U.S. patent application number 11/100664 was filed with the patent office on 2006-11-09 for cells exhibiting neuronal cell progenitor characteristics and methods of making them.
Invention is credited to Mari Dezawa.
Application Number | 20060251624 11/100664 |
Document ID | / |
Family ID | 35150551 |
Filed Date | 2006-11-09 |
United States Patent
Application |
20060251624 |
Kind Code |
A1 |
Dezawa; Mari |
November 9, 2006 |
Cells exhibiting neuronal cell progenitor characteristics and
methods of making them
Abstract
Disclosed are cells exhibiting neuronal progenitor cell
characteristics, and methods of making them from marrow adherent
stem cells by regulating cellular pathways in the marrow adherent
stem cells that are associated with glial transdifferentiation of
the marrow adherent stem cells.
Inventors: |
Dezawa; Mari; (Kyoto,
JP) |
Correspondence
Address: |
Innovation Law Group
1165 Rosefield Way
Menlo Park
CA
94025
US
|
Family ID: |
35150551 |
Appl. No.: |
11/100664 |
Filed: |
April 7, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60561613 |
Apr 12, 2004 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/368 |
Current CPC
Class: |
C12N 2501/70 20130101;
C12N 2501/60 20130101; C12N 2501/727 20130101; C12N 2501/155
20130101; C12N 5/0619 20130101; A61K 35/12 20130101; C12N 2506/1353
20130101; C12N 2501/115 20130101; A61P 25/00 20180101; C12N 2501/01
20130101; C12N 2501/415 20130101; A61P 25/02 20180101; C12N 2501/13
20130101 |
Class at
Publication: |
424/093.2 ;
435/368 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 5/08 20060101 C12N005/08 |
Claims
1. A method of producing cells exhibiting neuronal progenitor cell
characteristics from material comprising marrow adherent stem
cells, the method comprising: regulating cellular pathways in the
marrow adherent stem cells that are associated with glial
transdifferentiation of the marrow adherent stem cells; wherein the
cellular pathways are sufficiently regulated to induce at least a
portion of the marrow adherent stem cells to transdifferentiate
into cells exhibiting neuronal progenitor cell characteristics; and
with the proviso that the regulating does not comprise transfection
of the marrow adherent stem cells with notch intracellular
domain.
2. The method of claim 1, wherein the marrow adherent stem cells
are selected from the group consisting of human marrow adherent
stem cells, rat marrow adherent stem cells, mouse marrow adherent
stem cells, primate marrow adherent stem cells, pig marrow adherent
stem cells, cow marrow adherent stem cells, and sheep marrow
adherent stem cells.
3. The method of claim 1, wherein the regulating comprises
incubation of a glial regulating agent with the marrow adherent
stem cells.
4. The method of claim 3, wherein the incubation comprises
transfection of a glial regulating agent into the marrow adherent
stem cells.
5. The method of claim 3, wherein the glial regulating agent
comprises inhibitors or antagonists or agents that interfere with
signaling pathways for gliogenic factors.
6. The method of claim 3, wherein the glial regulating agent
comprises agonists for neurogenesis.
7. The method of claim 3, wherein the glial regulating agent
comprises a JAK/STAT inhibitor; an inhibitor of STAT1 or STAT3;
4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline; antagonists of
bone morphogenic protein 2 or bone morphogenic protein; whole or
portions of gene products from genes expressing Noggin, Chordin,
Follistatin, sonic hedgehog (SHH), or agonists of these genes; Hes
inhibitors; Hes 1 or Hes 5 inhibitors; inhibitors of Id-1;
inhibitors of mammalian homologs of Drosophila glide/gcm;
inhibitors of Sox9; inhibitors of Neurogenin3; inhibitors of CNTF;
whole or portions of gene products from genes expressing Wnt1,
Neurogenin1, Mash1, Math1, Math6, or NeuroD, or their agonists.
8. The method of claim 1, further comprising: isolating the cells
exhibiting neuronal progenitor cell characteristics; and
administering them to a patient.
9. The method of claim 1, wherein the marrow adherent stem cells
are derived from cord blood.
10. The method of claim 1, wherein the marrow adherent stem cells
are derived from bone marrow.
11. A method for producing cells exhibiting neuronal progenitor
cell characteristics comprising: incubating marrow adherent stem
cells with a glial regulating agent in an amount sufficient to
induce at least a portion of the marrow adherent stem cells to
transdifferentiate into cells exhibiting neuronal progenitor cell
characteristics; with the proviso that the interacting does not
comprise transfection of the marrow adherent stem cells with notch
intracellular domain.
12. The method of claim 11, wherein the marrow adherent stem cells
are selected from the group consisting of human marrow adherent
stem cells, rat marrow adherent stem cells, mouse marrow adherent
stem cells, primate marrow adherent stem cells, pig marrow adherent
stem cells, cow marrow adherent stem cells, and sheep marrow
adherent stem cells.
13. The method of claim 11, wherein the incubation comprises
transfection of the glial regulating agent into the marrow adherent
stem cells.
14. The method of claim 11, wherein the glial regulating agent
comprises inhibitors or antagonists or agents that interfere with
signaling pathways for gliogenic factors.
15. The method of claim 11, wherein the glial regulating agent
comprises agonists for neurogenesis.
