U.S. patent application number 10/181508 was filed with the patent office on 2004-02-19 for multipotent neural stemcells from peripheral tissues and uses thereof.
Invention is credited to Akhavan, Mahnaz, Fernandes, Karl J. L., Fortier, Mathieu, Golster, Andrew, Miller, Freda, Toma, Jean.
Application Number | 20040033597 10/181508 |
Document ID | / |
Family ID | 27050050 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033597 |
Kind Code |
A1 |
Toma, Jean ; et al. |
February 19, 2004 |
Multipotent neural stemcells from peripheral tissues and uses
thereof
Abstract
This invention relates to multipotent neural stem cells,
purified from the peripheral nervous system of mammals, capable of
differentiating into neural and non-neural cell types. These stem
cells provide an accessible source for autologous transplantation
into CNS, PNS, and other damaged tissues.
Inventors: |
Toma, Jean; (Toronto
Ontario, CA) ; Akhavan, Mahnaz; (Toronto Ontario,
CA) ; Fernandes, Karl J. L.; (Toronto Ontario,
CA) ; Fortier, Mathieu; (Orford, CA) ; Miller,
Freda; (Toronto Ontario, CA) ; Golster, Andrew;
(Saskatoon Sakatchewan, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
27050050 |
Appl. No.: |
10/181508 |
Filed: |
April 1, 2003 |
PCT Filed: |
January 24, 2001 |
PCT NO: |
PCT/CA01/00047 |
Current U.S.
Class: |
435/368 ;
435/371 |
Current CPC
Class: |
C12N 5/0619 20130101;
A61K 35/12 20130101; C12N 2506/03 20130101; C12N 2501/11 20130101;
C12N 5/0607 20130101; A61P 43/00 20180101; C12N 2501/235 20130101;
A61P 25/00 20180101; C12N 5/0623 20130101; C12N 2510/00 20130101;
C12N 2501/195 20130101; A61P 25/28 20180101; A61P 25/14 20180101;
C12N 2501/70 20130101; C12N 2501/115 20130101; A61P 25/16
20180101 |
Class at
Publication: |
435/368 ;
435/371 |
International
Class: |
C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 1999 |
KR |
P99-34362 |
Claims
1. A multipotent stem cell substantially purified from skin of a
mammal.
2. The multipotent stem cell of claim 1, wherein said skin
comprises the dermal layer.
3. The multipotent stem cell of claim 1, wherein said skin is from
a postnatal mammal.
4. The multipotent stem cell of claim 1, wherein said skin is from
an adult mammal.
5. The multipotent stem cell of claim 1, wherein said skin is from
a juvenile mammal.
6. The multipotent stem cell of claim 1, wherein said mammal is a
human.
7. The multipotent stem cell of claim 1, wherein said cell
expresses nestin.
8. The multipotent stem cell of claim 1, wherein said cell
expresses fibronectin.
9. The multipotent stem cell of claim 1, which under appropriate
conditions can be differentiated into a neuron, an astrocyte, a
Schwann cell, or an oligodendrocyte.
10. The multipotent stem cell of claim 1, which can be
differentiated into a non-neural cell.
11. The multipotent stem cell of claim 9, wherein said non-neural
cell is a smooth muscle cell or an adipocyte.
12. The multipotent stem cell of claim 1, wherein said cell
contains a heterologous gene in an expressible genetic
construction.
13. The multipotent stem cell of claim 11, wherein said gene
encodes a therapeutic protein.
14. The multipotent stem cell of claim 11, wherein said gene
encodes a cell fate-determining protein.
15. A population of at least ten cells, wherein at least 30% of the
cells are multipotent stem cells substantially purified from skin
of a mammal.
16. The population of cells of claim 15, wherein said skin is from
a human.
17. A composition comprising (i) a multipotent stem cell
substantially purified from skin of a mammal, and (ii) a
pharmaceutically acceptable carrier, auxiliary or excipient.
18. A cell aggregate comprising at least ten multipotent stem cells
derived from skin of a mammal, said multipotent stem cells capable
of differentiating as dopaminergic neurons.
19. The aggregate of claim 17, wherein said cells grow detached
from the culture substrate.
20. The aggregate of claim 18, wherein said aggregate comprises at
least one hundred multipotent neural stem cells derived from skin
of a mammal.
21. A method of producing a population of at least ten cells,
wherein at least 30% of the cells are multipotent stem cells
substantially purified from skin of a postnatal mammal, or progeny
of said multipotent stem cells, said method comprising: (a)
providing said skin from said mammal; (b) culturing said skin under
conditions in which multipotent stem cells proliferate and in which
at least 25% of the cells that are not multipotent stem cells die
or attach to the culture substrate; and (c) continuing culture step
(b) until at least 30% of the cells are multipotent stem cells or
progeny of said multipotent stem cells.
22. A method of producing a population of at least ten cells,
wherein at least 30% of the cells are multipotent stem cells
substantially purified from skin of a postnatal mammal or progeny
of said multipotent stem cells, said method comprising: (a)
providing said skin from said mammal; (b) culturing said skin under
conditions in which multipotent stem cells proliferate and in which
at least 25% of the cells that are not multipotent stem cells die
or attach to the culture substrate; (c) separating said multipotent
stem cells from said cells that attach to said culture substrate;
and (d) repeating steps (b) and (c) until at least 30% of the cells
are multipotent stem cells or progeny of said multipotent stem
cells.
23. The method of claim 22, wherein said population is at least one
hundred cells.
24. A multipotent stem cell in the central nervous system of a
mammal, said multipotent stem cell produced by a method of
comprising transplanting a multipotent stem cell substantially
purified from the skin of a mammal into the central nervous system
of said mammal.
25. The multipotent stem cell of claim 24, wherein said mammal from
which said cell is substantially purified is said mammal into which
said cell is transplanted.
26. The multipotent stem cell of claim 25, wherein said mammal from
which said cell is substantially purified is immunologically
similar to said mammal into which said cell is transplanted.
27. A kit comprising a multipotent stem cell substantially purified
from skin of a mammal.
28. The kit of claim 27, said kit comprising a population of cells,
wherein at least 30% of said cells are multipotent stem cells
substantially purified from said skin.
29. A kit for the substantial purification of multipotent stem
cells from skin of a mammal.
30. A method of treating a patient having a disease characterized
by failure of a cell type, said method comprising administering to
said patient a multipotent stem cell derived from a peripheral
tissue, said multipotent stem cell capable of producing neurons and
glia.
31. The method of claim 30, wherein said peripheral tissue is
skin.
32. A kit for use in performing the method of claim 30.
33. A method for treating a patient having a disease characterized
by failure of a cell type, said method comprising admininstering to
said patient a differentiated cell that is the progeny of a
multipotent stem cell derived from skin.
34. The method of claim 33, wherein said skin is from a postnatal
mammal.
35. A method for making a neuron that expresses dopamine, said
method comprising: (a) providing a multipotent stem cell derived
from skin; and (b) culturing said cell under conditions whereby
said cell differentiates into a neuron that expresses dopamine.
36. A method for making a cell other than a skin cell, said method
comprising culturing a multipotent stem cell substantially purified
from peripheral tissue of a mammal under conditions in which said
cell differentiates as a cell other than a skin cell.
37. The method of claim 36, wherein said cell is a neuron, a glial
cell, a cardiac cell, a pancreatic islet cell, an adipocyte or a
smooth muscle cell.
38. The method of claim 36, wherein said peripheral tissue is
skin.
39. A substantially pure composition of multipotent stem cells,
which multipotent stem cell differentiates into neural cell types
and non-neural cell types.
40. The stem cells of claim 39, wherein said non-neural cell type
is an adipocyte, a smooth muscle cell or a cardiac cell.
41. An ex vivo cellular composition wherein at least 30% of the
cells are progeny of the multipotent stem cells of claim 39.
42. A cellular composition of claim 41, wherein said progeny is
undifferentiated progeny.
43. A cellular composition of claim 42, wherein said progeny is
capable of differentiating into a neural or non-neural cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
priority from U.S. application Ser. No. 09/670,049, filed Sep. 25,
2000, which application is a continuation-in-part of application
Ser. No. 09/490,422, filed Jan. 24, 2000, each of which is hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to multipotent stem cells
(MSCs), or multipotent neural stem cells (MNSCs), purified from
peripheral tissues including peripheral tissues containing sensory
receptors such as the skin, olfactory epithelium, and tongue.
[0003] There are a number of diseases of the central nervous system
("CNS") which have a devastating effect on patients. These diseases
are debilitating, often incurable, and include, for example,
Alzheimer's disease, Huntington's disease, Parkinson's disease, and
Multiple Sclerosis.
[0004] By way of example, Parkinson's disease is a progressive
degenerative disorder of unknown cause. In healthy brain tissue,
dopaminergic neurons extend from the substantia nigra of the brain
into the neighboring striatum. In Parkinson's disease, these
dopaminergic neurons die.
[0005] There are a number of methods to treat Parkinson's disease.
One method is to treat humans having Parkinson's disease with
L-DOPA. A second method is to transplant cells into the substantia
nigra or striatum. Transplanted cells replace endogenous cells that
are lost as a consequence of disease progression. An animal model
of Parkinson's disease is an MPTP-treated non-human primate. The
MPTP-treated animals have been transplanted with dopamine-rich
embryonic neurons with some success.
[0006] To date, the cells used for neural transplant have been
collected from the developing brains of aborted fetuses. Aside from
the ethical considerations, the method from a practical standpoint
is unlikely to provide a sufficient amount of neural tissue to meet
the demands. Thus, another source of cells for transplantation is
desirable.
