factor, epidermal
growth factor and nicontinamide.
[0060] Progressive differentiation of the nestin-positive,
partially differentiated endodermal/neuronal precursor cells to
insulin-secreting pancreatic islet cells corresponds with a
decrease in nestin expression and an increase insulin production,
indicating that differentiated insulin-secreting pancreatic islet
cells are produced. Further characterization can be accomplished
using known immunocytochemical and antibody techniques. For
example, immunocytochemical analysis of insulin-secreting
pancreatic islet cells generated in vitro reveals that the cells
also express other proteins that are associated with
naturally-differentiated pancreatic islet cells.
[0061] The invention also includes a method for producing an
isolated pancreatic islet cell from isolated marrow stromal cells.
The method comprises differentiating an isolated marrow stromal
cell in the same general manner as recited above, thereby producing
an isolated insulin-secreting pancreatic islet cell.
[0062] The differentiated insulin producing pancreatic cells
generated as described herein may be further tested to determine
their viability in vivo. The insulin producing islet cells may be
transplanted, using sterile technique and known and accepted
procedures, into individual experimental animals, typically
implanted beneath the kidney capsule as disclosed in U.S. Pat. No.
6,001,647.
[0063] The invention further includes a method of treating a human
patient having a disease, disorder, or condition of the pancreatic
endocrine system by administering the differentiated
insulin-secreting pancreatic islet cells of the invention to the
body of the patient.
[0064] The methods disclosed herein encompass introduction of
differentiated insulin-secreting pancreatic islet cells and/or
other endodermal cells in cell-based therapeutics where a patient
is in need of the administration of such cells. The differentiated
cells are preferably administered to a human. When isolated
insulin-secreting pancreatic islet cells are administered to a
patient with diabetes, the insulin-secreting pancreatic islet cells
will beneficially influence cells which are already present in the
pancreatic system. For example, insulin-secreting pancreatic islet
cells which are introduced into the body of the patient may be used
to replace non-functioning and/or damaged pancreatic islet
cells.
[0065] Thus, the methods disclosed herein induce marrow stromal
cell differentiation into endodermal cells, particularly
insulin-secreting pancreatic islet cells. Such methods are crucial
in the development of cell-based therapeutics for treatment of
diabetes. Indeed, prior to the present invention, the lack of
sources of pancreatic cells that can be introduced into the
pancreas of a human patient, has severely impeded the development
of Type I and Type II diabetes therapeutics. The adult marrow stem
cells constitute a unique and novel source for the production of
pancreatic cells for the treatment of diabetes.
[0066] The cells produced by the methods disclosed herein are
useful in the treatment of Type I diabetes which afflicts two
million patients due to pancreatic islet cell damage and requiring
insulin injection. It is estimated that thirty-five million
patients with Type II diabetes associated with non-responsiveness
of target tissues to insulin are treated by insulin injection, a
significant portion of which may be replaced through the use of the
generated pancreatic islet cells.
[0067] The ability to grow functioning islet cells in vitro from
the bone marrow stromal cells of an individual represents a major
technical breakthrough and facilitates the use of new strategies
for treating and studying insulin-dependent diabetes. For example,
in accordance with the subject invention, new cultured islets from
diabetic individuals can be implanted in a patient as a way to
control or eliminate the patient's need for insulin therapy because
the cultured islets and/or islet cells are able to produce insulin
in vivo.
[0068] Induction of Other Endodermal Phenotypes
[0069] Additional methods employ cultures optimized to induce
differentiation of the nestin-positive endodermal/neuronal
precursor cells of the invention into other cells of endodermal
lineage including but not limited to gut epithelium and liver
cells.
[0070] By employing various combinations of factors at different
stages, different seeding densities and at different times, MSC
cultures may be optimized to preferentially induce differentiation
to various desired cell fates. In addition, factors produced by the
MSC cultures in the course of differentiation which augment growth
of a particular cell type can be isolated, sequenced, cloned,
produced in mass quantities, and added to MSC cultures to
facilitate growth and targeted differentiation of those cultures.
The relevant factors are identified by concentrating MSC culture
supernates from early, intermediate and late stages of
differentiation and testing for the ability of these concentrates
to augment MSC growth and differentiation into the target
population. Positive effects for the desired cell type are
correlated with molecular constituents in the concentrates by
two-dimensional gel electrophoresis of positive and negative
supernates, purification and N-terminal sequencing of spots present
only in the positive concentrates and subsequent cloning and
expression of the genes encoding these factors.
[0071] The invention further includes a method of treating a human
patient having a disease, disorder, or condition associated with
tissues of endodermal origin by administering particular
differentiated endodermal cells of the invention to the body of the
patient. The methods disclosed herein encompass introduction of
differentiated endodermal cells in cell-based therapeutics where a
patient is in need of the administration of such cells. The
differentiated cells are preferably administered to a human. The
precise site of administration of the differentiated cells will
depend on any number of factors, including but not limited to, the
site of a lesion to be treated, the type of disease being treated,
the age of the human and the severity of the disease, and the like.
Determination of the site of administration is well within the
skill of the arstisan versed in the administration of cells to
animals.
[0072] As a result, these MSC derived precursor cells create a
potential therapeutic treatment for a variety of other diseases
associated with of endodermal origin, including but not limited
inflammatory bowel disease, stomach cancer, colon cancer,
colo-rectal cancer and liver disease. Determination of the site of
administration is well within the skill of the artisan versed in
the administration of cells to mammals. Cells may be introduced by
direct injection, by using a shunt, or by any other means used in
the art for the introduction of compounds into any of several organ
systems.
[0073] Tissue Engineering
[0074] The subject invention also greatly facilitates genetic
engineering of endodermal cells, particularly islet cells, to
resist subsequent immunological destruction. For example, cultured
islet cells can be transformed to express a protein or peptide
which will inhibit or prevent the destructive immune process. Other
useful proteins or peptides may be expressed. In addition,
expression of specific autoantigens, such as GAD, 64 kD islet cell
surface antigens (see Payton et al., 1995), or any other markers
identified on the differentiated pancreatic cells, can be
eliminated by standard gene knock-out or selection procedures to
produce differentiated pancreatic cells which are not susceptible
or are less susceptible to auto-immune attack. Methods for
producing such mutant or knock out cell lines are well known in the
art and include, for example, homologous recombination methods
disclosed in U.S. Pat. Nos. 5,286,632; 5,320,962; 5,342,761; and in
WO 90/11354; WO 92/03917; WO 93/04169; WO 95/17911, all of which
are herein incorporated by reference for this purpose.