16. The method of claim 11, wherein the glial regulating agent
comprises a JAK/STAT inhibitor; an inhibitor of STAT1 or STAT3;
4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline; antagonists of
bone morphogenic protein 2 or bone morphogenic protein; whole or
portions of gene products from genes expressing Noggin, Chordin,
Follistatin, sonic hedgehog (SHH), or agonists of these genes; Hes
inhibitors; Hes 1 or Hes 5 inhibitors; inhibitors of Id-1;
inhibitors of mammalian homologs of Drosophila glide/gcm;
inhibitors of Sox9; inhibitors of Neurogenin3; inhibitors of CNTF;
whole or portions of gene products from genes expressing Wnt1,
Neurogenin1, Mash1, Math1, Math6, or NeuroD, or their agonists.
17. The method of claim 11, further comprising: isolating the cells
exhibiting neuronal progenitor cell characteristics; and
administering them to a patient.
18. The method of claim 11, wherein the marrow adherent stem cells
are derived from cord blood.
19. The method of claim 11, wherein the marrow adherent stem cells
are derived from bone marrow.
20. Cells exhibiting neuronal progenitor cell characteristics made
according to the method of claim 1.
21. Cells exhibiting neuronal progenitor cell characteristics made
according to the method of claim 11.
22. A method comprising: administering the cells exhibiting
neuronal progenitor cell characteristics of claim 20 to a
patient.
23. A method comprising: administering the cells exhibiting
neuronal progenitor cell characteristics of claim 21 to a
patient.
24. A method comprising: providing the cells exhibiting neuronal
progenitor cell characteristics of claim 1; and combining the cells
exhibiting neuronal progenitor cell characteristics with at least
one neurotrophic factor, wherein the at least one neurotrophic
factor is present in an amount effective to promote the
differentiation of the cells exhibiting neuronal progenitor cell
characteristics into cells that exhibit one or more characteristics
of neurons.
25. The method of claim 24, further comprising: isolating the cells
that exhibit one or more characteristics of neurons.
26. The method of claim 24, wherein the at least one neurotrophic
factor comprises basic-fibroblast growth factor, ciliary
neurotrophic factor, or forskolin.
27. Cells that exhibit one or more characteristics of neurons
produced according to the method of claim 24.
28. A method comprising: administering the cells that exhibit one
or more characteristics of neurons of claim 24 to a patient.
29. A method comprising: providing the cells exhibiting neuronal
progenitor cell characteristics of claim 11; and combining the
cells exhibiting neuronal progenitor cell characteristics with at
least one neurotrophic factor, wherein the at least one
neurotrophic factor is present in an amount effective to promote
the differentiation of the cells exhibiting neuronal progenitor
cell characteristics into cells that exhibit one or more
characteristics of neurons.
30. The method of claim 29, further comprising: isolating the cells
that exhibit one or more characteristics of neurons.
31. The method of claim 29, wherein the at least one neurotrophic
factor comprises basic-fibroblast growth factor, ciliary
neurotrophic factor, or forskolin.
32. Cells that exhibit one or more characteristics of neurons
produced according to the method of claim 29.
33. A method comprising: administering the cells that exhibit one
or more characteristics of neurons of claim 29 to a patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. provisional patent
application 60/561,613, filed Apr. 12, 2004, which is incorporated
herein by reference in its entirety
FIELD OF THE INVENTION
[0002] The invention relates to cells exhibiting neuronal
progenitor cell characteristics, and methods of making them from
marrow adherent stem cells by regulating cellular pathways in the
marrow adherent stem cells that are associated with glial
transdifferentiation of the marrow adherent stem cells.
BACKGROUND OF THE INVENTION
[0003] A limitation in the research and treatment of Central
Nervous System (CNS) or Peripheral Nervous System (PNS) diseases is
the conventional recognition that terminally differentiated neurons
are significantly limited in their ability to proliferate.
Accordingly, any treatment of CNS or PNS diseases that requires
transplant of terminally differentiated neurons is difficult to
accomplish.
[0004] One proposed approach to overcoming this difficulty has been
to culture large numbers of mitotic cells exhibiting neuronal
progenitor cell characteristics ("CPCs"). Such cells could
theoretically differentiate in vivo into neurons that could
function in the treatment of CNS and/or PNS diseases.
Alternatively, CPCs might be differentiated in vitro into neurons
and then transplanted into patients. However, such CPCs are rare
and difficult to isolate from donors. Therefore, conventionally,
researchers have attempted to obtain CPCs from treated embryonic
and fetal stem cells (collectively referred to as "embryonic stem
cells" hereinafter).
[0005] Embryonic stem cells, which are pluripotent cells, have been
used to generate a large variety of tissue types, and could be a
source of CPCs. I. Weissman, Stem cells: units of development,
units of regeneration, and units in evolution (Review). Cell 100,
157-168 (2000). However, the use of embryonic stem cells raises a
number of ethical concerns, and so is a disfavored source of stem
cells for production of CPCs. Additionally, embryonic stem cells
can be tumorigenic, which generates safety concerns as to any
transplant procedure that could potentially result in the delivery
of embryonic stem cells to a patient such as creation of a CPC
graft from embryonic stem cells.
[0006] Some researchers have attempted to utilize other types of
stem cells, such as mesenchymal stem cells in the production of
CPCs. United States Patent Application 20030003090 of Prockop, et
al., filed Jan. 2, 2003, and entitled "Directed in vitro
differentiation of marrow stromal cells into neural cell
progenitors" discloses that the expression levels of both NSE and
vimentin were increased in human mesenchymal stem cells after their
incubation with 0.5 millimolar IBMX and 1 millimolar dbcAMP. The
increase in NSE and vimentin mRNAs coincided with the appearance of
neural cells in the cultures. However, Prockop et al. reported that
there was no change in the expression level of either MAP1B or
TuJ-1. Since NSE, MAP1B, and TuJ-1 are early neuron-characteristic
markers, and vimentin is an early marker for glia, Prockop et al.
suggested that the hMSCs transdifferentiated in vitro into some
early progenitors of either neurons or glia. However, the early
progenitor cells of Prockop may be undesirable for use because they
seem to display a very immature neuronal phenotype whose clinical
efficacy is not well understood.