[0007] Stem cells are undifferentiated cells that exist in many
tissues of embryos and adult mammals. In embryos, blastocyst stem
cells are the source of cells which differentiate to form the
specialized tissues and organs of the developing fetus. In adults,
specialized stem cells in individual tissues are the source of new
cells, replacing cells lost through cell death due to natural
attrition, disease, or injury. Stem cells may be used as substrates
for producing healthy tissue where a disease, disorder, or abnormal
physical state has destroyed or damaged normal tissue.
[0008] MSCs, or MNSCs, may be used as a source of cells for
transplantation. The stem cells may themselves be transplanted or,
alternatively, they may be induced to produce differentiated cells
(e.g., neurons, oligodendrocytes, Schwann cells, or astrocytes) for
transplantation. Transplanted stem cells may also be used as
vectors for the expression of therapeutic molecules, such as growth
factors, cytokines, anti-apoptotic proteins, and the like. Thus,
stem cells are a potential source of cells for alternative
treatments of diseases involving loss of cells or tissues.
[0009] The safest type of tissue graft (using stem cells or
otherwise) is one that comes from self (an autologous tissue
source). Autologous tissue sources are widely used in procedures
such as bone transplants and skin transplants because a source of
healthy tissue is readily accessible for transplant to a damaged
tissue site. In brain diseases, such as Parkinson's disease,
healthy dopaminergic neuronal brain tissue may exist at other sites
in the brain, but attempts to transplant these neurons may harm the
site where the healthy neurons originate. Multipotent stem cells
that can be differentiated into dopaminergic neurons may be
available at other sites from which they may be transplanted, but
the CNS, particularly the brain, is physically difficult to
access.
[0010] In several tissues, stem cells have been purified and
characterized. For example, neural stem cells have been purified
from the mammalian forebrain (Reynolds and Weiss, Science
255:1707-1710, 1992) and these cells were shown to be capable of
differentiating into neurons, astrocytes, and oligodendrocytes. PCT
publications WO 93/01275, WO 94/16718, WO 94/10292 and WO 94/09119
describe uses for these cells. It could be impractical or
impossible, however, to first access brain or other CNS tissue for
biopsy and then again for transplant in patients with weakened
health. It would be very useful if there were accessible stem cells
capable of differentiating into CNS cell types, such as
dopaminergic neurons; such cells would be a source of cells for
autologous transplants.
[0011] Thus, there is a clear need to develop methods for
identifying from accessible tissues multipotent stem cells that can
act as a source of cells that are transplantable to the CNS, PNS,
or other tissues in vivo in order to replace damaged or diseased
tissue.
SUMMARY OF THE INVENTION
[0012] We have substantially purified multipotent stem cells, or
MNSCs, from the peripheral tissue of postnatal mammals, including
juvenile and adult mammals. We have identified skin as a source of
MNSCs and provide methods for the purification of skin-derived
MNSCs, thus simplifying the harvesting of cells for transplantation
relative to previous methods. The MNSCs possess desirable features
in that they are multipotent and self-renewing. The cells can be
repeatedly passaged and differentiated into numerous cell types of
the CNS, including astrocytes, oligodendrocytes, and neurons. The
MSCs of this invention express nestin, an immunological marker of
stem cells and progenitor cells, as well as fibronectin. The cells
are capable of differentiating as dopaminergic neurons, and thus
are useful source of dopaminergic neurons for homotypic grafts into
Parkinson's Disease patients. The MNSCs have been demonstrated to
make smooth muscle cells and adipocytes, and may also make other
non-neural cells such as cardiac muscle cells, pancreatic islet
cells (e.g., alpha, beta, phi, delta cells), hematopoietic cells,
hepatocytes, and the like. The cells may also be used for
autologous or heterologous transplants to treat, for example, other
neurodegenerative diseases, disorders, or abnormal physical
states.
[0013] Accordingly, in a first aspect, the invention features MNSCs
substantially purified from a peripheral tissue of a postnatal
mammal. In one embodiment the peripheral tissue includes a sensory
receptor.
[0014] In a second aspect, the invention features a cell that is
the progeny of a MNSC substantially purified from a peripheral
tissue of a postnatal mammal. The cell may be a mitotic cell or a
differentiated cell (e.g., a neuron, an astrocyte, an
oligodendrocyte, a Schwann cell, or a non-neural cell). Preferred
neurons include neurons expressing one or more of the following
neurotransmitters: dopamine, GABA, glycine, acetylcholine,
glutamate, and serotonin. Preferred non-neural cells include
cardiac muscle cells, pancreatic cells (e.g., islet cells (alpha,
beta, phi and delta cells,)), exocrine cells, endocrine cells,
chondrocytes, osteocytes, skeletal muscle cells, smooth muscle
cells, hepatocytes, hematopoietic cells, and adipocytes.
[0015] In a third aspect, the invention features a population of at
least ten cells, wherein at least 30% of the cells are MNSCs
substantially purified from a peripheral tissue of a postnatal
mammal or progeny of the MNSCs. In one embodiment the peripheral
tissue includes a sensory receptor.
[0016] Preferably, at least 50% of the cells are MNSCs
substantially purified from the peripheral tissue or progeny of the
MNSCs. More preferably, at least 75% of the cells are MNSCs
substantially purified from the peripheral tissue or progeny of the
MNSCs. Most preferably, at least 90%, 95%, or even 100% of the
cells are MNSCs substantially purified from the peripheral tissue
or progeny of the MNSCs. The MNSCs may be cultured for extended
periods of time. Thus, the population of cells may have been in
culture for at least thirty days, sixty days, ninety days, or
longer (e.g., one year or more). Preferably, the population is at
least twenty cells, and may be more than fifty cells, a thousand
cells, or even a million cells or more.
[0017] In a fourth aspect, the invention features a pharmaceutical
composition including (i) a mitotic or differentiated cell that is
the progeny of a MNSC substantially purified from a peripheral
tissue of a postnatal mammal, and (ii) a pharmaceutically
acceptable carrier, auxiliary or excipient
[0018] In a fifth, related aspect, the invention features a
pharmaceutical composition including (i) a MNSC substantially
purified from a peripheral tissue of a postnatal mammal, and (ii) a
pharmaceutically acceptable carrier, auxiliary or excipient.
[0019] Preferably, the composition of the fourth or fifth aspect
includes a population of cells, wherein at least 30%, 50%, 75%,
90%, 95%, or even 100% of the cells are MNSCs substantially
purified from the peripheral tissue or progeny of the MNSCs. The
composition may include one or more types of cells selected from a
group consisting of MNSCs, or neurons, oligodendrocytes, Schwann
cells, astrocytes, adipocytes, smooth muscle cells, cardiomyocytes,
chondrocytes, osteocytes, skeletal muscle cells, hepatocytes,
hematopoietic cells, exocrine cells endocrine cells and alpha,
beta, phi and delta cells, which are progeny of MNSCs.
[0020] In a sixth aspect, the invention features a method of
producing a population of at least ten cells, wherein at least 30%
of the cells are MNSCs substantially purified from a peripheral
tissue of a postnatal mammal or progeny of the MNSCs, wherein the
peripheral tissue includes a sensory receptor, the method
including: (a) providing the peripheral tissue from the mammal; (b)
culturing the tissue under conditions in which MNSCs proliferate
and in which at least 25% of the cells that are not MNSCs die; and
(c) continuing culture step (b) until at least 30% of the cells are
MNSCs or progeny of the MNSCs.
[0021] In a seventh aspect, the invention features another method
of producing a population of at least ten cells, wherein at least
30% of the cells are MNSCs substantially purified from skin tissue
of a postnatal mammal or progeny of the MNSCs, the method
including: (a) providing the skin tissue from the mammal; (b)
culturing the tissue under conditions in which MNSCs proliferate
and in which at least 25% of the cells that are not MNSCs die; (c)
separating the MNSCs from cells that are not MNSCs; and (d)
repeating steps (b) and (c) until at least 30% of the cells are
MNSCs or progeny of the MNSCs.
[0022] Suitable culture conditions for step (b) of the sixth and
seventh aspects are preferably as follows: (i) triturating or
otherwise separating tissue into single cells or cell clusters and
placing into culture medium; (ii) culturing the cells in culture
medium and under conditions (e.g., DMEM: Ham's F-12 medium
containing B-27 supplement, antibacterial and antifungal agents,
5-100 ng/ml bFGF, and 2-100 ng/ml EGF) that allows for the
proliferation of MNSCs but does not promote, to the same extent,
proliferation of cells that are not MNSCs; and (iii) culturing the
separated tissue for three to ten days, during which time the MNSCs
proliferate in suspension but non-MNSCs do not proliferate in
suspension (these cells either attach to the plastic or they die).
Preferably, at least 50% of the cells in suspension surviving after
the period in culture are MNSCs or progeny of the MNSCs, more
preferably, at least 75% of the cells are MNSCs or progeny of the
MNSCs, and, most preferably, at least 90% or even 95% of the
surviving cells are MNSCs or progeny of the MNSCs. In preferred
embodiments tissue is separated mechanically.
[0023] In an eighth aspect, the invention features a method of
treating a patient having a disease associated with cell loss. The
method includes the step of transplanting the multipotent stem
cells of the invention into the region of the patient in which
there is cell loss. Preferably, prior to the transplanting step,
the method includes the steps of providing a culture of peripheral
tissue, preferably containing sensory receptors, from the patient
and isolating a multipotent neural stem cell from the peripheral
tissue. After transplanation, the method may further include the
step of differentiating (or allowing the differentiation of) the
MNSCs into a desired cell type to replace the cells that were lost.
Preferably, the region is a region of the CNS or PNS, but can also
be cardiac tissue, pancreatic tissue, or any other tissue in which
cell transplantation therapy is possible.