[0075] Moreover, the subject invention contemplates the in vivo use
of in vitro grown MSCs to produce pancreas-like structures or an
"ecto-pancreas" organ that exhibits functional, morphological and
histological characteristics similar to those observed in a normal
pancreas. Thus, the ability to produce a functional "ecto-pancreas"
in vivo from in vitro grown pancreatic cells can be used to treat,
reverse or cure a wide variety of pancreatic diseases.
[0076] Definitions
[0077] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e. to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0078] As used herein, "stromal cells", "isolated marrow stromal
cells" and "MSCs" are used interchangeably and are meant to refer
to the small fraction of cells in bone marrow which can serve as
stem cell-like precursors of osteocytes, chondrocytes, adipocytes
and various other cell types and which are isolated from bone
marrow by their ability adhere to plastic dishes. Marrow stromal
cells may be derived from any animal. In some embodiments, stromal
cells are derived from primates, preferably humans.
[0079] As used herein, the term "isolated" referring to a cell is
meant to refer to a cell which has been separated from its natural
environment. This term includes gross physical separation from its
natural environment, e.g., removal from the donor animal, e.g., a
rat or human. When used to refer to a population of cells, the term
"isolated" includes populations of cells which result from
proliferation of the isolated cells of the invention. For uses
requiring a pure population of the endodermal cells produced by the
present invention, particularly insulin-secreting pancreatic islet
cells, the differentiated cells of the invention can be isolated
from a mixture of cells by several methods known in the art.
[0080] As used herein, the term "anti-oxidant" is meant to refer to
those substances that inhibit oxidation or reactions promoted by
oxygen or peroxides. Examples of anti-oxidants include, but are not
limited to, beta-mercaptoethanol, dimethylsulfoxide, butylated
hydroxytoluene, butylated hydroxyanisole, ascorbic acid,
dimethylfumarate, and n-acetylcysteine.
[0081] As used herein, the terms "beneficial protein or peptide"
and "therapeutic protein or peptide" are used interchangeably and
are meant to refer to a protein or peptide, for example insulin,
which can compensate for the protein encoded by a defective gene
and/or insufficient gene expression that is causally linked to the
disease or symptoms of the disease, disorder or condition
characterized by a gene defect. The presence of the protein
alleviates, reduces, prevents or causes to be alleviated, reduced
or prevented, the causes and/or symptoms that characterize the
disease, disorder or condition.
[0082] As used herein, a disease, disorder or condition which can
be treated with a beneficial or therapeutic protein or peptide is
meant to refer to a disease, disorder or condition that can be
treated or prevented by the presence of a protein which alleviates,
reduces, prevents or causes to be alleviated, reduced or prevented,
the causes and/or symptoms that characterize the disease, disorder
or condition. Diseases, disorders and conditions which can be
treated with a beneficial protein or peptide include diseases,
disorders and conditions characterized by a gene defect as well as
those which are not characterized by a gene defect but which
nonetheless can be treated or prevented by the presence of a
protein which alleviates, reduces, prevents or causes to be
alleviated, reduced or prevented, the causes and/or symptoms that
characterize the disease, disorder or condition.
[0083] The term "isolated nucleic acid" should be construed to
refer to a nucleic acid sequence, or segment, or fragment which has
been purified from the sequences which flank it in a naturally
occurring state, e.g., a DNA fragment which has been removed from
the sequences which are normally adjacent to the fragment e.g., the
sequences adjacent to the fragment in a genome in which it
naturally occurs. The term also applies to nucleic acids which have
been substantially purified from other components which naturally
accompany the nucleic acid, e.g., RNA or DNA or proteins which
naturally accompany it in the cell.
[0084] As used herein, "transfected cells" is meant to refer to
cells to which a gene construct has been provided using any
technology used to introduce nucleic acid molecules into cells such
as, but not limited to, classical transfection (calcium phosphate
or DEAE dextran mediated transfection), electroporation,
microinjection, liposome-mediated transfer, chemical-mediated
transfer, ligand mediated transfer or recombinant viral vector
transfer.
[0085] The term "differentiation" as used herein, should be
construed to mean the induction of a differentiated phenotype in an
undifferentiated cell by coculturing the undifferentiated cell in
the presence of a substantially homogeneous population of
differentiated cells, in the presence of products of differentiated
cells or in the presence of an inducer of cell differentiation.
[0086] The term "endodermal cell" as used herein should be
construed to mean a cell expressing a phenotype characteristic of a
cell normally associated with a tissue derived from the endodermal
embryonic germ layer.
[0087] The term "endodermal/neuronal precursor cell" as used herein
should be construed to mean an MSC partially differentiated such
that it expresses nestin and has the potential to further
differentiate into an endodermal and/or a neuronal cell
phenotype.
[0088] The term "endodermal/neuronal precursor-inducing compound"
as used herein is meant to refer to those compounds capable of
inducing differentiation of a stromal cell into a nestin-positive
endodermal/neuronal precursor cell.
[0089] The term "insulin-secreting pancreatic islet cell" as used
herein should be construed to mean an MSC differentiated such that
it expresses the pancreatic islet marker insulin.
[0090] The term "neuron" as used herein should be construed to mean
a nerve cell capable of receiving and conducting electrical
impulses from the central nervous system. A nerve cell or "neuron"
typically comprises a cell body, an axon, axon terminals, and
dendrites and is derived from the ectodermal embryonic germ
layer.
[0091] The term "endodermal differentiation-inducing compound" is
meant to refer to those compounds capable of inducing
differentiation of a nestin-positive endodermal/neuronal precursor
cell into an endodermal cell. These compounds include, but are not
limited to antioxidants, trophic factors, and growth factors.
[0092] The term "pancreatic islet differentiation-inducing
compound" is meant to refer to those compounds capable of inducing
differentiation of a nestin-positive endodermal/neuronal precursor
cell into a pancreatic islet cell. These compounds include, but are
not limited to antioxidants, trophic factors, and growth
factors.
[0093] The invention is further described in detail by reference to
the following specific examples. These examples are provided for
purposes of illustration only, and are not intended to be limiting
unless otherwise specified. Thus, the invention should in no way be
construed as being limited to the following examples, but rather,
should be construed to encompass any and all variations which
become evident as a result of the teaching provided herein.