[0007] Accordingly, there is a scarcity of conventionally available
and suitable sources of CPCs for use, for example, in the research
and treatment of CNS or PNS diseases. Further, there is a scarcity
of methods that can be used to produce such CPCs in a suitable
manner suitable for use. What are needed are methods and
compositions that overcome such problems.
SUMMARY OF THE INVENTION
[0008] In an aspect, the invention relates to a method of producing
cells exhibiting neuronal progenitor cell characteristics from
material comprising marrow adherent stem cells, the method
comprising: regulating cellular pathways in the marrow adherent
stem cells that are associated with glial transdifferentiation of
the marrow adherent stem cells; wherein the cellular pathways are
sufficiently regulated to induce at least a portion of the marrow
adherent stem cells to transdifferentiate into cells exhibiting
neuronal progenitor cell characteristics; and with the proviso that
the regulating does not comprise transfection of the marrow
adherent stem cells with notch intracellular domain.
[0009] In another aspect, the invention relates to a method for
producing cells exhibiting neuronal progenitor cell characteristics
comprising: incubating marrow adherent stem cells with a glial
regulating agent in an amount sufficient to induce at least a
portion of the marrow adherent stem cells to transdifferentiate
into cells exhibiting neuronal progenitor cell characteristics;
with the proviso that the interacting does not comprise
transfection of the marrow adherent stem cells with notch
intracellular domain.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The inventor has unexpectedly and surprisingly discovered
that the problems and limitations noted above can be overcome by
practicing the invention disclosed herein. The present invention
addresses producing CPCs from marrow adherent stem cells (MASCs) by
regulating cellular pathways in MASCs that are associated with
glial transdifferentiation of the MASCs. Ways to make and use the
invention are disclosed herein.
[0011] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. The discussion of
references herein is intended merely to summarize the assertions
made by their authors and no admission is made that any reference
constitutes prior art. Applicants reserve the right to challenge
the accuracy and pertinence of the cited references.
[0012] Cells exhibiting neuronal progenitor cell characteristics
("CPCs") are defined as, for the purposes of this invention, being
cells that are mitotic, express nestin and other cell markers
specific for neural precursor/neural progenitor cells, and are
derived from MASCs. CPCs can differentiate into neurons, glia, and
oligodendrocytes, and precursors of any of the foregoing. CPCs can
be derived from MASCs according to methods disclosed herein. In an
embodiment, human CPCs are EfnB2+, CD90-, and PDGF receptor beta-.
These markers may be used to separate CPCs from MASCs using FACS
following glial transdifferentiation of the MASCs according to the
present invention. Suitable methods of handling CPCs are known
conventionally, including those methods disclosed, for example, in
published United States patent application 20020012903 to Goldman
et al.
[0013] Generally, CPCs according to the invention may be produced
by regulating cellular pathways in MASCs that are associated with
glial transdifferentiation of the MASCs, with the cellular pathways
being sufficiently regulated to induce at least a portion of the
MASCs to transdifferentiate into CPCs.
[0014] A wide variety of regulating methods may be useful in the
practice of this invention. These include, but are not limited to,
modification of the medium and conditions in which cells are grown,
if grown ex vivo; modifying the tissue environment in which the
MASCs are present, if grown in vivo; or incubation of the MASCs
with glial regulating agents. The precise manner of regulation does
not matter for the purposes of this invention, so long as glial
transdifferentiation of the MASCs is effectively regulated, thus
allowing differentiation of the MASCs into CPCs. Generally, the
regulation of cellular pathways in MASCs that are associated with
glial transdifferentiation of the MASCs takes place under
conditions that are appropriate to maintain any MASCs or CPCs in a
mitotic and viable state. Such conditions are known to one of skill
in the art, and may be found in, for example, M. Kallos et al.,
Large-scale expansion of mammalian neural stem cells: a review. Med
Biol Eng Comput. 2003 May;41(3):271-82. Suitable conditions and
techniques also can be found elsewhere in the literature both for
cell culture and in vivo environments.
[0015] In preferred embodiments of the invention, regulation of the
cellular pathways in MASCs that are associated with glial
transdifferentiation of the MASCs may be accomplished by incubating
the MASCs with glial regulating agents. In a more preferred
embodiment, regulation of the cellular pathways in MASCs that are
associated with glial transdifferentiation of the MASCs may be
accomplished by incubating the MASCs with glial regulating agents
in amounts sufficient to induce at least a portion of the MASCs to
transdifferentiate into CPCs. Incubations in the context of the
present invention may involve culturing MASCs in the presence of
glial regulating agents with the intent that the glial regulating
agents either interact with MASC cell surface receptors or are
transported into the interior of the MASCs to interact with
internal cellular pathways. Such transportation may be passive,
such as diffusive transport, or active, such as through active
transporters or a mixture of the two. In vitro incubations may be
performed in a conventional manner, for instance incubating
cultures of MASCs in alpha-MEM, or similar media, to which glial
regulating agent(s) are added. Suitable incubation techniques may
be found generally in the literature, including for example M
Kallos et al., Large-scale expansion of mammalian neural stem
cells: a review. Med Biol Eng Comput. 2003 May;41(3):271-82.