[0024] In a ninth aspect, the invention features a kit including
MNSCs substantially purified from peripheral tissue of a postnatal
mammal, or a mitotic or differentiated cell that is the progeny of
the MNSC, preferably wherein the peripheral tissue from which the
MNSC is purified includes a sensory receptor. Preferably, the kit
includes a population of cells, wherein at least 30%, 50%, 75%,
90%, or even 95% of the cells are MNSCs substantially purified from
the peripheral tissue or progeny of the MNSCs.
[0025] In a tenth aspect, the invention features a kit for
purifying MNSCs from peripheral tissue containing sensory
receptors. The kit includes media or media components that allow
for the substantial purification of MNSCs of the present invention.
The kit may also include media or media components that allow for
the differentiation of the MNSCs into the desired cell type(s).
Preferably, the kit also includes instructions for its use.
[0026] In one preferred embodiment of each of the foregoing aspects
of the invention, the peripheral tissue is skin tissue. In another
preferred embodiment, the peripheral tissue is olfactory
epithelium, tongue tissue, hair follicles, sweat glands, or
sebaceous glands. In still another embodiment, the peripheral
tissue of the first aspect specifically excludes olfactory
epithelium and tongue tissue. In another preferred embodiment of
each of the foregoing aspects of the invention, the stem cells do
not express detectable levels of p75.
[0027] The peripheral tissue can be from a newborn mammal, a
juvenile mammal, or an adult mammal. Preferred mammals include, for
example, humans, non-human primates, mice, pigs, and rats. The
MNSCs can be derived from peripheral tissue of any individual,
including one suffering from a disease or from an individual
immunologically compatible to an individual suffering from a
disease. In a preferred embodiment, the cells, or progeny of the
cells, are transplanted into the CNS or PNS of an individual having
a neurodegenerative disease and the individual is the same
individual from whom the MNSCs were purified. Following
transplantation, the cells can differentiate into cells that are
lacking or non-functional in the disease.
[0028] Preferably, the MNSCs express nestin and finronectin and may
also express glutamic acid decarboxylase. The MNSCs of the present
invention can, under appropriate conditions, differentiate into
neurons, astrocytes, Schwann cells, oligodendrocytes, and/or
non-neural cells (e.g., cardiac cells, pancreatic cells, smooth
muscle cells, adipocytes, hepatocytes, etc.).
[0029] MNSCs can be stably or transiently transformed with a
heterologous gene (e.g., one encoding a therapeutic protein, such
as a protein which enhances cell divisions or prevents apoptosis of
the transformed cell or other cells in the patient, or a cell
fate-determining protein).
[0030] By "multipotential stem cell" or "multipotent neural stem
cell" is meant a cell that (i) has the potential of differentiating
into at least two cell types selected from a neuron, an astrocyte,
and an oligodendrocyte, and (ii) exhibits self-renewal, meaning
that at a cell division, at least one of the two daughter cells
will also be a stem cell. The non-stem cell progeny of a single MSC
or MNSC are capable of differentiating into neurons, astrocytes,
Schwann cells, and oligodendrocytes. Hence, the neural stern cell
is "multipotent" because its progeny have multiple differentiative
pathways. The MSC or MNSC also has the potential to differentiate
as another cell type (e.g., a skin cell, a hematopoietic cell, a
smooth muscle cell, a cardiac muscle cell, a skeletal muscle cell,
or a pancreatic cell). The term MSC or MNSC is not intended to be
limited to cells that are developmentally dervied from neural
lineages.
[0031] By "substantially purified" is meant that the desired cells
(e.g., MNSCs) are enriched by at least 30%, more preferably by at
least 50%, even more preferably by at least 75%, and most
preferably by at least 90% or even 95%.
[0032] By "therapeutic protein" is meant a protein that improves or
maintains the health of the cell expressing the protein or a that
of a cell in proximity to the expressing cell. Example therapeutic
proteins include, without limitation, growth factors (NGF, BDNF,
NT-3, NT4/5, HGF, TGF-family members, PDGF, GDNF, FGF, EGF family
members, IGF, insulin, BMPs, Wnts, hedgehogs, and heregulins)
cytokines (LIF, CNTF, TNFm interleukins, and gamma-interferon), and
anti-apoptotic proteins (LAP proteins, Bcl-2 proteins, Bcl-X.sub.L,
Trk receptors, Akt PI3 kinase, Gab, Mek, E1B55K, Raf, Ras, PKC,
PLC, FRS2, rAPs/SH2B, and Np73).
[0033] By "peripheral tissues" is meant a tissue that is not
derived from neuroectoderm, for example peripheral tissue
containing senory receptors, and specifically includes olfactory
epithelium, tongue, skin (including dermis and/or epidermis), and
mucosal layers of the body (e.g., mouth, reproductive system).
[0034] By a "population of cells" is meant a collection of at least
ten cells. Preferably, the population consists of at least twenty
cells, more preferably at least one hundred cells, and most
preferably at least one thousand or even one million cells. Because
of the MNSCs of the present invention exhibit a capacity for
self-renewal, they can be expanded in culture to produce
populations even billions of cells.
[0035] By "postnatal" is meant an animal that has been born at
term.
[0036] By "a disease characterized by failure of a cell type" is
meant one in which the disease phenotype is the result of loss of
cells of that cell type or the loss of function of cells of that
cell type.
[0037] Other features and advantages of the present invention will
become apparent from the following detailed description and the
claims. It will be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of example only, and
various changes and modifications within the spirit and scope of
the invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIGS. 1A-1G are photographs showing that mouse skin-derived
MNSCs are nestin-positive and are capable of differentiating into
neurons, glia, and smooth muscle cells.
[0039] FIG. 2 is a series of photographs showing that neonate and
adult mouse skin-derived MNSCs express both nestin (middle row) and
fibronectin (bottom row).
[0040] FIG. 3A is a series of photographs showing western blot
analysis for nestin, neurofilament M (NF-M) and GFAP in cells
differentiated from neonate and adult mouse skin-derived MNSCs.
[0041] FIG. 3B is a series of photographs showing that human
skin-derived MNSCs express nestin.
[0042] FIG. 3C is a series of photographs showing that a subset of
morphologically complex cells expressed nestin and -tubulin, a
profile typical of newly-born neurons.
[0043] FIG. 3D is a series of photographs showing that GFP positive
cells are also positive for neuron-specific enolase.
[0044] FIG. 4A is a photograph showing the expression of A2B5, a
marker for oligodendrocyte precursors, on undifferentiated mouse
skin-derived MNSCs.
[0045] FIG. 4B is a photograph showing the expression of the
oligodendrocyte marker galactocerebroside (GalC) on cells
differentiated from mouse skin-derived MNSCs.
[0046] FIG. 5 is a series of photographs showing that the fate of
mouse skin-derived MNSCs can be manipulated by controlling plating
conditions.
[0047] FIG. 6 is a series of photographs showing that neonate and
adult mouse skin-derived MNSCs can differentiate as adipocytes.
[0048] FIGS. 7A and 7B are photographs showing that
nestin-positive, fibronectin-positive MNSCs can be derived from
mouse dermis.
[0049] FIGS. 8A and 8B are photographs showing that individual
MNSCs are multipotent. Clones derived from single cells contained
NF-M-positive cells (arrowheads) and CNPase-positive cells (arrows)
(FIG. 3A). In FIG. 3B, arrowheads indicate cells that only express
GFAP, while arrows indicate cells expressing both GFAP and
CNPase.
[0050] FIGS. 9A and 9B are photographs of western blot analysis of
cells differentiated from mouse skin-derived MNSCs (FIG. 9A) or of
MNSCs themselves (FIG. 9B).
[0051] FIG. 10 is a series of photographs showing the effect of
various pharmacological agents on mouse skin-derived MNSCs.
[0052] FIGS. 11A-11E are photographs of immunoprocessed sections of
rat brains into which mouse skin-derived MNSCs were
transplanted.
DETAILED DESCRIPTION OF THE INVENTION
[0053] We have substantially purified multipotent stem cells, e.g.,
neural stem cells (MNSCs), from peripheral tissues of mammals,
including skin, olfactory epithelium, and tongue. These cells
proliferate in culture, so that large numbers of stem cells can be
generated. These cells can be induced to differentiate, for
example, into neurons, astrocytes, and/or oligodendrocytes by
altering the culture conditions. They can also be induced to
differentiate into non-neural cells such as smooth muscle cells and
adipocytes. The substantially purified neural stem cells are thus
useful for generating cells for use, for example, in autologous
transplants for the treatment of degenerative disorders or trauma
(e.g., spinal cord injury). In one example, MNSCs may be
differentiated into dopaminergic neurons and implanted in the
substantia nigra or striatum of a Parkinson's disease patient. In a
second example, the cells may be used to generate oligodendrocytes
for use in autologous transplants for the treatment of multiple
sclerosis. In still another example, the MNSCs may be used to
generate Schwann cells for treatment of spinal cord injury, cardiac
cells for the treatment of heart disease, or pancreatic islet cells
for the treatment of diabetes. If desired, in any of the foregoing
examples, the cells may be genetically modified to express, for
example, a growth factor, an anti-apoptotic protein, or another
therapeutic protein.
[0054] The MNSCs display some similarities to stem cells derived
from mammalian forebrain, but also possess some distinctive
differences. In particular, when the MNSCs of the present invention
differentiate in the presence of serum, about 5-20% of the
differentiated cells express neuronal markers, whereas
differentiated forebrain stem cells generate only a small
percentage of neurons. Moreover, significant numbers of
dopaminergic neurons are found in differentiated cultures of MNSCs
of the present invention, whereas such neurons have not been
observed in cultures of forebrain stem cells differentiated in
serum.