EXAMPLES
[0094] In a particular embodiment set forth as follows, adult rat
stromal cells are expanded as undifferentiated cells in culture for
a sufficient number of passages to indicate their proliferative
capacity. This exemplary treatment protocol induces the stromal
cells to exhibit a pancreatic phenotype including pancreatic islet
cell morphology and expression of various pancreatic-specific
markers.
[0095] Human marrow stromal cells treated using the novel protocol
exemplified herein differentiate into pancreatic cells similarly to
rat MSCs. Thus, the protocol is not limited to rodent stromal
cells. By use of the strategies exemplified herein, mammalian
marrow stromal cells can be induced to differentiate into
insulin-secreting pancreatic islet cells.
[0096] Cultures
[0097] Rat MSCs are originally cultured in alpha-Modified Eagle's
Medium (alpha-MEM) supplemented with 20% FBS, 2 mM L-glutamine, 100
units per milliliter penicillin, 100 milligrams per milliliter
streptomycin and 25 nanograms per milliliter amphotericin B. For
each passage the cells are plated at about 8,000 cells per square
centimeter and grown to confluency. At passage 6 the cells are
transferred to DMEM (pH 8.0)/20% FBS without additional
supplementation, and maintained beyond passage 14. Subconfluent
cultures of rat and human MSCs are maintained in DMEM/20% FBS.
[0098] Twenty-four hours before induction of partial
differentiation to nestin-positive precursor cells, medium is
replaced with a pre-induction medium consisting of DMEM, 20% FBS
and 1 millimolar B -mercaptoethanol. The cells are then transferred
to a serum free endodermal/neuronal precursor induction medium
composed of DMEM and 1-10 millimolar 13-mercaptoethanol. Under
these circumstances, the cells begin to express nestin indicating
that they are partially differentiated, potentially endodermal
and/or neural precursors. To effect further differentiation to
pancreatic islet cell phenotype, the partially differentiated
nestin-positive endodermal/neuronal precursor cells are transferred
to serum-free DMEM containing 1-10 mM B-mercaptoethanol with and
without 10 nanograms/ml basic fibroblast growth factor (bFGF).
[0099] In parallel preparations, the precursor induction medium is
composed of DMEM and 200 micromolar butylated hydroxyanisole and
the nestin-positive endodermal/neuronal precursor cells are
transferred to serum-free DMEM containing 1-10 mM butylated
hydroxyanisole with and without 10 nanograms/ml bFGF.
[0100] Pancreatic/Neuronal Precursor Characterization
[0101] rMSCs are initially maintained in sub-confluent cultures in
pre-induction media supplemented with 1 mM beta-mercaptoethanol
(BME) for 24 hours. Under these conditions no changes in morphology
are evident. To effect differentiation into pancreatic/neuronal
precursor cells, the cells are transferred to serum-free medium
containing 1-10 millimolar BME (SFM/BME). Within 5 hours of
exposure to SFM/BME the cells begin to express nestin.
[0102] Insulin-Secreting Pancreatic Islet Cell Characterization
[0103] Nestin-positive endodermal/neuronal precursor cells from the
precursor induction medium are transferred to serum free medium
containing 1-10 mM mercaptoethanol with bFGF (SFM/BME/bFGF) and
without bFGF (SFM/BME). The expression of nestin decreases over
time as the cells mature and eventually differentiate to
insulin-secreting pancreatic islet cells. This is associated with a
concomitant increase in insulin secretion by the differentiated
cells.
[0104] To examine the effects of different anti-oxidants in
mediating the induction of differentiation in MSCs to
endodermal/neuronal progenitors, rMSCs may be treated with other
anti-oxidants, e.g., dimethylsulfoxide (DMSO), butylated
hydroxyanisole (BHA), or butylated hydroxytoluene (BHT), ascorbic
acid, dimethylfumarate, n-acetyleysteine, and the like, both alone
and in combination with each other.
[0105] Clonal Analysis
[0106] To determine whether individual rMSCs exhibit stem cell
characteristics of self-renewal and pluripotentiality, individual
clones are analyzed. To establish clones, rMSCs are plated at
approximately cells per square centimeters, grown to 50-150 cells
per colony, isolated with cloning cylinders, transferred to
separate wells and eventually to individual flasks. Stem cell
characteristics are confirmed when single cells replicate as
typical rMSCs and differentiate into insulin secreting pancreatic
islets cells after treatment.
[0107] Human Stromal Cells Differentiate into Insulin Secreting
Pancreatic Islet Cells
[0108] The pancreatic and/or neuronal cell potential of MSCs is not
unique to rodents as can can be demonstrated by previous
experiments using MSCs obtained from humans (hMSCs). In those
studies, hMSCs were isolated from a healthy adult donor and grown
in vitro (Bjornson et al., 1999, Science 283:534-537). hMSCs
resembled their rodent counterparts, growing as large flat cells in
the undifferentiated state. Human marrow stromal cells subjected to
the pancreatic differentiation protocols disclosed will attain
pancreatic islet cell characteristics in a time frame similar to
that observed for rMSCs.
[0109] Western Blot
[0110] Thirty milligrams of protein extract from untreated (U), BME
induced (I), and BME/bFGF-induced (II) rMSC pancreatic cultures are
separated on a 4%-20% gradient acrylamide gel and
electrophoretically transferred to a nylon membrane. The Western
blot is probed for insulin expression using an anti-insulin
monoclonal antibody followed by secondary antibody conjugated with
horse radish peroxidase (HRP). Color development is performed using
enhanced chemiluminescence reagents.
[0111] The blot is then stripped and probed for nestin expression
using anti-nestin polyclonal antibody. Again, the secondary
antibodies are BRP-conjugated, and color is developed using ECL
reagents.
[0112] Immunocytochemistry
[0113] Cultured rMSCs are fixed with 4% paraformaldehyde, incubated
with primary antibody overnight at 4.degree. C., incubated with
secondary antibody for one hour, followed by exposure to
avidin-biotin complex for one hour at 25.degree. C.
Diaminobenzidene (DAB) serves as chromogenic substrate for HRP.