Incubations may also take place in an in vivo environment, in which
case glial regulating agents according to the invention may be
administered either systemically or locally, and using conventional
methods.
[0016] In a preferred embodiment of incubation, if the glial
regulating agent is a protein or peptide, the method of incubation
may be a transfection of the DNA coding for that protein or peptide
into the MASCs. Transfections may be performed using commercially
available transfection protocols, such as the Lipofectamine.TM.
2000 system available from Invitrogen, or the Effectene.TM.
transfection system available from Qiagen, or other conventional
transfection protocols. In another preferred embodiment of
incubation, if the glial regulating agent is a protein or peptide,
the method of incubation may be viral delivery of the glial
regulating agent, using conventional viral vectors, such as
Lentiviral vector systems (BLOCK-iT.TM. Lentiviral RNAi Expression
System, Invitrogen) for stable expression and Adenoviral vector
systems (BLOCK-iT.TM. Adenoviral RNAi Expression System,
Invitrogen) for transient expression.
[0017] The incubations can take place at various times: serially,
in parallel or combinations of serial and parallel incubations of
the MASCs with various glial regulating agent(s).
[0018] In embodiments of the invention, there is the proviso that
regulating cellular pathways in the MASCs that are associated with
glial transdifferentiation of the MASCs does not comprise
transfection of the MASCs with the intracellular domain of the
Notch gene. In embodiments of the invention, there is the proviso
that incubating the MASCs with glial regulating agents does not
comprise transfection of the MASCs with the intracellular domain of
the Notch gene.
[0019] Marrow adherent stem cells (MASCs) are defined as being, for
the purposes of this invention, stem cells that are conventionally
recognized as differentiating into several types of cells found
primarily in connective tissues, including but not limited to,
osteoblasts, adipocytes, chondrocytes, and myocytes. MASCs
specifically exclude embryonic stem cells and fetal stem cells.
MASCs may be obtained from a wide variety of animals, including but
not limited to humans, and other mammals such as rats, mice,
primates, pigs, cows, and sheep. MASCs may be obtained from a
variety of tissues; preferred sources comprise bone marrow and cord
blood. Useful sources for MASCs, and methods of obtaining them are
described in Example 1 below, and elsewhere herein. In an
embodiment, human MASCs useful in the practice of this invention
express CD29, and CD90, but are negative for CD15, CD34, CD11 b/c,
CD31, CD45 and von Willebrand Factor.
[0020] In an embodiment, MASCs may be isolated from cord blood
using techniques described in the literature. For instance, C.
Campagnoli et al., Identification of mesenchymal stem/progenitor
cells in human first-trimester fetal blood, liver, and bone marrow.
1: Blood. 2001 October 15;98(8):2396-402., describes methods
generally useful in obtaining fetal blood MASCs. In A. Erices et
al., Mesenchymal progenitor cells in human umbilical cord blood. 1:
Br J Haematol. 2000 April;109(1):235-42., there was described
methods generally useful in obtaining MASCs from cord blood. L. Hou
et al., Induction of umbilical cord blood mesenchymal stem cells
into neuron-like cells in vitro. Int J Hematol. 2003
October;78(3):256-61, describes methods generally useful in
obtaining purifying, and expanding human umbilical cord blood
MASCs.
[0021] Glial regulating agents are defined as being, for the
purposes of this invention, substances that, among other
characteristics, possess the characteristic of inhibiting
transdifferentiation of MASCs into glial cells and promoting their
transdifferentiation into CPCs. Glial regulating agents may act
through a variety of different mechanisms to direct MASCs away from
the glial fate. For instance, pro-neural basic helix-loop-helix
transcription factors such as Mash 1, Math 1 and neurogenin 1 are
believed to be activators of neuronal gene expression.
[0022] Proneural genes are believed to drive neuronal
transdifferentiation of MASCs while inhibiting glial
transdifferentiation. One mechanism by which glial
transdifferentiation may be inhibited is through the regulation of
STAT-mediated signal transduction. Signal transduction by STAT is
believed to be triggered by phosphorylation which is believed to be
catalyzed by the Janus family of tyrosine kinases (JAK). Inhibition
of the JAK-STAT signal transduction therefore may regulate glial
transdifferentiation pathways and promote the neuronal fate of
MASCs.
[0023] Glial regulating agents according to the invention may
comprise inhibitors or antagonists or agents that interfere with
the signaling pathways for gliogenic factors. Glial regulating
agents may also comprise agonists for neurogenesis, including
neurogenic factors. Use of these agonists or factors may negatively
control gliogenesis of MASCs in the practice of this invention.
Glial regulating agents according to the practice of this invention
may comprise conventional forms of therapeutic molecules, including
but not limited to small molecules, peptides, and whole or portions
of gene products.
[0024] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, JAK/STAT inhibitors,
including inhibitors of STAT1 and STAT3. In certain embodiments,
such JAK/STAT inhibitors may comprise RNAi for gene silencing of
the JAK/STAT pathway, antisense oligonucleotides to down regulate
the JAK/STAT pathway, or the small molecule JAK inhibitor
4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline. Additional
JAK/STAT inhibitors may be disclosed in United States Patent
Application 20040209799 of George Vasios, published Oct. 21, 2004;
and United States Patent Application 20040052762 of Hua Yu et al.,
published Mar. 18, 2004.
[0025] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, antagonists of BMP2 or 7
(bone morphogenic protein). Such antagonists may comprise whole or
portions of gene products from genes expressing Noggin, Chordin,
Follistatin, sonic hedgehog (SHH), or agonists of these genes.