[0055] Cell Therapy
[0056] The multipotent stem cells of this invention may be used to
prepare pharmaceutical compositions that can be administered to
humans or animals for cell therapy. The cells may be
undifferentiated or differentiated prior to administration. Dosages
to be administered depend on patient needs, on the desired effect,
and on the chosen route of administration.
[0057] The invention also features the use of the cells of this
invention to introduce therapeutic compounds into the diseased,
damaged, or physically abnormal CNS, PNS, or other tissue. The
MNSCs thus act as a vector to transport the compound. In order to
allow for expression of the therapeutic compound, suitable
regulatory elements may be derived from a variety of sources, and
may be readily selected by one with ordinary skill in the art.
Examples of regulatory elements include a transcriptional promoter
and enhancer or RNA polymerase binding sequence, and a ribosomal
binding sequence, including a translation initiation signal.
Additionally, depending on the vector employed, other genetic
elements, such as selectable markers, may be incorporated into the
recombinant molecule. The recombinant molecule may be introduced
into the stem cells or the cells differentiated from the stem cells
using in vitro delivery vehicles such as retroviral vectors,
adenoviral vectors, DNA virus vectors and liposomes. They may also
be introduced into such cells in vivo using physical techniques
such as microinjection and electroporation or chemical methods such
as incorporation of DNA into liposomes. The genetically altered
cells may be encapsulated in microspheres and implanted into or in
proximity to the diseased or damaged tissue.
[0058] In one embodiment, the MNSCs are used for the treatment of
neurological disease. In another aspect the MNSCs of the present
invention may also be used as a source of non-neural cells, for
example adipocytes and smooth muscle cells. As an example, PCT
publication WO99/16863 describes the differentiation of forebrain
MNSCs into cells of the hematopoietic cell lineage in vivo. The
MNSCs of the present invention appear to be more plastic and thus
are highly likely to also be capable of differentiating into
non-neural cells types, such as hematopoietic cells. Accordingly,
the invention features methods of treating a patient having any
disease or disorder characterized by cell loss by administering
MNSCs of the present invention (or cells derived from these cells)
to that patient and allowing the cells to differentiate to replace
the cells lost in the disease or disorder. For example,
transplantation of MNSCs and their progeny provide an alternative
to bone marrow and hematopoietic stem cell transplantation to treat
blood-related disorders. Other uses of the MNSCs are described in
Ourednik et al. (Clin. Genet. 56:267-278, 1999), hereby
incorporated by reference. MNSCs and their progeny provide, for
example, cultures of adipocytes and smooth muscle cells for study
in vitro and for transplantation. Adipocytes secrete a variety of
growth factors that mybe desirable in treating cachexia, muscle
wasting, and eating disorders. Smooth muscle cells may be, for
example, incorporated into vascular grafts, intestinal grafts,
etc.
EXAMPLE 1
[0059] Purification of MNSCs from Postnatal Mouse Olfactory
Epithelium
[0060] MNSCs from mouse olfactory epithelium were purified as
described below. Postnatal mice were stunned with a blow to the
head and then decapitated. The heads were sagitally sectioned with
a razor blade, and the olfactory epithelia of about six postnatal
(P1-P9) mouse pups were stripped from the conchae, nasal septum,
and vomeronasal organs using watch-maker forceps. This tissue was
placed into 3 mL of medium (DMEM/F-12 3:1) supplemented with 2%
B-27 (Gibco, Burlington, Ontario, Canada), 20 ng/mL epidermal
growth factor (EGF; Collaborative Research, Bedford, Mass.), 0.1%
fungizone, and 0.5 mL/100 mL penicillin/streptomycin (Gibco).
Following collection, the epithelia were teased apart with
watchmaker forceps, releasing a large number of single cells and
small cell clusters. The cell suspension was transferred to a 15 mL
tube, and 7 mL of additional medium was added. The clusters were
dissociated into single cells by titration with a 10 mL plastic
pipette and passed through a 60 micron filter (Gibco). Typically,
dissociated cells from the olfactory epithelia from six pups were
plated into two 50 mL tissue culture flasks and cultured in a 37
C., 5% CO.sub.2 tissue culture incubator. Two days later, most
cells in the cultures were dead or dying. A small number (less than
1% of the initial cell number) of large, phase bright cells were
present, however, most of which were attached to the flask bottom.
Over the next two to six days, these cells divided and produced
spherical clusters, which became larger over time. At four to five
days in culture, there were approximately 500 clusters of dividing
cells per pup used in the original purification. Most of these cell
clusters detached from the flask surface over the next few days.
These detached cell clusters continued to grow and fused together
to become macroscopic, reaching approximately 100 .mu.m in diameter
following 10 DIV. After 12 DIV, the cell clusters became
macroscopic, reaching approximately 200 .mu.m or greater in
diameter.
[0061] If EGF was not added to the medium, small clusters of
dividing cells were still seen by 4 DIV, and some cell were formed,
indicating that the cells themselves were producing trophic factors
in quantities that, in some cases, was sufficient to maintain their
proliferation.
[0062] Greater than 95% of the cells in the dividing clusters
expressed nestin, a marker for stem cells and neural stem cells.
These nestin-positive cells could be repeatedly passaged,
indicating that the cells were neural stem cells. Six days after
purification, the medium (5 mL) was removed from the flasks. This
medium contained many clusters of stem cells that had detached from
the flask surface. The detached cells were triturated with a
fire-polished pipette, thereby dissociating many of the cell
clusters into single cells. The medium containing the cells was
then placed in a second flask with an additional 15 mL of fresh
medium (total volume=20 mL). After a further six days, one quarter
of the medium was removed and the detached clusters of cells were
again triturated and transferred to a new flask with 15 mL fresh
medium. These cells have been successfully passaged more than
twenty times without losing their multipotency.
EXAMPLE 2
[0063] Differentiation of Mouse MNSCs into Neurons, Astrocytes and
Oligodendrocytes
[0064] After the cellular clusters of Example 1 had been generated,
they could be differentiated into neurons, astrocytes, and
oligodendrocytes. Clusters from cultures 7 to 14 days after
purification were plated onto polylysine coated 35 mm culture
dishes or 4 multiwell culture dishes, in DMEM/F12 media containing
2% fetal bovine serum (Hyclone, Logan, Utah) and 2% B-27
(containing no EGF). The medium was changed every three to four
days. Over the next six to nineteen days, cells migrated out of the
clusters onto the dish surface. Some of these cells had the
morphology of neurons, astrocytes, or oligodendrocytes. We
determined the phenotype of these cells using the following
antibodies: GFAP for astrocytes; neurofilament 160 (NF-160), MAP-2,
III tubulin, and NeuN for neurons; and GC for oligodendrocytes.
Antibodies to TH were used to identify dopaminergic, noradrenergic,
and adrenergic neurons. Dopamine -hydroxylase (DBH) was also used
for noradrenergic and adrenergic neurons.
[0065] Astrocytes, neurons, and oligodendrocytes were all found to
differentiate from the MNSCs of this invention, indicating that the
cells were multipotent We also cultured MNSCs from transgenic mice
which express -galactosidase off of the neuron specific T1-tubulin
promoter, which allowed us to use staining with the ligand X-gal
antibodies for -galactosidase as an additional neuronal marker. We
observed -galactosidase-positive cells.
[0066] Since the majority of differentiated cells remained in
clusters, it was not possible to determine the percentage of cells
expressing each marker. The majority of cells that migrated out of
the clusters were GFAP positive, while a large number of cells were
either NeuN or -galactosidase positive. A lower number of cells
were GC positive. Therefore the MNSCs could differentiate into
neurons, astrocytes and oligodendrocytes. TH-positive cells were
also identified. These TH-positive cells are most likely
dopaminergic neurons and not noradrenergic or adrenergic neurons,
since no cells were found to be DBH positive. Significantly, no TH,
GFAP or GC positive cells have ever been reported in vivo in the
nasal epithelium. Therefore the olfactory epithelium-derived
nestin-positive MNSCs are capable of differentiating into cell
types (e.g., oligodendrocytes, astrocytes, GABAergic neurons, and
dopaminergic neurons) never found in the olfactory epithelium.
[0067] Like the originally-purified olfactory MNSCs, MNSCs passaged
from two to twenty times could also differentiate into neurons,
astrocytes, and oligodendrocytes. MNSCs which had been passaged
were plated on polylysine-coated dishes. Cells migrated from the
clusters and spread out over the surface of the dish. After 16 DIV,
cells that were immunopositive for GC, GFAP, m tubulin, NeuN, lacZ,
or TH could be identified. Moreover, the proportion of cells
positive for the various markers was similar to that seen in the
differentiated cultures from the original cultures.
EXAMPLE 3
[0068] Purification of MNSCs from Olfactory Epithelial Tissue of
Adult Mice and Rats
[0069] Similar to the foregoing results, MNSCs were also purified
from adult mouse and rat olfactory epithelium and vomeronasal organ
using the methods described in Examples 1 and 2.
[0070] Adult mice and rats were anaesthetized with an overdose of
somnitol, and then decapitated. The olfactory and vomeronasal organ
epithelia were stripped from the conchae and nasal septum and
incubated in DMEM/F12 medium for one to two days after their
dissection and prior to the rest of the purification procedure.
After this incubation, the epithelia were dissociated in an
identical manner as the epithelia from juvenile mice. Two days
after the isolation, the majority of the cells were dead with the
exception of a very few large phase bright cells. These cells
divided over the next few days, forming small clusters of dividing
cells similar to those described in Example 1. These small clusters
grew to give rise to the large clusters that 10 detached from the
culture dish surface. After approximately six divisions, cells in
some of these clusters began to differentiate and spread out over
the flask's surface, while some other clusters, which had been
floating, reattached to the surface and then produced
differentiated cells. In some cases, cells multiplied to produce
small clusters of cells, but did not grow to form large cell
clusters like the postnatal cultures. We have passaged these cells
twenty times using the same procedure as that described above with
respect to the cells purified from juvenile olfactory epithelium.