[0114] FM1-43 Labeling
[0115] Cultures are treated with DMSO/BHA in serum-free media (SFM)
for approximately 4 hours. The cells are maintained for an
additional 30 minutes in artificial cerebral spinal fluid
(aCSF)/BHA. Cells are labeled in aCSF containing I millimolar
FM1-43 and 75 mM KCI for 60 seconds. The labeling mixture is
removed, the cultures are washed twice with aCSF, and the cells are
incubated in aCSF for 60 minutes to reduce background staining.
Cultures are fixed with 4% paraformaldehyde, and soaked for 24
hours in phosphate buffered saline (PBS) before analysis.
[0116] Stromal Cell Characterization
[0117] Rat mesenchymal stromal cells (rMSCs) are isolated from the
femurs of adult rats and propagated in vitro (Azizi et al., 1998,
Proc. Natl. Acad. Sci. USA 95:3908-3913). The data disclosed from
previous experiments shown in FIG. 9A demonstrate that the
distribution of cells stained with antibody to CD19B (unfilled)
does not differ from that of isotype control (filled), indicating
the rMSC cultures do not contain significant numbers of
contaminating CD11b-expressing cells. The absence of hematopoietic
precursors in the stromal cell cultures prepared according to the
method of the invention may also be verified. FIG. 9B also
demonstrates that in previous studies, the intensity of staining
does not differ between CD45 antibody (unfilled) and control
(filled) profiles, indicating that rMSCs cultured according to the
present invention methods are not contaminated by CD45-expressing
cells. Fluorescent cell sorting at passage 1 also demonstrates that
the cells are negative for CD11b (FIG. 9A), and CD45 (FIG. 9B),
which are cell surface markers associated with lymphohematopoietic
cells. Rat MSCs also express CD90 (FIG. 9C), consistent with their
undifferentiated state.
[0118] At the outset of the pancreatic differentiation disclosed
elsewhere herein, untreated rMS Cs are further characterized by
staining for the cell surface markers CD44 and CD71. Cells positive
for CD44 and CD71 expression, are consistent with previous reports
(Pittenger et al., 1999, Science 5 284:143-147; Bruder et al.,
1998, Clin. Orthop. Relat. Res. 355S:S247-S256). To the best of
Applicants' knowledge, this is the first report that peripheral
mesenchymal cells can differentiate into cells of endodermal
lineage in vitro. In particular, the present invention provides
methods of directing differentiation of MSCs into insulin producing
pancreatic islet cells in vitro. MSCs are useful in the treatment
of a wide variety of diseases disorders and conditions, and these
cells offer significant advantages over other so-called "stem"
cells. That is, bone marrow cells are readily accessible and
provides a renewable population which can be expanded in vitro
thereby allowing complex gene manipulations to be performed for ex
vivo gene therapy and/or for cell therapy for endodermal diseases,
disorders or conditions that require administering cells to the
site of an endodermal organ. Furthermore, autologous
transplantation overcomes the ethical and immunologic concerns
associated with the use of fetal tissue. Moreover, MSCs grow
rapidly in culture, precluding the need for immortalization, and
are capable of differentiating into cells normally derived from
multiple lineages including insulin producing pancreatic islet
cells produced according to the protocols disclosed herein.
[0119] The following observations suggest that untreated MSCs are
"multidifferentiated" and that further differentiation to specific
phenotypes, for example neuronal or pancreatic phenotypes,
comprises quantitative modulation of gene expression rather than
simple on-off switching of phenotypically specific genes.
[0120] MSCs normally differentiate into bone, cartilage, muscle,
tendon and fat, classical mesenchymal derivatives (Owen, 1988;
Beresford, 1989; Young et al., 1998; Pittenger et al., 1999). We
recently found that MSCs can be induced to overcome their
mesenchymal fate and differentiate into neurons in vitro (Woodbury
et al., 2000). Treatment with a relatively simple, fully defined
medium elicited neuronal differentiation of approximately 80% of
the cells. Within five hours, the MSCs formed characteristic
refractile spherical cell bodies, extended typical long neuritic
processes exhibiting terminal expansions and filopodia. The treated
MSCs expressed the neuroepithelial gene product, nestin,
transiently, the neuronal products neurofilament M (NF-M), tau and
Neu-N de novo, and increased expression of neuron-specific enolase
(NSE). These unanticipated observations indicate that MSCs may be
useful in the treatment of a number of neurological diseases, and
raise basic questions regarding stem cell biology and mechanisms of
differentiation. In addition, the discovery that developing islet
cells also express nestin (H. Zulewski et al., 2001, Diabetes 50:
521) points to the potential of MSCs as sources of insulin
producing pancreatic islet cells.
[0121] MSCs Express Diverse Genes
[0122] To begin characterizing the undifferentiated MSCs more
fully, we examined expression of a representative spectrum of genes
that included those specific for diverse lineages. Our initial
microarray screen of cultured dissociates revealed that the MSCs
expressed many genes associated with differentiated cells. The
expressed genes were not limited to mesodermal lineages, but
included genes representative of all germ layers.
[0123] To confirm this unexpected observation, we assayed
transcript levels by RT-PCR, focusing on prototypical genes
specific for different germ layers. In addition to expression of
expected mesodermal messages, such as SM22.alpha., RT-PCR revealed
mRNA for endodermal ceruloplasmin, ectodermal syntaxin, aldolase C,
glutamate receptor binding protein and APP (amyloid precursor
protein), and germline protamine2 (FIG. 1; Table 1). Omission of
RTase in controls eliminated PCR products, confirming that the
signals observed in the experimental groups were derived from RNA
transcripts and not contaminating genomic DNA (FIG. 1).
[0124] Expression of additional mRNA species identified by
microarray, including mesodermal myosin and leptin, and neural
N-methyl-D-aspartate receptor subunit 1 (NMDA RI) was also
confirmed by RT-PCR.
[0125] Gene Expression During Neuronal Differentiation
[0126] While the expression of diverse gene products characteristic
of distinct lineages by "undifferentiated" MSCs was unexpected, it
may reflect extensive plasticity intrinsic to this stem cell
population. Differentiation to specialized cell types, in turn,
might be expected to differentially alter transcription of subsets
of these messages. To examine this contention, we subjected MSCs to
the neuronal differentiation protocol, harvested RNA after 48
hours, and assessed expression of the foregoing prototypical target
genes by RT-PCR. In fact, neuronal differentiation significantly
altered the pattern of gene expression. Induction decreased the
transcription of germline protamine2 to undetectable levels.