[0026] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, Hes inhibitors,
including but not limited to Hes 1 and/or Hes 5 inhibitors. In
certain embodiments, such Hes inhibitors may comprise RNAi for gene
silencing of Hes, or antisense oligonucleotides to down regulate
Hes.
[0027] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, inhibitors of Id-1. See
S. Tzeng et al., Id1, Id2, and Id3 gene expression in neural cells
during development. Glia. 1998 December;24(4):372-81. In certain
embodiments, such Id-1 inhibitors may comprise RNAi for gene
silencing of Id-1, or antisense oligonucleotides to down regulate
Id-1.
[0028] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, inhibitors of mammalian
homologs of Drosophila glide/gcm (glial cells missing), including
but not limited to Gcm1 (murine) or GCMB (human). See Y. Iwasaki et
al., The potential to induce glial differentiation is conserved
between Drosophila and mammalian alial cells missing genes.
Development. 2003 December;130(24):6027-35. Epub 2003 Oct. 22; and
M. Kammerer et al., GCMB, a second human homolog of the fly
glide/gcm gene. Cytogenet Cell Genet. 1999;84(1-2):43-7.). In
certain embodiments, such glide/gcm homolog inhibitors may comprise
RNAi for gene silencing of glide/gcm homologs (such as Gcm1
(murine) or GCMB (human)), or antisense oligonucleotides to down
regulate glide/gcm homologs (such as Gcm1(murine) or GCMB
(human)).
[0029] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, inhibitors of Sox9,
which may be a transcription factor for oligodendrocyte lineage.
See C. Stolt et al., The Sox9 transcription factor determines glial
fate choice in the developing spinal cord. Genes Dev. 2003 Jul.
1;17(13):1677-89.). In certain embodiments, such Sox9 inhibitors
may comprise RNAi for gene silencing of Sox9, or antisense
oligonucleotides to down regulate Sox9.
[0030] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, inhibitors of
Neurogenin3, which may be a transcription factor for gliogenesis.
In certain embodiments, such Neurogenin3 inhibitors may comprise
RNAi for gene silencing of Neurogenin3, or antisense
oligonucleotides to down regulate Neurogenin3.
[0031] In an embodiment, glial regulating agents according to the
invention include, but are not limited to, inhibitors of ciliary
neurotrophic factor (CNTF). In certain embodiments, such CNTF
inhibitors may comprise RNAi for gene silencing of CNTF, or
antisense oligonucleotides to down regulate CNTF.
[0032] In certain embodiments, glial regulating agents may comprise
whole or portions of gene products from genes expressing Wnt1,
which strongly inhibits gliogenesis. See K. Tang et al., Wnt-1
promotes neuronal differentiation and inhibits gliogenesis in P19
cells. Biochem Biophys Res Commun. 2002 Apr. 26;293 (1):167-73.
Whole or portions of gene products from genes expressing Wnt1 may
be administered by transfection or other conventional methods, such
as gene therapy methods including viral vectors.
[0033] In certain embodiments, glial regulating agents may comprise
whole or portions of gene products from genes expressing a subset
of neural basic helix-loop-helix (bHLH) factors that play
instructive roles during neurogenesis or are expressed in
proliferating CPCs. Such glial regulating agents may comprise whole
or portions of gene products from genes expressing Neurogenin1,
Mash1, Math1, Math6, or NeuroD. Whole or portions of gene products
from genes expressing the subset of neural basic helix-loop-helix
(bHLH) factors, including but not limited to Neurogenin1, Mash1,
Math1, Math6, or NeuroD, may be administered by transfection or
other conventional methods, such as gene therapy methods including
viral vectors.
[0034] Additionally, glial regulating agents may be administered
singly or in combination. In a preferable embodiment, if a
combination of glial regulating agents is used in the practice of
the invention, then glial regulating agents that act on different
glial regulating pathways may be selected. This may serve to
enhance the overall glial regulating effect of the glial regulating
agents.
[0035] For the purposes of this invention, isolating CPCs comprises
isolating CPCs from non-CPC cells in a sample, such as MASCs that
have not transdifferentiated into CPCs. Such isolation may comprise
a single isolation or multiple isolations. If multiple isolations
are to be performed, different types or techniques of isolation may
be preferably used, as such different types or techniques of
isolation may enhance isolation results. A wide variety of
isolation methods are useful in the practice of this invention.
Examples of such isolation methods include, but are not limited to
flow cytometry (aka FACS sorting), magnetic separation techniques,
and visual sorting. Immunocytochemistry may also be used in
instances where cell viability is not critical.
[0036] FACS sorting can be performed using conventional FACS
equipment and protocols with antibodies that are specific to
epitopes associated with one or more characteristics of CPCs. One
such epitope may be EfnB2 in the case of human CPCs. N. Ivanova et
al., A stem cell molecular signature. Science 298(5593):601-4 (Oct.
18, 2002). Antibodies additionally useful in the practice of the
invention, although not necessarily for FACS sorting, comprise
anti-CD15, anti-CD29, anti-CD34, anti-CD90, anti-CD31, anti-CD45,
anti-CD11b/c, and anti-von Willebrand factor. Cell populations FACS
equipment useful in the practice of this invention include, but are
not limited to, a FACScalibur.TM. analyzer with CellQuest.TM.
software (Becton Dickinson, Franklin Lakes, N.J.), or FACS
equipment available from Guava Technologies (Hayward, Calif.).