These proliferating cells from the adult were also
nestin-positive.
[0071] After the cell clusters derived from adult tissue had been
generated, the cells could be differentiated into neurons,
astrocytes, and oligodendrocytes. Seven days after isolation,
clusters were plated onto polylysine-coated 35 mm culture dishes or
multi-well culture dishes, in medium containing 2% fetal bovine
serum and 2% B-27, but no EGF. Over the next month, cells migrated
from the cell clusters and onto the dish surface. We determined the
phenotype of these cells using antibodies to astrocytes, neurons,
dopaminergic neurons, and oligodendrocytes as described above.
[0072] Neurons (including dopaminergic neurons), astrocytes, and
oligodendrocytes were found, although the number of these cells was
much lower than the number obtained from the juvenile. The cells
purified from adult olfactory epithelia are self-renewing and
multipotent, and thus are MNSCs.
EXAMPLE 4
[0073] Purification of MNSCs from Mouse Tongue
[0074] We derived MNSCs from the tongue, another peripheral tissue
that contains sensory receptors. The tongue was dissected to remove
the epithelial layer that contains the sensory receptors and their
underlying basal cells. This layer of tissue was triturated to
produce single cells and the single cells were plated in flasks
containing DMEM/F12 media supplemented with B-27 and EGF, TGF,
and/or bFGF, as described for the olfactory epithelium. After two
to three days in a 37.degree. C., 5% CO.sub.2 tissue culture
incubator, greater than 99% of the cells in the culture were dead
or dying. A small number (less than 1%) of large phase-bright cells
were present, however, most of which attached to the flask bottom.
Over the next two to six days, these cells divided and produced
spherical clusters that became larger over time and detached from
the flask surface. The cells in these clusters were
nestin-positive.
[0075] These nestin-positive MNSCs can be passaged using the same
techniques as used for the multipotent stem cells derived from the
olfactory epithelium. Similarly, the MNSCs can be differentiated
into neurons, astrocytes and oligodendrocytes using the techniques
described herein.
EXAMPLE 5
[0076] Purification of MNSCs from Mouse Skin
[0077] Skin from neonatal mice aged 3-15 days was dissociated and
cultured in uncoated flasks containing 20 mg/mL EGF and 40 mg/mL
bFGF. Over the subsequent one to five days, many (>90%) of the
cells die. A small population of cells hypertrophy and proliferate
to form small cell clusters growing in suspension. Some of thee
cells first attach to the tissue cluster plastic, hypertrophy and
proliferate, and then detach as the clusters become of sufficient
size. Other cells never attach to the tissue culture plastic and
instead proliferate in suspension from the beginning. After four to
five days, the cell clusters are small but easily distinguishable
as clusters of proliferating cells. By seven to ten days, many of
the cell clusters reach diameters of as much as 100 .mu.m, while by
two weeks, the cell clusters are macroscopic if left unperturbed
Many cells adhered to the plastic, and many died, but by about
three to seven days, suspended clusters of up to about 20 cells
formed. These suspended or floating cells were transferred to a new
flask seven days after initial culturing; again, many cells
adhered, but the cells in the floating clusters proliferated to
generate larger clusters of more than about 100 cells (FIG. 1A, top
panel). These larger clusters were then isolated, dissociated and
passaged. By this process of selective adhesion, substantially pure
populations of floating clusters were obtained after 3 to 4 weeks.
Cells that generated these clusters were relatively abundant; 1.5
to 2 cm.sup.2 of abdomen skin was sufficient to generate six 25
cm.sup.2 flasks of floating clusters over this period of time.
[0078] To determine whether clusters contained MNSCs, we
dissociated the clusters and plated the cells onto
poly-D-lysine/laminin-coated dishes or chamber slides without
growth factors and, 12 to 24 hours later, immunostained them for
the presence of the neural precursor-specific marker nestin. After
three passages, the majority of the cells expressed nestin (FIG.
1B, top panel), a property they maintained over subsequent
passages. They did not, however, express the p75 neurotrophin
receptor, a marker for neural crest stem cells, as detected either
by immunocytochemistry or western blots.
[0079] We also determined whether the skin-derived MNSCs expressed
fibronectin. Four lines of skin-derived MNSCs cultured from either
adult (FIG. 2; left two columns) or neonatal (right two columns)
mouse skin, cultured for either long term (first and third columns)
or short term (second and fourth columns) were each dissociated,
plated for two days in DMEM/F12 (3:1) containing 2% B-27 supplemen,
and then immunostained for nestin and fibronectin. As is
demonstrated in FIG. 2, the majority of cells expressed both
markers.
[0080] To determine whether clusters of cells could be generated
from adults, skin of adult mice was dissociated and cultured as
described above. Similar to neonatal mouse skin, most cells adhered
to the flask or died when first cultured. After three to seven
days, however, clusters of up to approximately 20 cells were
observed that subsequently increased in size. When these cells were
passaged at least three times (FIG. 1A, bottom panel), and plated
onto poly-D-lysine/laminin overnight in the absence of growth
factors, they too were immunopositive for nestin (FIG. 1B, bottom
panel) and fibronectin (FIG. 2). The nestin-positive cells from
adults and neonates have been passaged in this manner for over 30
passages, during which time the number would have theoretically
expanded at least 10.sup.9-fold (assuming a doubling time of
approximately one week).
[0081] To determine whether these nestin-positive,
fibronectin-positive cells from skin could generate neural cell
types, we analyzed neonatal skin-derived cells after three or more
passages and greater by plating them on poly-D-lysine/laminin in
the absence of growth factors. Immunostaining (FIGS. 1C and 1D) and
western blot analysis (FIG. 3A) revealed that the skin-derived
cells expressed neuronal markers. At seven days, a subpopulation of
morphologically-complex cells coexpressed nestin and
neuron-specific .beta.III-tubulin, a profile typical of newly-born
neurons (FIG. 1C). At later time points of 7-21 days, cells also
expressed neurofilament-M (NF-M)(FIGS. 1D, 3A) and neuron-specific
enolase, two other neuron-specific proteins. Finally, some
neurofilament-positive cells expressed GAD (FIG. 1D), a marker for
GABAergic neurons, which are not found in the PNS. Similar results
were obtained for adult skin-derived MNSCs, although at early
passages some of the the.beta.Illtubulin and neurofilament-positive
cells were less typically neuronal in morphology.
[0082] Immunostaining and western blots revealed that both neonatal
and adult MNSCs generated cells expressing the glial markers GFAP
and CNPase at seven to twenty-one days after plating (FIGS. 1D-1F,
2A. Double-labeling for these proteins demonstrated the presence of
(i) cells that were GFAP-positive but not CNPase-positive
(potentially astrocytes), (ii) cells that expressed CNPase but not
GFAP (potentially oligodendrocytes or their precursors), and (iii)
a small subpopulation that were bipolar and expressed both CNPase
and GFAP (potentially Schwann cells) (FIG. 1E). A subpopulation of
GFAP-positive cells also expressed nestin, a finding previously
reported for developing CNS astrocytes. Additionally, some cells
were positive for A2B5, a marker for oligodendrocyte precursors
(FIG. 4)). Like GAD-positive neurons, astrocytes and
oligodendrocytes are normally found only in the CNS.
[0083] Double-labeling studies supported the following additional
conclusions. First, glial versus neuronal markers were expressed in
distinct subpopulations of MNSCs progeny. Second, after twenty
passages, skin-derive MNSCs were still able to differentiate into
neurons and glial cells. Finally, skin-derived MNSCs were able to
generate smooth muscle cells (as determined by both expression of
smooth muscle actin (SMA) and morphology; FIG. 1G) and adipocytes
(FIGS. 5 and 6).
EXAMPLE 6
[0084] MNSCs Originate from the Dermal Layer of the Skin
[0085] The two major layers of the skin are the epidermis and the
dermis. To determine the origin of the skin-derived MNSCs, we
dissected and cultured P7, P14, and P18 mouse epidermis and dermis.
The two layers of the skin were separated by incubating the skin
pieces (1.times.2 cm.sup.2) in 0.2% trypsin at 40.degree. C. for
about 24-36 hours, or until the dermis could be separated from the
epidermis. The cells in each layer were dissociated separately and
then cultured in DMEM/F12 (3:1) with B-27 supplement, EGF (20
ng/mL) and FGF (40 ng/mL). Only the cells derived from the dermis
generated cluster of cells similar to those derived from whole skin
(FIG. 7A). No viable cells were obtained from the epidermis. To
characterize the dermis-derived cell clusters, the clusters were
cultured for four weeks and then plated onto tissue culture chamber
slides coated with poly-D-lysine and laminin. After 24 hours, the
cells were then processed for immunocytochemistry. Like MNSCs
derived from whole mouse skin, the dermis-derived cells coexpressed
nestin and fibronectin (FIG. 7B).
EXAMPLE 7
[0086] Clonal Analysis Indicates that Skin-derived MNSCs are
Multipotent
[0087] To determine whether skin-derived MNSCs are multipotent, we
isolated single cells by limiting dilution of cells from clusters
that three months prior had been derived from neonatal mice. We
cultured the cells for five weeks in medium from the same culture
line and containing growth factor, and then differentiated the
cells for two weeks in medium lacking growth factor but containing
3% rat serum. The cells were then processed for
immunocytochemistry. As is demonstrated in FIG. 8, single clones of
cells contained NF-M- and CNPase-positive cells (FIG. 8A), and
GFAP- and CNPase-positive cells (FIG. 8B).