Similarly, endodermal ceruloplasmin dramatically decreased. Perhaps
unexpectedly, NMDA receptor binding protein also decreased.
Expression of the neural genes aldolase C and syntaxin exhibited
modest decreases, as did muscle-specific SM22.alpha.. Among the
genes surveyed, only APP expression remained unchanged as the MSCs
assumed neuronal morphologic characteristics, allowing this signal
to serve as an internal control, confirming that equal amounts of
cDNA were present in each reaction.
[0127] We had previously found that the neuron-specific
intermediate filament, NF-M is expressed by MSC-derived neurons,
but not by undifferentiated MSCs (Woodbury et al., 2000). Here, we
employed NF-M expression as a temporal, quantitative index of
neuronal differentiation to help place the present observations in
context. NF-M expression is associated with initiation of
neuritogenesis, neural process outgrowth and assumption of the
characteristic mature neuronal morphology (Carden et al.,
1987).
[0128] MSCs maintained in serum free medium for 24 hours exhibited
extremely low, but detectable levels of NF-M mRNA by RT-PCR,
consistent with adoption of neuronal morphologies by a small number
of cells under these conditions. However, incubation with the
neural induction medium for 24 hours greatly enhanced NF-M message
levels, consistent with differentiation of the vast majority of
MSCs into neurons (FIG. 3A). As a positive control, we detected
NF-M in the cerebellum of 32-day-old rats, generating a single band
of the expected size (FIG. 3A). The encephalizing gene noggin
(Smith and Harland, 1992) was unchanged 24 hours after neural
differentiation, establishing the specificity of the increase in
NF-M (FIG. 3). To define the temporal profile of differentiation,
we examined NF-M transcript levels 5 and 24 hours after initiation
of induction. NF-M was undetectable after 5 hours of incubation in
serum free medium, and was just above the level of detection after
5 hours in neuronal induction medium (FIG. 3B). Transcript
abundance increased dramatically at 24 hours, consistent with
previous observations. In aggregate, our observations indicate that
assumption of neuronal morphologies by MSCs is accompanied by a
dramatic increase in the prototypical neuronal gene, NF-M, complex
modulation of other neuronal genes, and decreased transcription of
germline and mesodermal genes.
[0129] Expression of Neuroglial Genes
[0130] Having established the utility of RT-PCR in defining the
unexpected expression of diverse differentiated genes in the
"undifferentiated" MSCs and in neuronal differentiation, we
examined transcriptional regulation of genes specific to the
neuroglial lineage. APP served as a control, ensuring that equal
amounts of target cDNA were used in each reaction (FIG. 4). We
examined GFAP (glial fibrillary acidic protein), the classical
astrocyte marker (Eng et al., 197 1), which is also expressed in
neuroglial precursor cells (Laywell et al., 2000; Doetsch et al.,
1999). Uncommitted MSCs expressed GFAP (FIG. 4A). With neuronal
differentiation, the gene product was detectable at 24 hours, but
48 hours post-neuronal induction was no longer discernible,
consistent with neuronal, but not glial differentiation.
Nevertheless, expression of GFAP by the MSCs is consistent with a
growing body of evidence indicating that neural precursors in vivo
express neuronal and glial markers and can differentiate into
either lineage.
[0131] To begin assessing mechanisms regulating neuronal
differentiation, we examined NeuroD, a transcription factor
transiently expressed in neuronal precursor cells, known to
regulate neuronal fate decisions (Lee, 1997; Morrow et al., 1999).
Robust expression of NeuroD was detected in the uncomitted MSCs,
suggesting that these undifferentiated cells were already "primed"
for neural differentiation (FIG. 4A). With differentiation, NeuroD
progressively decreased, and was markedly diminished by 48 hours,
consistent with transient expression of the transcription factor in
differentiating neurons (Lee et al., 1995).
[0132] We used NF-M and tau as neuron-specific marker prototypes to
examine translation with neuronal differentiation. Consistent with
our previous findings, NF-M mRNA was undetectable in uncommitted
MSCs, but was present after 48 hours of neuronal induction. Tau
transcripts were not detectable in the uncommitted MSCs, but were
present in MSC-derived neurons (bracketed bands) consonant with
early stages of neuronal differentiation. This finding is
consistent with our previous studies indicating that tau is not
expressed by MSCs but is present in MSC-derived neurons (Woodbury
et al., 2000). Analysis of the tau transcripts revealed that the
known tau mRNA isoforms, generated by alternative splicing of exons
2 and 3 (Goedert et al., 1991), were present in the MSC-derived
neurons (FIG. 4B). Collectively, these data indicate that
incubation in NIM decreases the expression of neural precursor
messages (GFAP, NeuroD), while simultaneously increasing the
expression of specific neuronal markers (NF-M, tau), consistent
with ongoing neuronal differentiation.
[0133] Expression of tau was confirmed at the protein level
immunocytochemically. MSC-derived neurons, maintained in NIM for 10
days were probed with anti-tau antibody. There was significant
heterogeneity in the level of tau expression in the neurons even
after 10 days, which often correlated with the degree of neuronal
morphologic differentiation. For example, abundant intensely
tau-positive neurons (>) with long varicose (arrows) processes
were evident, whereas neighboring cells exhibiting immature,
transitional morphologic features displayed weak staining (FIG.
5A). Heterogeneity of staining is further exemplified in FIG. 5B.
Cells in the field exhibit neuronal morphological characteristics
and definitive tau positivity, yet staining intensity varies from
cell to cell. Many cells in this culture (>) have elaborated
long processes which terminate in growth cone-like structures
(arrow).
[0134] We examined additional neuronal gene products that are
expressed in more mature neurons or that are associated with
functional neuronal communication. At 10 days, the MSC-derived
neurons uniformly expressed TOAD-64, a neuron-specific protein
thought to play a role in axonal pathfinding (Minturn et al., 1995)
(FIG. 6A). Similarly, .beta.-tubulin 111, an intermediate filament
characteristic of mature neurons (Menezes and Luskin, 1994) was
present in virtually all cells (FIG. 6B). In contrast to these
neuronal markers, O-4, a classic oligodendrocyte gene product, and
MBP, a marker for mature oligodendrocytes, were not detectable in
MSC-derived neurons (data not shown).