[0037] Alternatively, isolation may be performed using magnetic
separation techniques, such as the BioMag.TM. protocols and
reagents, available in kit form from Qiagen. Immunocytochemistry
may be another separation technique useful in the practice of this
invention; useful Immunocytochemical methods are described in M.
Dezawa et al., Sciatic nerve regeneration in rats induced by
transplantation of in vitro differentiated bone-marrow stromal
cells. Eur. J. Neurosci. 14,1771-1776 (2001). Immunocytochemical
inspections may be made under a confocal laser scanning microscope,
such as the Radians 2000 (Bio-Rad, Hertfordshire, UK). Conventional
visual cell sorting techniques may be used in the practice of this
invention.
[0038] Neurons are defined as, for the purposes of this invention,
being any of the impulse-conducting cells that constitute the
brain, spinal column, and nerves, consisting of a nucleated cell
body with one or more dendrites and a single axon. Biochemically,
neurons are characterized by reaction with antibodies for
neurofilament-M, beta3-tubulin, and TuJ-1. These reactions may be
used to isolate neurons or cells exhibiting one or more
characteristics of neurons using techniques such as FACS sorting.
Neural cells are also characterized by secreting neurotransmitters,
neurotransmitter synthetases or neurotransmitter-related proteins,
for example neuropeptide Y and substance P.
[0039] Neurotrophic agents are defined as being, for the purposes
of this invention, substances that, among other characteristics,
possess the characteristic of causing or promoting the
differentiation of CPCs into neurons or cells that exhibit one or
more characteristics of neurons. Neurotrophic agents useful in the
practice of this invention comprise but are not limited to
basic-fibroblast growth factor (bFGF), ciliary neurotrophic factor
(CNTF), and forskolin (FSK). Neurotrophic agents may be combined
with the CPCs of the present invention using cell handling
techniques known in the art. Preferred methods may be found
generally in PCT/JP03/01260 of Dezawa et al. In a preferred
embodiment, bFGF, CNTF and FSK are combined with CPCs in cell
culture in amounts effective to cause or promote the
differentiation of CPCs into neurons or cells that exhibit one or
more characteristics of neurons.
[0040] Glial cells are defined as, for the purposes of this
invention, being any of the cells that make up the network of
branched cells and fibers that support the tissue of the central
nervous system. Glial cells include, but are not limited to
astrocytes, Schwann cells, oligodendrocytes, and microglia.
[0041] Genes are defined as, for the purposes of this invention,
being a set of connected transcripts, wherein a transcript is a set
of exons produced via transcription followed (optionally) by
pre-mRNA splicing. Gene products are defined as, for the purposes
of this invention, being proteins translated from genes. Portions
of genes are defined as, for the purposes of this invention, being
a subset of a gene. Portions of gene products are defined as, for
the purposes of this invention, being a subset of a gene
product.
[0042] Patient means an animal, typically a mammal, and more
typically, a human, that is the subject of medical observation or
study.
[0043] CPCs produced according to the invention may be administered
to patients through a variety of methods, including but not limited
to infusion through an injection cannula, needle or shunt, or by
implantation within a carrier, e.g., a biodegradable capsule, but
other routes of administration, are also within the scope of the
invention. Inventive routes of administration comprise local and
systemic routes. Local administration may preferable include
administration to targeted potions of the CNS or PNS, and
preferably includes intraparenchymal routes. Systemically routes of
administration comprise parenteral routes, with intravenous (i.v.),
or intra-arterial (such as through internal or external carotid
arteries) administration being preferred routes of systemic
administration. Systemic administration techniques can be adapted
from techniques used to administer precursor cells generally, such
as those disclosed in D Lu et al., Intraarterial administration of
marrow stromal cells in a rat model of traumatic brain injury. J
Neurotrauma. 2001 August;18(8):813-9.
[0044] Amounts of CPCs administered to a patient may be determined
clinically, using conventional dose ranging techniques, and
clinical assessments of a particular patient's disease.
[0045] The present invention is not to be limited in terms of the
particular embodiments described in this application, which are
intended as single illustrations of individual aspects of the
invention. Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will be
apparent to those skilled in the art. Functionally equivalent
methods within the scope of the invention, in addition to those
enumerated herein, will be apparent to those skilled in the art
from the foregoing description. Such modifications and variations
are intended to fall within the scope of the appended claims. The
present invention is to be limited only by the terms of the
appended claims, along with the full scope of equivalents to which
such claims are entitled.
[0046] The Examples set forth below are meant to be illustrative,
and in no way limiting, of the scope of the present invention.
EXAMPLES
Materials and Methods:
[0047] MASCs: Rat MASCs (Wistar strain) are isolated and cultured
as described in M. Dezawa et al., Sciatic nerve regeneration in
rats induced by transplantation of in vitro differentiated
bone-marrow stromal cells. Eur. J. Neurosci. 14, 1771-1776 (2001).
As for human MASCs, commercially purchased MASCs (PT-2501,
BioWhittaker. Walkersville, Md.) and MASCs obtained from healthy
donors are used. Cells may be maintained in alpha-MEM (Sigma,
M-4526) with 10% fetal bovine serum (FBS).
[0048] In the case of obtaining MASCs from healthy donors, an
initial step is to obtain bone marrow aspirate from healthy donors
using conventional aspiration techniques. The cell aspirate is then
transferred into a 50 ml tube. 13 ml Histopaque is then carefully
underlayed, using a 10 ml pipette. The tube is then centrifuged
@200 rpm for 20 minutes. Cells at the interphase are then
harvested. PBS is then added (at least 3.times. the volume of the
interphase) and the mixture centrifuged @1200 rpm. The cells are
washed twice more with PBS. The cell pellet is then resuspended in
DMEM +10% FCS, and the cells counted. 5.times.10 6 cells are
replated per T-75 tissue culture flask, and incubated for 3 days.