EXAMPLE 8
[0088] Western Blot Analysis of Skin-derived MNSCs
[0089] For western blot analysis of skin-derived MNSCs, four
cultures (one adult-derived line and three neonate-derived lines)
that had been passaged from seven to 40 times were analyzed either
as clusters or following differentiation by plating in medium
containing 1% FBS, B-27 supplement, and fungizone for 14 days in 60
mm dishes coated with poly-lysine and laminin. Cell lysates were
prepared, and equal amounts (50-100 .mu.g) of protein from each
culture were separated on 7.5% or 10% polyacrylamide gels,
transferred to membrane, and then probed with anti-nestin
monoclonal antibody (1:1000; Chemicon), anti NF-M polyclonal
antibody (1:1000; Sigma), anti GFAP polyclonal antibody (1:1000,
Dako), or antifibronectin polyclonal antibody (1:1000; Sigma). As
positive controls, we used cortical progenitor cells cultured in
the presence of CNTF (which results in astrocytic differentiation)
or in the absence of CNTF (which results in neuronal
differentiation) and adult mouse cortex. As negative controls, we
used sympathetic neurons and liver. As illustrated in FIG. 9A,
western blotting confirmed the expression of GFAP and NF-M in
cultures differentiated from both adult and neonate skin-derived
MNSCs. Similarly, FIG. 9B illustrates the expression of both nestin
and fibronectin in adult and neonate skin-derived MNSC
clusters.
EXAMPLE 9
[0090] MNSC Differentiation can be Modulated by Plating
Conditions
[0091] As is illustrated above, when clusters of skin-derived MNSCs
are dissociated and plated in medium containing FGF and EGF, most
of the nestin-positive cells become neurofilament-positive. We have
found that when the cells are plated in medium containing 10% FBS,
the cells adopt a morphology similar to that displayed by
adipocytes. The adoption of the adipocyte cell fate was confirmed
by staining with Oil Red O (FIG. 5). The ability of 10% FBS to
induce adipocyte differentiation was true for both adult and
neonate skin-derived MNSCs (FIG. 6).
EXAMPLE 10
[0092] Pharmacological Inhibitors Affect Survival and Proliferation
of Skin-derived MNSCs
[0093] When skin-derived MNSCs are plated for three days in
proliferation medium containing FGF, they typically exhibit a
spherical morphology characteristic of their proliferative state
(FIG. 10). We tested the ability of pharmacological agents to alter
this phenotype. Supplementing the medium with PD098059 (an
inhibitor of the ERK MAPK pathway) caused proliferating cells to
flatten and differentiate (FIG. 10), while supplementing witht
LY294002 (an inhibitor of the PI-3-K pathway, caused the cells to
die (FIG. 10). The p38 MAPK inhibitor SB203580 had no observed
effect on the proliferating skin-derived MNSCs.
EXAMPLE 11
[0094] Purification of Nestin-positive Cells from Adult Human
Skin
[0095] We have purified nestin-positive cells from human scalp. To
purify MNSCs from human skin, we utilized tags of scalp tissue
generated by placement of a stereotactic apparatus during
neurosurgery. Scalp tags totalling 1 cm.sup.2 or less from each of
eight individuals were used. The skin included dermal and epidermal
tissue. Tissue was cut into smaller pieces that were then
transferred into HBSS containing 0.1% trypsin for forty minutes at
37.degree. C. Following trypsinization, tissue samples were washed
twice with HBSS and once with DMEM:F12 (3:1) supplemented with 10%
rat serum to inactivate the trypsin. Trypsinized tissue was then
mechanically dissociated by trituration in a pipette and the
resulting dispersed cell suspension was poured through a 40 .mu.m
cell strainer into a 15 mL tube. The tube was then centrifuged for
five minutes at 1000 rpm (.about.1200.times.g). The cells were
resuspended in DMBM:F12 medium containing 40 ng/mL bFGF, 20 ng/nL
EGF, 2% B-27 supplement, and antibacterial and antifungal agents,
and then cultured in 12 well plastic tissue culture plates. Every
seven days, the cell clusters are harvested by centrifugation,
triturated with a fire-polished pasteur pipette, and cultured in
fresh medium.
[0096] As for the use of rodent skin, most cells (>75%) adhered
to the plastic or died, but after seven days, small floating
clusters of cells were observed. These clusters were then partially
dissociated and transferred to new wells, where they slowly
increased in size. After additional passaging, clusters were plated
on poly-D-lysine/laminin in 3% FBS with no growth factors, and
analyzed for the presence of neural markers.
[0097] Within two weeks, greater than 30% of the cells within the
cell clusters were nestin-positive (FIG. 3B). Immunolabeling of
four to six week old cultures also revealed that many of the cells
in the clusters were nestin-positive with the percentage varying
from less than 50% to greater than 80% two to three days after
plating. Double-label immunocytochemistry at the same or longer
time-points revealed that, in all cultures, some nestin-positive
cells also expressed .beta.III-tubulin and displayed elongated
neurites (FIG. 3C). Thus, adult human skin is a source for
nestin-positive MNSCs cells that, when differentiated, can express
neuron-specific proteins.
EXAMPLE 12
[0098] Purification and Differentiation of MNSCs Derived from other
Human Peripheral Tissues Containing Sensory Receptors
[0099] MNSCs can be purified from human olfactory epithelium using
the same procedures as described for the purification of stem cells
from rodent olfactory epithelium. Source material is acquired by
surgical removal of olfactory epithelial tissue from the donor.
Because the MNSCs are capable of proliferation and self-renewal,
little source tissue is required. Preferably, the amount is at
least about 1 mm.sup.3. Conditions for culturing human cells are
described in Example 6, above. Other conditions are known to those
skilled in the art, and can be optimized for proliferation or
differentiation of neural stem cells, if desired.
[0100] We can purify MNSCs from other peripheral tissues containing
sensory receptors, other than the olfactory epithelium, tongue, and
skin, using techniques described herein. Passaging and
differentiation of these cells is also performed using the same
techniques described herein. Other peripheral tissues containing
sensory receptors include, for example, mucosal membranes from the
mouth or reproductive system.
EXAMPLE 13
[0101] Transformation of MNSCs
[0102] In therapy for neurodegenerative diseases, it may be
desirable to transplant cells that are genetically modified to
survive the insults that caused the original neurons to die. In
addition, MNSCs may be used to deliver therapeutic proteins into
the brain of patients with neurodegenerative disorders to prevent
death of host cells. Examplary therapeutic proteins are described
herein. In still another example, MNSCs can be induced to
differentiate into a desired cell type by transforming the cells
with nucleic acid molecules encoding proteins that regulate cell
fate decisions (e.g., transcription factors such as Is1-1, en-1,
en-2 and nurr-1, implicated in regulating motorneuron and striatal
phenotypes). Using such a method, it is possible to induce the
differentiation of the specific cell types required for transplant
therapy. Therefore, it would be advantageous to transform MNSCs
with nucleic acid molecules encoding desired proteins. We have
previously used recombinant adenovirus to manipulate both
postmitotic sympathetic neurons and cortical progenitor cells, with
no cytotoxic effects. We now have established that olfactory
epithelial-derived MNSCs and skin-derived MNSCs can each be
successfully transfected with high efficiency and low toxicity.
EXAMPLE 14
[0103] Differentiation of MNSCs into the Appropriate Cell Type in
Vivo following Transplantion into Adult Rodent Brain
[0104] One therapeutic use for the MNSCs of the present invention
is autologous transplantation into the injured or degenerating CNS
or PNS to replace lost cell types and/or to express therapeutic
molecules. We demonstrate below that the MNSCs can differentiate
into neurons when transplanted into the adult brain.
[0105] If desired, the dopaminergic innervation of the adult
striatum can be unilaterally destroyed by a local infusion of
6-hydroxydopamine under conditions in which noradrenergic neurons
are spared. Several weeks later, MNSCs are transplanted into both
the intact and lesioned striatum. Altenatively, the cells can be
transplanted into unlesioned animals. The fate of the transplanted
MNSCs is then determined by immunohistochemistry. Exemplary
transplantation studies are described below. These studies
demonstrate that transplanted MNSCs can differentiate into neurons
in vivo, as they can in vitro. In the former case, differentiation
and cell fate choice is controlled by the local environment into
which each cell is placed. Both in vitro-differentiated and
undifferentiated cells are useful, therapeutically in the
treatment, for example, of neurodegenerative disease (e.g.,
Parkinson's disease and multiple sclerosis) or spinal cord injury.
For example, dopamingeric neurons differentiated from MNSCs, or the
MNSCs themselves, may be transplanted into the substantia nigra or
the striatum of patients having Parkinson's disease. If desired,
the MNSCs may also be genetically-modified to express a desired
protein.
[0106] In one example, the dopaminergic innervation to adult rat
striatum was first unilaterally lesioned with the chemotoxin
6-hydroxydopamine, and the efficacy of the lesions was tested two
weeks later by amphetamine-induced rotational behavior. Two days
prior to transplantation, rats were immunosuppressed with
cyclosporin. MNSCs, produced from olfactory epithelia as described
herein, were then stereotactically injected into the
caudate-putamen complex on both the lesioned and unlesioned sides.
Sixteen days following transplantation, animals were sacrificed,
and sections of the striatum were analyzed for nestin- and
TH-immunoreactivity. Five of eight animals received successful
injections of MNSCs in the striatum. Of these, four animals showed
evidence of a nestin-positive tract on both the lesioned and
unlesioned sides, although tracts on the lesioned side appeared to
be more intensely nestin-immunoreactive. On adjacent sections,
TH-positive cells were observed confined to an area close to the
transplant tract on both the lesioned and unlesioned side. As many
as 25-30 TH-positive cells were identified on each section. Cell
morphology varied from small, round cells lacking processes to
neurons that were morphologically complex with multiple fine
processes. In some cases, the processes of these TH-positive
neurons extended into the striatum for distances of up to 300
.mu.m.