[0135] Genes Associated with Neurotransmission
[0136] To begin assessing the developing ability for functional
communication, we initially examined synaptophysin, which is
associated with synaptic vesicles and transmission. The protein was
detected in cell bodies as well as varicose, putative transmitter
release sites along processes, reflecting an immature pattern of
distribution (FIG. 6). Analysis by RT-PCR indicated that
synaptophysin m. RNA was not present in undifferentiated MSCs, but
was detectable after 24 hours of neuronal differentiation, and
continued to increase thereafter (48 hrs.) (FIG. 6D).
[0137] To further assess the developing capability for
communicative function, we examined the expression of
neurotransmitter enzymes. At 10 days, a large population of the
neurons expressed choline acetyltransferase (ChAT), which catalyzes
the synthesis of the excitatory transmitter, acetylcholine (FIGS.
7A, 7B). A similar percentage of ChAT-positive cells was seen when
a monoclonal ChAT antibody from a different commercial source was
used for staining, validating this staining pattern (data not
shown). Interestingly, the majority of neurons (>85%) derived
from the multipotent P19 embryonal carcinoma cell line are also
cholinergic (Parnas and Linial, 1995). A smaller subpopulation of
MSC-derived neurons expressed tyrosine hydroxylase, the
rate-limiting enzyme in the synthesis of catecholamines, dopamine,
norepinephrine and epinephrine (FIGS. 7C, 7D). In aggregate, these
observations indicate that the MSC-derived neurons were developing
the structural apparatus for synaptic communication and the
machinery for transmitter signal biosynthesis. The expression of
enzymes for different transmitters suggests that the MSC-derived
neurons are capable of differentiating into multiple subtypes.
[0138] Morphologic Reversion and Gene Expression
[0139] Neuronal differentiation of MSCs exhibited notable
plasticity at its earliest stages, when it was partially
reversible. Cells exposed to NIM assumed characteristic neuronal
morphologies, displaying refractile cell bodies and long processes
by 24 hours post-induction (FIG. 8A). Withdrawal of NIM from
MSC-derived neurons elicited process retraction and reversion of
morphology within 24 hours to flat cells, which shared
characteristics with uncommitted MSCs but generally displayed a
more stellate morphology (FIG. 8B). To assess changes in gene
expression associated with reversion, MSCs were differentiated to
neurons by exposure to NIM for 24 hours. At this time, NIM was
removed from half of the cells and replaced with SFM, while the
remaining cells were maintained in NIM. Twenty-four hours later (48
hours in total) we harvested RNA from neuronal and reverted cells
and assessed changes in gene expression by RT-PCR. We again assayed
expression of the archetypal targets from various germ layers
expressed at high levels in uncommitted MSCs. Reversion from the
neuronal to MSC phenotype was associated with striking changes in
expression of a subset of the target genes. Germline protamine2
mRNA, which decreased dramatically with neuronal conversion,
increased markedly in the reverted MSCs. Similarly, endodermal
ceruloplasmin message increased in MSCs from depressed levels in
the neurons. Strikingly, NeuroD mRNA reappeared in the MSCs,
presumably indicating the potential for re-differentiation into
neurons. GFAP message was also re-expressed, consistent with,
reversion to a primitive neuroglial precursor state. In sum, the
reverted MSCs appear to re-express multipotentiality, consistent
with plasticity at the time of differentiation. In contrast,
expression of several genes was unchanged by reversion to MSCs;
message levels for APP, muscle specific SM22 cc (FIG. 8C),
syntaxin, and aldolase C (data not shown) remained the same.
[0140] Multidifferentiation of MSCs
[0141] Far from being undifferentiated, blank slates, the MSCs
actively transcribe genes specific for all the classical embryonic
germ layers. As expected, the stromal cells express prototypical
mesodermal genes, including SM22a, myosin and leptin. In addition,
however, MSCs express protamine2, which is germline-specific
(Domenjoud et al., 1991), indicative of an early, uncommitted
state. Simultaneously, endodermal ceruloplasmin, expressed at high
levels in the fetal and adult liver and lung (Fleming and Gitlin,
1990), is transcribed in uncommitted MSCs. Likewise, ectodermal
syntaxin 13, highly enriched in the brain (Advanti et al., 1998),
and brain-specific aldolase C (Mukai et al., 1991) are expressed by
MSCs. NMDA glutamate binding protein (Kumar et al., 1991) and APP
(Shivers et al., 1988) represent additional neural genes
transcribed by MSCs. These observations are consistent with our
previous finding that the MSCs express low levels of
neuron-specific enolase (Woodbury et al., 2000).
[0142] It may be concluded that MSCs are not "undifferentiated",
but rather "multidifferentiated". Recently Labat and co-workers
(2000) have proposed the existence of a monocytoid ectomesenchymal
stem/progenitor cell that expresses both neural and mesenchymal
gene products. Previous work by Enver and colleagues has
demonstrated that lymphohematopoietic marrow stem cells express
genes characteristic of multiple hematopoietic lineages prior to
unilineage commitment (Hu et al., 1997; Cross and Enver, 1997;
Enver and Greaves, 1998). Moreover, MSCs co-express genes specific
for a number of mesenchymal lineages, including adipocytes,
osteoblasts, fibroblasts, and muscle (Seshi et al., 2000). The
present work extends these observations, indicating that MSCs
transcribe germline, endodermal and ectodermal genes, in addition
to mesodermal genes. Our observations imply, consequently, that
genes specific for multiple lineages are accessible for
transcription in the MSCs, allowing for diverse differentiative
fates. Indeed, bone marrow cells have already been shown to give
rise to skeletal muscle, hepatocytes, glia and neurons, in addition
to the aforementioned mesenchymal derivatives (see Morrison, 2001
for review, and references therein). One might anticipate that MSCs
are capable of generating a far larger spectrum of cell types. In
the case of neuronal differentiation, prior multidifferentiation
may help elucidate aspects of the process, and begin to approach
underlying mechanisms. One striking feature of MSC neuronal
differentiation is rapidity: Within 5 hours of exposure to the
induction medium, the cells assume typical neuronal morphological
features and express a variety of neuron-specific genes (Woodbury
et al., 2000; present studies). The prior expression of neuronal
genes by the MSCs may explain this rapid response. Presumably,
quantitative alteration in genes already being transcribed obviates
the need for elaboration of new transcription factors or histone
acetylation, for example.