On day 4, the non-adherent cells are removed and the flask washed
three times with medium. The adherent cells are allowed to grow in
the flask. When the cells reach 20-30% confluence, the content of
2-3 flasks are pooled and re-plated in one T-75 flask. When the
cells in this pooled reach confluence, the cells are trypsinized
using 0.05% trypsin and 0.02% EDTA. The cells are then washed and
counted. The cells are then resuspended in Sigma alpha MEM+10% FBS
(M-4526). In experiments where lipofection is to be used, it is
important to insure that the medium contains no I-glu. Glutamine is
not added. The cells are expanded for 2-4 weeks and are frozen in
early passages.
[0049] Cell surface markers in rat and human MASCs are analyzed
with fluorescence activated cell analysis (FACS). In an embodiment,
the MASCs express CD29, and CD90, but are negative for CD34, CD31,
CD45, CD11b/c, and von Willebrand Factor consistent with M.
Pittenger et al., Multilineage potential of adult human mesenchymal
stem cells. Science 284, 143-147 (1999); and J. Kohyama et al.,
Brain from bone: efficient "meta-differentiation" of marrow
stroma-derived mature osteoblasts to neurons with Noggin or a
demethylating agent. Differentiation 68, 235-244 (2001) (FIG. 1A).
The same result is obtained by immunocytochemistry. Adipogenic,
chondrogenic and osteogenic differentiation of both rat and human
MASCs are confirmed according to the method described by M.
Pittenger et al., Multilineage potential of adult human mesenchymal
stem cells. Science 284, 143-147.
[0050] FACS analysis. Cells at a final concentration of 1.times.10
7/ml are incubated with 1 mg of a monoclonal antibody in phosphate
buffered saline (PBS). Incubations may be performed in the presence
of 10 mg of mouse immunoglobulin to prevent nonspecific antibody
binding. In rat MASCs, mouse anti-CD34 (Santa Cruz Antibodies) and
hamster anti-CD29 (PharMingen, San Diego, Calif.) may be labeled
with FITC, and controls may be incubated either with FITC-labeled
anti-mouse or hamster IgG. Mouse anti-CD54 and CD11b/c may be all
purchased from PharMingen. Mouse anti-von Willebrand factor and
other antibodies needed in the practice of this invention may be
obtained commercially. Controls may include cells stained either
with non-immune mouse serum. If these antibodies are conjugated to
FITC, the cells may be subsequently incubated with 1 mg of
FITC-conjugated anti-mouse IgG. In human MASCs, phycoerythrin
labeled mouse anti-CD34, CD29, CD54, CD11b/c and von Willebrand
factor may be used, and controls may include cells stained with
phycoerythrin labeled anti-mouse IgG. Data may be acquired and
analyzed on a FACScalibur with CellQuest software (Becton
Dickinson, Franklin Lakes, N.J.).
[0051] Immunocytochemistry. The general procedure is described in
M. Dezawa et al., Sciatic nerve regeneration in rats induced by
transplantation of in vitro differentiated bone-marrow stromal
cells. Eur. J. Neurosci. 14, 1771-1776 (2001). After the fixation
of cells with 4% paraformaldehyde in phosphate-buffered saline
(PBS), they are incubated with primary antibodies for overnight at
4 Deg. C. Antibodies to nestin may be purchased commercially from
PharMingen. Cells may be then incubated with secondary antibodies
to Alexa Fluor 488 or 546 conjugated anti-mouse IgG, IgM, or rabbit
IgG (Molecular Probes, Eugene, Oreg.) for 1 hour at room
temperature, and TOTO-3 iodide (Molecular Probes) counter staining
may be performed. Inspections may be made under a confocal laser
scanning microscope (Radians 2000, Bio-Rad, Hertfordshire, UK).
Example 1
[0052] Human MASCs (PT-2501, BioWhittaker, Walkersville, Md.) were
allowed to grow in culture in alpha-MEM containing 10% FBS
generally according to E. Sudbeck et al., Structure-based design of
specific inhibitors of Janus kinase 3 as apoptosis-inducing
antileukemic agents. Clin. Cancer Res. 5, 1569-1582 (1999). The
MASCs were incubated with 40 ug/ml
4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-131,
Calbiochem, San Diego, Calif.) for two days. The WHI-131 was washed
off after 2 days.
Example 2
[0053] Human MASCs, prepared according to the Materials and Methods
section, are allowed to grow in culture in alpha-MEM containing 10%
FBS generally according to E. Sudbeck et al., Structure-based
design of specific inhibitors of Janus kinase 3 as
apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5,
1569-1582 (1999). Once the culture has reached 90% confluence,
several RNAs, designed using the BLOCK-iT.TM. RNAi Designer
(Invitrogen) are incubated with the culture for a period of time
sufficient to silence Sox9 expression, using BLOCK-iT.TM. protocols
available from Invitrogen. Resulting CPCs are isolated from
untransdifferentiated MASC's by sequential selection using magnetic
beads coated with appropriate antibodies such as anti-EfnB2
(positive selection for CPCs), anti-CD90 (negative selection for
CPCs), and anti-PDGF receptor beta (negative selection for CPCs).