[0107] To confirm that these TH-positive neurons derived from the
MNSCs, we performed two sets of experiments in which the
transplanted cells were detectably-labeled. In one set of
experiments, transplanted MNSCs were derived from T 1:n1acZ
transgenic mice, in which the neuron-specific T 1-tubulin promoter
drives expression of a nuclear-localized .beta.-galactosidase
marker gene. Immunohistochemical analysis of animals receiving the
transgenic MNSCs revealed the presence of
.beta.-galactosidase-positive neurons within the transplant tract,
confirming that the transplanted MNSCs generated neurons in vivo,
as they did in vitro. In a second set of experiments, MNSCs were
labelled with BrdU for 18 hours, washed to remove the BrdU label,
and then transplanted unilaterally into the
6-hydroxydopamine-lesioned striatum of animals (10 rats, 4 mice)
prepared as described herein. Immunohistochemical analysis with
anti-BrdU revealed that all animals showed evidence of
BrdU-positive transplant tracts. Immunocytochemistry with anti-GFAP
revealed that, in both xenografts and allografts, GFAP-positive
cells with heterogeneous morphology were concentrated at the
transplant site, but were also present in moderate amounts over the
entire ipsilateral hemisphere, with additional scattered reactive
astrocytes seen in the contralateral hemisphere. GFAP-BrdU
double-labelled cells were present mainly within or close to the
transplant tract, and varied in morphology from small, round cells
with only a few processes, to large polygonal or fusiform cells
with multiple processes. Immunohistochemistry with anti-TH revealed
that TH-BrdU double-labeled cells were also present, although these
were few in number relative to GFAP-BrdU positive cells. BrdU-TH
double-labeled cells were mainly small to medium-sized without
processes, although some examples of double-labeled cells with
processes were found within and adjacent to, the transplant tract.
Thus, MNSCs generated astrocytes and neurons in vivo, and a
subpopulation of the latter were TH-positive. Together, these
findings show that peripheral tissue-derived MNSCs are capable of
generating cell types that are never found within olfactory tissue,
including oligodendrocytes and TH-positive neurons.
[0108] To determine whether skin-derived MNSCs also generate
differentiated neural cell types in vivo, we tagged adult mouse
skin-derived MNSCs with (i) BrdU, and (ii) a recombinant adenovirus
expressing GFP, and then transplanted them as cell clusters of
about 20 to about 100 cells into the lateral ventricles of P2 rats.
Immunostaining fourteen days later revealed that, in all animals
analyzed (n=8), transplanted cells had migrated extensively (FIG.
11A). In particular, tagged cells had integrated into the cortex,
the hypothalamus and the amygdala in all, and into the hippocampus
in two of the transplanted brains (FIG. 11A). In the cortex,
GFP-positive cells were located in patches (FIGS. 11A, 11B) or
occasionally as single cells (FIG. 11C), including some that had
integrated into and adopted the morphology of layer V pyramidal
neurons (FIGS. 11B, 11C). These cells had triangular-shaped soma,
and projected a presumptive apical dendrite from layer V towards
layer I, in a manner similar to the endogenous layer V neurons.
That these cells were neurons was demonstrated by double-labeling
for neuron-specific enolase (FIG. 3D). Immunocytochemical analysis
also confirmed that these were transplanted cells, as BrdU-positive
cells were present in the same locations as GFP-positive cells in
all brains (FIG. 11B).
[0109] In both the amygdala and hippocampus, transplanted cells
also displayed neuronal morphology. In the amygdala, GFP and
BrdU-positive cells were large, with prominent nuclei, and
extensive processes (FIG. 11E). In the hippocampus, transplanted
cells had integrated into both the dentate gyrus and pyramidal cell
layers, and their morphology was typical of the endogenous granule
and pyramidal cells, respectively (FIGS. 11A, 11E). GFP-positive
staining was also seen within the molecular layer. Finally, GFP-
and BrdU-positive cells were observed in other locations, such as
the hypothalamus, where the morphology of many cells was not
typically neuronal.
[0110] Skin-derived MNSCs tranplanted into adult rats also survive
and integrate. We labeled adult mouse skin-derived MNSCs that had
been passaged more than thirty times with the nuclear dye 33258,
washed extensively, and then injected the cells stereotactically
into the brains of adult rats that were immunosuppressed with
cyclosporin. Four weeks later, we sacrificed the animals by
perfusion and processed the brains for histological examination.
Hoeschst-labeled cells were present in the hippocampus, olfactory
bulb, and striatum. From these data, we conclude that the
transplanted skin-derived MNSCs are capable of survival following
transplantation. Moreover, cells are capable of migrating from the
site of injection to numerous brain regions.
[0111] Skin-derived MNSCs are also capable of survival, migration,
and integration following transplantation into a hemisected adult
mouse spinal cord. In this example, the cells were injected into
the injured sides of hemisected spinal cords. Eight days later, the
animals were sacrificed and the spinal cords processed for
histological analysis. Hoechst-labeled cells were present at the
site of the initial injection, and had also migrated extensively
into the injured spinal cord.
EXAMPLE 15
[0112] Differentiation of Non-neural Cells from MNSCs
[0113] In addition to being capable of differentiating as neural
cells (i.e., neurons, oligodendrocytes, astrocytes, and Schwann
cells), the peripheral tissue-derived MNSCs are capable of
differentiating as non-neural cells that are normally not found in
the tissue from which the cells were derived. For example, we have
demonstrated that the skin-derived MNSCs can differentiate as
smooth muscle cells and adipocytes. It is likely that the cells
described herein have even greater potential. Conditions for the
differentiation of the MNSCs into smooth muscle cells is described
herein.
[0114] Signals or conditions sufficient for inducing MNSCs to
differentiate as other cell types (e.g., lymphocytes, cardiac
muscle cells, skeletal muscle cells, melanocytes, and pancreatic
cells) are known in the art. For example, unique signals induce
neural crest-derived stem cells to become melanocytes, cartilage,
smooth muscle cells, or bone (for review, see LaBonne and
Bronner-Fraser, J. Neurobiol., 36:175-189, 1998; Sieber-Blum, Intl.
Rev. Cytol. 197:1-33, 2000). Conditions for inducing CNS-derived
neural stem cells to differentiate as non-neural cells such as
smooth muscle cells, skeletal muscle cells, hepatocytes,
hematopoietic cells, osteocytes, and chondrocytes have similarly
been elucidated (Bjornson et al., Science 283:534-537, 1999; Tsai
and McKay, J. Neurosci. 20:3725-3735, 2000; Keirstead et al., J.
Neurosci. 19:7529-7536, 1999; Mujtaba et al., Dev. Biol. 200:1-15,
1998; Clark et al., Science 288:1660-1663, 2000).
[0115] Our recent discovery that MNSCs maintain the potential to
produce both neural and non-neural cell types has been accompanied
by the discovery that non-neural stem cells such as bone
marrow-derived stem cells (i.e., stromal cells or mesenchymal stem
cells) also have the potential to produce a wide variety of neural
and non-neural stem cells (Ferrari et al., Science 279:1528-1530,
1998; Gussoni et al., Nature 401:390-394, 1999; Peterson et al.,
Science 284:1168-1170, 1999; Pereira et al., Proc. Natl. Acad. Sci.
USA 92:4857-4861, 1995; Prockop, Science 276:71-74, 1997; Kessler
and Byrne, Annu. Rev. Physiol. 61:219-242, 1999; Pittenger et al.,
Science 284:143-147). The conditions under which these bone
marrow-derived cells differentiate as, for example, skeletal muscle
cells, cardiac muscle cells, hepatocytes, adipocytes, osteocytes,
or chrondrocytes are likely to be conditions under which the
peripheral tissue-derived MNSCs would differentiate similarly.
Thus, the peripheral tissue-derived MNSCs described herein can be
induced to differentiate into both neural and non-neural cells that
are not normally found in the tissue from which the MNSCs were
derived.
[0116] The foregoing experiments were performed using the following
methods.
[0117] Skin-derived MNSC Culture
[0118] For neonatal (three to 14 days) and adult (two months to one
year) mice, skin from abdomen and back was carefully dissected free
of other tissue, cut into 2-3 mm.sup.3 pieces, washed three times
in HBSS, and then digested with 0.1% trypsin for 40 minutes at 37
C., followed by 0.1% DNAase for one minute at room temperature.
Tissue pieces were then washed twice with HBSS, once with media
(DMEM-F12, 3:1, 1 g/ml fungizone, 1% penicillin/streptomycin)
containing 10% rat serum (Harlan Bioproducts), and twice with
serum-free media. Skin pieces were then mechanically dissociated in
media, and the suspension poured through a 40 M cell strainer
(Falcon). Dissociated cells were centrifuged, and resuspended in 10
ml media containing B-27 supplement, 20 ng/ml EGF and 40 ng/ml bFGF
(both Collaborative Research). Cells were cultured in 25 cm.sup.2
tissue culture flasks (Corning) in a 37 C., 5% CO.sub.2 tissue
culture incubator.
[0119] To culture human skin-derived MNSCs, two to three pieces of
scalp tissue ranging between 4-9 mm.sup.2 (generated by placement
of the stereotaxic apparatus for neurosurgery), were washed with
HBSS, any subcutaneous tissue was removed, and the skin was cut
into small pieces 1-2 mm.sup.3 in size. Tissue pieces were
transferred to 15 mL Falcon tubes, washed three times with HBSS,
and enzymatically digested in 0.1% trypsin for 40 minutes at 37 C.,
and then washed as for mouse tissue. Dissociated cells were
suspended in 5 mL of the same media used for mouse cultures, with
the addition of 20 ng/ml LIF (R&D Systems Inc.). The cell
suspension was placed in Falcon 6-well tissue culture plates and
maintained in a 37 C., 5% CO.sub.2 tissue culture incubator. Cells
were subcultured by partial dissociation of the clusters that
formed every 7 to 10 days.