[0143] Expression of NeuroD by MSCs
[0144] Similarly, the expression of NeuroD by the MSCs may also
account for the speed of differentiation, and may provide insight
into regulatory mechanisms. NeuroD family members, bHLH
transcription factors, are transiently expressed in neuronal
precursors, and initiate neuronal differentiation (Lee, 1997).
These factors appear to function as master regulators of mammalian
neurogenesis, as transfection of murine embryonic carcinoma cells
with NeuroD2 transcripts initiates neural differentiation in
non-neural cells (Farah et al., 2000). In the neural retina, NeuroD
plays a role in multiple developmental functions, including retinal
cell fate determination, differentiation and neuron survival
(Morrow et al., 1999). In this model system NeuroD induces
withdrawal from the cell cycle, regulates neuronal vs. glial cell
fate decisions, and favors amacrine vs. bipolar differentiation.
The expression of NeuroD by MSCs and its decrease with neuronal
differentiation is consistent with a role in stromal conversion to
neurons, a contention that we are presently examining
experimentally. Moreover, the reappearance of NeuroD in neurons
that have reverted to the MSC phenotype is particularly
provocative, suggesting that the neuronal potential is an intrinsic
property even of stromal stem cells de-differentiated from
neurons.
[0145] MSCs as Neuroglial Precursors
[0146] Unexpectedly, the undifferentiated MSCs expressed glial as
well as neuronal genes. GFAP, the traditional astrocytic marker, is
expressed in the MSCs, but decreased with neuronal differentiation.
The gene product was detectable 24 hours after neural induction,
but by 48 hours was undetectable, consistent with neuronal, but not
glial differentiation. Consequently, neuronal differentiation of
MSCs exhibits commonalities with neuronal differentiation in the
normal adult rodent brain in which neurons derive from neuroglial
precursors that express GFAP as well as neuronal characters
(Laywell et al., 2000; Doetsch et al., 1999). Although the MSCs
expressed the astrocyte marker GFAP, 0-4, a traditional
oligodendrocyte gene product, and MBP (myelin basic protein),
specific for mature oligodendrocytes, were not present. These
observations complement our previous work, demonstrating that the
primitive intermediate filament, nestin, characteristic of
neuroepithelial precursors, is expressed in MSC-derived neurons at
5 hours, but decreases progressively, and is undetectable 6 days
after neuronal differentiation, mimicking normal neuronal
differentiation in vivo (Woodbury et al., 2000). We tentatively
conclude that neuronal differentiation from MSCs exhibits many
sequential features of normal neuronal differentiation in vivo.
[0147] Plasticity of MSCs and Neurons
[0148] Plasticity is apparently maintained for a period of time
after MSCs differentiate into neurons. Removal of the neural
inducing medium after initial conversion at 48 hours resulted in
the striking reversion of neurons to the MSC phenotype within 24
hours. Neuritic processes rapidly contracted and disappeared, cell
bodies lost refractility and flattened, and the cells assumed a
typical MSC morphology. With reversion, germline protamine2 was
re-expressed after becoming undetectable in the neurons. Endodermal
ceruloplasmin increased significantly in the reverted MSCs. Most
dramatically, in the present context, NeuroD mRNA reappeared in the
reverted MSCs, after having disappeared 48 hours after neuronal
differentiation, suggesting that the revertants retained the
potential for re-differentiation. The reverted MSCs also
re-expressed GFAP mRNA after disappearance in the neurons,
suggesting that the MSCs have, indeed, regressed to a primitive
neuroglial precursor state. The re-expression of a number of
functionally critical MSC genes indicates that the cells revert to
the multidifferentiated, multipotential state. Our More generally,
these observations indicate that fate determination and
differentiation are not necessarily irrevocable and unidirectional,
but rather may be multidirectional under appropriate
circumstances.
[0149] Materials and Methods
[0150] Marrow Stromal Cell Isolation and Culture. MSCs were
isolated from the femurs of adult rats as previously described
(Azizi et al., 1998) and maintained in Dulbecco's Modified Eagles
Medium (DMEM, Life Technologies) supplemented with 20% Fetal Bovine
Serum (FBS, Atlanta Biologicals).
[0151] Neuronal Induction. Neuronal differentiation was performed
as described (Woodbury et al., 2000) with modification. Briefly,
prior to neuronal induction rMSCs were grown in DMEM, 20% FBS, 10
ng/ml bFGF. The monolayer was rinsed twice with PBS and transferred
to Neuronal induction media (NIM) consisting of 100 gM BHA, 10 gM
forskolin, 2% DMSO, 5 U/ml heparin, 5 nM K252a, 25 mM KCI, 2 mM
valproic acid, 1.times.N2 supplement (Life Technologies), 10 ng/ml
bFGF, 10 ng/ml PDGF in a base of DMEM. After induction cells were
maintained at 30.degree. C. without further additions. For
reversion studies NIM was removed and replaced with unsupplemented
DMEM.
[0152] RNA Isolation. RNA was isolated from induced and control
rMSCs using Trizol reagent according to manufacturer's
recommendations (Life Technologies). The resulting RNA pellet was
subjected to a chloroform extraction and two ethanol
precipitations. Yield was determined spectrophotometrically.
[0153] cDNA Synthesis. Two .mu.g of RNA were reverse-transcribed
using Superscript 11 Reverse Transcriptase (Life Technologies) in a
50 .mu.l volume containing 1 .mu.g oligo dT primer, 200 .mu.M
dNTPs, and buffers supplied by the manufacturer. The reaction was
carried out in a Perkin-Elmer 9600 PCR machine with the following
parameters: 25.degree. C., 5 minutes; 37.degree. C., 5 minutes;
42.degree. C., 60 minutes; 48.degree. C. 10 minutes. A 5 minute
ramp time was employed between each temperature. In control
reactions the Superscript 11 reverse transcriptase was omitted.
[0154] Polymerase Chain Reaction. 2 to 5 .mu.l of cDNA target was
amplified by PCR using specific primer pairs (listed below and in
Table 1). Tfl polymerase (Epicentre) and PCR Optimization Kit
(Epicentre) were utilized following manufacturer's recommendations.
To ensure equal distribution of target a master mix containing all
components except specific primers was generated and then aliquoted
to each reaction tube. PCR reactions were performed in a
Perkin-Elmer 9600 as follows. Initial 3 minute denaturing step at
92.degree. C., followed by 30-35 cycles of 94.degree. C., 5
seconds; 55-65.degree. C., 10 seconds, 68.degree. C., 30 seconds;
74.degree. C, 30 seconds. All reactions were performed in 20 .mu.gl
volume.