The antibodies and coated beads may be obtained from commercial
suppliers. The cells in PBS are incubated with coated beads for 1
hr. @ room temperature. The cell-bound beads are removed using a
magnet. The CPCs are washed free of the antibody and re-suspended
in alpha-MEM containing 10% FBS and allowed to proliferate.
Example 3
[0054] Human MASCs, prepared according to the Materials and Methods
section, are allowed to grow in culture in alpha-MEM containing 10%
FBS generally according to E. Sudbeck et al., Structure-based
design of specific inhibitors of Janus kinase 3 as
apoptosis-inducing antileukemic agents. Clin. Cancer Res. 5,
1569-1582 (1999). Antisense oligomers to Hes 1 are generated
according to techniques disclosed in any one of H. Moulton et al.,
Peptide-assisted delivery of steric-blocking antisense oligomers.
Curr Opin Mol Ther. 2003 April;5(2):123-32; C. Stein et al.,
Antisense oligonucleotides as therapeutic agents--is the bullet
really magical? Science. 1993 Aug. 20;261(5124):1004-12; or C.
Helene, The anti-gene strategy: control of gene expression by
triplex-forming-oligonucleotides. Anticancer Drug Des. 1991
December;6(6):569-84. Once the MASC culture reaches 90% confluence,
the Hes-1 antisense oligomers are incubated with the MASCs for a
period sufficient to downregulate Hes-1 expression, according to
techniques disclosed in any of the three references cited in this
example. Resulting CPCs are isolated from untransdifferentiated
MASC's by sequential selection using magnetic beads coated with
appropriate antibodies such as anti-EfnB2 (positive selection for
CPCs), anti-CD90 (negative selection for CPCs), and anti-PDGF
receptor beta (negative selection for CPCs). The antibodies and
coated beads may be obtained from commercial suppliers. The cells
in PBS are incubated with coated beads for 1 hr. @ room
temperature. The cell-bound beads are removed using a magnet. The
CPCs are washed free of the antibody and re-suspended in alpha-MEM
containing 10% FBS and allowed to proliferate.
Example 4
[0055] Wnt-1 expression plasmids are generated according to M. Sen
et al., Regulation of fibronectin and metalloproteinase expression
by Wnt signaling in rheumatoid arthritis synoviocytes. Arthritis
Rheum. 2002 November;46(11):2867-77. Human MASCs, prepared
according to the Materials and Methods section, are allowed to grow
in culture in alpha-MEM containing 10% FBS generally according to
E. Sudbeck et al., Structure-based design of specific inhibitors of
Janus kinase 3 as apoptosis-inducing antileukemic agents. Clin.
Cancer Res. 5, 1569-1582 (1999). Once the culture reaches 90%
confluence, the MASCs are incubated with the Wnt-1 expression
plasmids for two days at 37 deg C. and 5% CO2 using the
Lipofectamine.TM. 2000 reagent and protocols available from
Invitrogen. After the two days of incubation, the culture is
selected for transfected cells using conventional selection
techniques for a period of 10 days. Resulting CPCs are isolated
from untransdifferentiated MASC's by sequential selection using
magnetic beads coated with appropriate antibodies such as
anti-EfnB2 (positive selection for CPCs), anti-CD90 (negative
selection for CPCS), and anti-PDGF receptor beta (negative
selection for CPCs). The antibodies and coated beads may be
obtained from commercial suppliers. The cells in PBS are incubated
with coated beads for 1 hr. @ room temperature. The cell-bound
beads are removed using a magnet. The CPCs are washed free of the
antibody and re-suspended in alpha-MEM containing 10% FBS and
allowed to proliferate.
Example 5
[0056] The cells produced according to Example 1 were placed in
Minimum Essential Mediam Alpha Eagle Modification (M4526, Sigma
Co.) containing 20% fetal bovine serum (14-501 F, Lot #61-1012,
BioWhittaker Co.). 5 microM of forskolin (344273, Calbiochem, La
Jolla, Calif.), 10 ng/ml of recombinant human basic fibroblast
growth factor (100-18B, Peprotech EC, Ltd., London, UK) and 10
ng/ml of ciliary neurotrophic factor (557-NT, R&D Systems,
Minneapolis, Minn.) were added. The culture was grown for 3 days,
at which point cells exhibiting neuronal progenitor cell
characteristics were recognized, with the result of 29.46.+-.3.0%
of MAP-2ab-positive cells. MAP-2ab was analyzed for using Western
blotting, with cell lysates prepared from incubated cells, and 50
ug of lysate proteins electrophorased on 5% and 10%
SDS-polyacrylamide gel. Antigens to MAP-2 (1:500, Chemicon) were
detected using alkaline phosphatase.
Example 6
[0057] The cells exhibiting neuronal progenitor cell
characteristics of Example 5 are harvested, and grown to 90%
confluence in culture in alpha-MEM containing 10% FBS generally
according to E. Sudbeck et al., Structure-based design of specific
inhibitors of Janus kinase 3 as apoptosis-inducing antileukemic
agents. Clin. Cancer Res. 5, 1569-1582 (1999). Next, 5 mM of
forskolin (344273, Calbiochem), 10 ng/ml of basic fibroblast growth
factor (100-18B, Peprotech EC, Ltd.) and 50 ng/ml of ciliary
neurotrophic factor (557-NT, R&D Systems) are added to the cell
culture.
[0058] The cells are grown for ten days in the presence of the
neurotrophic agents, and then are analyzed for the characteristic
morphology of neural cells and for positive reaction for antibodies
against MAP-2 (MAB364, Chemicon), neurofilament (814342, Boehringer
Manheim) and nestin (BMS4353, Bioproducts)
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