[0120] To passage floating clusters of cells, the medium containing
the cell clusters was centrifuged, the cell pellet mechanically
dissociated with a fire-polished Pasteur pipette, and the cells
reseeded in fresh media containing B-27 supplement and growth
factors as above. Cells were passaged every 6 to 7 days. For
induction of differentiation into neural cells, the cell clusters
were centrifuged, the growth factor-containing supernatant removed,
and the clusters resuspended in fresh media containing B-27
supplement and either 3% rat serum or 1-3% fetal bovine serum. The
clusters were then plated onto 4-well Nunclon culture dishes coated
with poly-D-lysine/laminin, and the medium was changed every 3 to 7
days.
[0121] Transplantation of Olfactory Epithelium-derived MNSCs
[0122] Olfactory epithelium-derived MNSCs were purified and
cultured as described herein. Female Sprague-Dawley rats or CD1
albino mice (Charles River, Montreal, Quebec, Canada) weighing
180-200 g or 25-30 g, respectively, were anaesthetized with a
mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg)
(intraperitoneal) prior to stereotactic injections of 24 .mu.g of
6-hydroxydopamine hydrobromide (dissolved in 5 .mu.L of 0.9% saline
containing 0.2 mg/ml ascorbate) into the right medial forebrain
bundle (Tooth bar:-2.4 mm; A:-4.4 mm; L:1.0 mm; V:7.5 mm). Two
weeks after the lesion, animals were tested for rotational
behavior. Animals were immunosuppressed with cyclosporine (40
mg/kg, intraperitoneal) once a day until the day of sacrifice. For
MNSC transplantation, anaesthetized animals were mounted in a Kopf
stereotactic apparatus, and 2.times.2.5 .mu.L aliquots of MNSCs
were injected unilaterally into the lesioned caudate putamen or
bilaterally in some animals. The injections were made using a 5
.mu.L Hamilton syringe at the following coordinates: Tooth bar,
-2.4 mm; A: 0.2; L: 3.0; V: 5.5-6.0. Injections were performed over
a period of three minutes, a further five minutes was allowed for
diffusion, and the needle was then retracted. These 5 .mu.L
injections contained MNSCs derived from one neonatal pup cultured
for 7 to 14 days. For the BrdU experiments, BrdU (10 .mu.M) was
added to culture media for 18 hours, after which the MNSCs were
washed three times with fresh media to remove the BrdU, and then
transplanted one day later.
[0123] Transplantation of Skin-derived MNSCs
[0124] Labeling of skin-derived MNSCs was performed as follows.
Three days prior to transplantation, free-floating cell clusters
were partially dissociated by gentle trituration, and then exposed
to 50 MOI of a recombinant adenovirus expressing GFP, using
standard techniques. Twenty-four hours later, the MNSCs were
centrifuged, washed, and resuspended in fresh medium containing 2
.mu.M BrdU for an additional two days. Prior to transplantation,
MNSCs were rinsed five times with fresh medium and resuspended to a
concentration of 50,000 cells/.mu.l. At the time of
transplantation, approximately 75% of the MNSCs expressed GFP,
while 95% were BrdU positive.
[0125] MNSCs labeled with BrdU and GFP were stereotaxically
injected into the right lateral ventricle of cryoanaesthetized two
day old rat pups (co-ordinates from Bregma: lateral 1.5 mm, ventral
3.3 mm). Approximately 50,000 cells were injected over a three
minute period in a volume of 1 .mu.L. Fourteen days following
transplantation, mice were perfused with 50 mL 4% formaldehyde
buffered with PBS. Fifty micron coronal sections through the
forebrain were cut using a freezing microtome and analyzed
immunocytochemically. All eight animals receiving cell transplants
showed extensive labeling for tagged cells. No evidence of tumor
formation was observed.
[0126] Immunostaining
[0127] Immunostaining of olfactory epithelium-derived MNSCs was
performed as follows. With the exception of GC immunocytochemistry,
culture dishes were washed twice with Tris-buffered saline (TBS; 10
mM Tris, 150 mM NaCl, pH 8), then fixed with 4% formaldehyde,
washed three times with TBS, blocked with TBS plus 2% goat serum
(Jackson ImmunoResearch, Mississuagua, Ontario, Canada), and 0.1%
Triton-X (Sigma Chemicals, St. Louis Mo.) for 30 minutes, then
incubated with primary antibody in TBS plus 2% goat serum.
Following primary antibody incubation, the dishes were washed three
times with TBS, incubated in secondary antibody in TBS plus 2% goat
serum, washed three times, and then viewed with a fluorescence
inverted microscope. The antibodies to GFAP (Boehringer
Mannheim,Laval, Quebec, Canada), III tubulin (Sigma), NeuN (Dr. R.
Mullen), MAP-2 (clone AP-20; Sigma), and NF-160 (American Tissue
Culture Collection, Manassas Va.) were monoclonal antibodies used
at concentrations of 1:200; 1:25; 1:10, and 1:1 respectively.
Antibodies to nestin (a gift from Dr. Ron MacKay National
Institutes of Health), TH (Eugenetech Eugene, OR), and DBH
(Eugenetech) were rabbit polyclonal antibodies used at
concentrations of 1:1000, 1:200, and 1:200 respectively. Secondary
antibodies Cy3 conjugated goat anti-mouse (Jackson ImmunoResearch)
and Cy3 conjugated goat anti-rabbit (Jackson ImmunoResearch), and
were used at 1:1500. For double-labelling experiments, we used FITC
goat anti-mouse (Jackson ImmunoResearch).
[0128] For GC immunocytochemistry, living cultures were incubated
in DMEM containing HEPES, 5% heat inactivated horse serum (HS), and
1:10 GC antibody for 30 min at 37 C., washed three times with the
medium/HEPES/HS, fixed with 4% formaldehyde for 15 minutes, rinsed
three times in TBS, incubated in Cy3 conjugated goat anti-mouse
antibody (1:1500) for two hours, and finally washed three times in
TBS. Cultures processed for immunocytochemistry without primary
antibodies revealed no staining.
[0129] Immunocytochemical analysis of cultured skin-derived MNSCs
was performed as follows. The primary antibodies that were used
were: anti-nestin polyclonal (1:250, Dr. Ron McKay, NINDS),
anti-nestin monoclonal (1:400, PharMingen Inc.),
anti-.beta.III-tubulin monoclonal (1:500, Tuj1 clone, BabCo),
anti-neurofilament-M polyclonal (1:200, Chemicon Intl.), anti-GAD
polyclonal (1:800, Chemicon Intl.), anti-NSE polyclonal (1:2000,
Polysciences Inc.), anti-GFAP polyclonal (1:200, DAKO), anti-CNPase
monoclonal (1:400, Promega), anti-p75NTR polyclonal (1:500,
Promega), anti-SMA monoclonal (1:400, Sigma-Aldrich), and anti-A2B5
monoclonal (Dr. Jack Snipes, M.N.I.). The secondary antibodies were
Cy3-conjugated goat anti-mouse (1:200), Cy3-conjugated goat
anti-rabbit (1:400), FITC-conjugated goat anti-mouse (1:50-1:100),
and FITC-conjugated goat anti-rabbit (1:200) (all from Jackson
Immunoresearch Laboratories).
[0130] Immunocytochemical analysis of free-floating brain sections
was performed by DAB immunohistocheniistry. For GFP, sections were
incubated in 0.3% H.sub.2O.sub.2 for one hour to inhibit endogenous
tissue peroxidase activity prior to blocking. For BrdU
immunohistochemistry, sections were pre-incubated in 0.5% sodium
borohydride for 20 minutes prior to blocking of endogenous
peroxidase activity in 0.03% H.sub.2O.sub.2 for 30 minutes. To
permeabilize the nuclei for BrdU immunohistochemistry, sections
were incubated in 1% DMSO for 10 minutes, the DNA denatured with 2N
HCl for 60 minutes, and the HCl neutralized with 0.1M borate buffer
for 5 minutes. All sections were blocked for one hour in 10% BSA,
and then incubated for 48 hours at 4.degree. C. with either
anti-GFP (1:1000, Clontech) or anti-BrdU (1:100, Becton-Dickinson).
Primary antibodies were detected using a biotinylated horse
anti-mouse secondary antibody (1:200, Vector Laboratories) for one
hour at room temperature, and visualized using the Vectastain kit
(Vector Laboratories) and a nickel-enhanced DAB reaction containing
0.05% DAB, 0.04% nickel chloride, and 0.015% H.sub.2O.sub.2.
Sections were mounted onto slides, dehydrated through a series of
ethanols and Histoclear (Fisher Scientific), and coverslipped using
Permount (Fisher Scientific).
[0131] Fluorescence immunohistochemistry was performed to
co-localize GFP expression with NSE. Free-floating sections were
blocked in 10% BSA for one hour at room temperature, and then
incubated 48 hours at 4.degree. C. in a solution containing mouse
anti-GFP and rabbit anti-NSE. Sections were incubated with Cy3
conjugated anti-mouse and FITC conjugated anti-rabbit secondary
antibodies for one hour at room temperature, and coverslipped using
Sigma Mounting Medium.
Other Embodiments
[0132] The present invention has been described in terms of
particular embodiments found or proposed by the present inventors
to comprise preferred modes for the practice of the invention. It
will be appreciated by those of skill in the art that, in light of
the present disclosure, numerous modifications and changes can be
made in the particular embodiments exemplified without departing
from the intended scope of the invention. All such modifications
are intended to be included within the scope of the appended
claims.
[0133] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
* * * * *