1 TABLE 1 Neurofilament-M (NF-M) Accession # Z12152 F:
AGGTGGCCTTCCTGCGGAGCAATC SEQ. ID NO.1 R: GCCTCAGGAGACTTCACGGGAGAC
SEQ. ID NO.2 tau Accession#X79321 F: GGCTTTGAAGCAGCATGGCTGAAC SEQ.
ID NO.3 R: GGCCTGATCACAAACCCTGCTTGG SEQ. ID NO.4 NeuroD Accession
#D82945 F: TGACCAAATCATACAGCGAGAGC SEQ. ID NO.5 R:
AGAAGTTGCCATTGATGCTGAGCG SEQ. ID NO.6 GFAP Primer sequences were
based on those re- ported in Condorelli et al. (1999) F:
GAGACGTATCACCTCTGCAC SEQ. ID NO.7 R: GGAAGCAACGTCTGTGAGGT SEQ. ID
NO.8 SE- GENE/ SYM- LAY- QUENCE ACCESS# BOL PRIMERS ER ID CERULO-
CERU F: CTACAGTTGCTCCAACGTTGCCAGG ENDO SEQ. PLASMIN DERM ID L33869
NO.9 R: AGTAACCAGCTTCCAGGCGTTTGG SEQ. ID NO.10 SM22a SM22 F:
TCTCCTTCCAGTCCACAAACGACC MESO- SEQ. L41154 DERM ID NO.11 R:
CTTCCCTTTCTAACTGATGATCTG, SEQ. ID NO.12 PRO- PROT F:
ACTATGGITCGCTACCGAATGAGG GERMI- SEQ. TAMINE2 NAL ID X14674 NO.13 R:
ATCAACATGGAATGGTGTTGTGGC SEQ. ID NO.14 ALDO- ALDO F:
TTGGACTGAGCTACTGTCTGTTGC ECTO- SEQ. LASE C DERM ID M63656 NO.15 R:
TTTCAGCACACAGCGCCATTTGGC SEQ. ID NO.16 AMYLOID APP F:
CTCAGAGAACCCTGTGGATGTCCG ECTO- SEQ. PRE- DERM ID CURSOR NO.17
PROTEIN R: GCATCTCGCTCCAGGTATTTGTAGG SEQ. X07648 ID NO.18 NMDA GLUT
F: AGTTTCTTGGTCTCTGGGGACAGC ECTO- SEQ. GLUTA- DERM ID MATE NO.19
BINDING R: AACTGATGGTCAGGATCGACAGGG SEQ. SUBUNIT ID S61973 NO.20
SYN- SYN F: CTTCAACAGCATCATCCAGACATC ECTO- SEQ. TAXIN DERM ID
AF044581 NO.21 R: CACCTTGGTCGTGGATCATCATAGC SEQ. ID NO.22 RT-PCR
Targets and Representative Germ Layer. Gene targets amplified by
RT-PCR, Genbank accession numbers, and representative germ layer
are shown. The sequences used to generate primers were specific for
the targeted genes. The symbol used to identify targeted genes in
the accompanying figures is displayed.
[0155] Immunocytochemistry. Cells were fixed in 4% paraformaldehyde
and stored under PBS at 4.degree. C. until stained.
[0156] Primary antibodies: polyclonal anti-tau, Sigma [1:1000];
polyclonal. anti-TOAD64, Chemicon [1:5000]; monoclonal
anti-.beta.-tubulin III, Chemicon [1:200]; monoclonal
anti-synaptophysin, Chemicon [1:200]; polyclonal anti-choline
acetyltransferase, Chemicon [1:1000]; monoclonal anti-tyrosine
hydroxylase, Chemicon [1:1000]. Biotinylated secondary antibodies
and peroxidase ABC kit were obtained from Vector. CoCl.sub.2
enhanced DAB was used as the chromagen.
[0157] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety.
[0158] While the invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
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Sequence CWU 1
1
22 1 24 DNA Artificial Sequence Description of Artificial Sequence
nucleic acid primer 1 aggtggcctt cctgcggagc aatc 24 2 24 DNA
Artificial Sequence Description of Artificial Sequence nucleic acid
primer 2 gcctcaggag acttcacggg agac 24 3 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 3 ggctttgaag
cagcatggct gaac 24 4 24 DNA Artificial Sequence Description of
Artificial Sequence nucleic acid primer 4 ggcctgatca caaaccctgc
ttgg 24 5 23 DNA Artificial Sequence Description of Artificial
Sequence nucleic acid primer 5 tgaccaaatc atacagcgag agc 23 6 24
DNA Artificial Sequence Description of Artificial Sequence nucleic
acid primer 6 agaagttgcc attgatgctg agcg 24 7 20 DNA Artificial
Sequence Description of Artificial Sequence nucleic acid primer 7
gagacgtatc acctctgcac 20 8 20 DNA Artificial Sequence Description
of Artificial Sequence nucleic acid primer 8 ggaagcaacg tctgtgaggt
20 9 25 DNA Artificial Sequence Description of Artificial Sequence
nucleic acid primer 9 ctacagttgc tccaacgttg ccagg 25 10 24 DNA
Artificial Sequence Description of Artificial Sequence nucleic acid
primer 10 agtaaccagc ttccaggcgt ttgg 24 11 24 DNA Artificial
Sequence Description of Artificial Sequence nucleic acid primer 11
tctccttcca gtccacaaac gacc 24 12 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 12
cttccctttc taactgatga tctg 24 13 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 13
actatggttc gctaccgaat gagg 24 14 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 14
atcaacatgg aatggtgttg tggc 24 15 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 15
ttggactgag ctactgtctg ttgc 24 16 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 16
tttcagcaca cagcgccatt tggc 24 17 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 17
ctcagagaac cctgtggatg tccg 24 18 25 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 18
gcatctcgct ccaggtattt gtagg 25 19 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 19
agtttcttgg tctctgggga cagc 24 20 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 20
aactgatggt caggatcgac aggg 24 21 24 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 21
cttcaacagc atcatccaga catc 24 22 25 DNA Artificial Sequence
Description of Artificial Sequence nucleic acid primer 22
caccttggtc gtggatcatc atagc 25
* * * * *