Title: Pharmaceuticals containing multipotential precursor cells from tissues containing sensory receptors
Abstract: Current sources of neural stem and progenitor cells for neural transplantation are essentially inaccessible in living animals. This invention relates to neural precursor cells (stem cells, progenitor cells or a combination of both types of cells) isolated from the olfactory epithelium of mammals that can be passaged and expanded, and that will differentiate into cell types of the central nervous system (CNS), including astrocytes, oligodendrocytes, and tyrosine-hydroxylase-positive neurons. These precursor cells provide an accessible source for autologous transplantation in CNS, PNS, spinal cord and other damaged tissues.
Patent Number: 6,969,608 Issued on 11/29/2005 to Miller, et al.
| Inventors:
|
Miller; Freda (Montreal, CA);
Gloster; Andrew (Saskatoon, CA)
|
| Assignee:
|
McGill University (Montreal, CA)
|
| Appl. No.:
|
920272 |
| Filed:
|
August 22, 1997 |
| Current U.S. Class: |
435/325; 435/368; 435/352; 435/353; 435/354; 424/93.1; 424/93.7 |
| Intern'l Class: |
C12N 005/00 |
| Field of Search: |
435/368,325,353,354
424/931,937
|
References Cited [Referenced By]
U.S. Patent Documents
| 5318907 | Jun., 1994 | Ronnette et al.
| |
| 5338839 | Aug., 1994 | McKay et al.
| |
| 5753506 | May., 1998 | Johe.
| |
| 5824489 | Oct., 1998 | Anderson et al.
| |
| 5912175 | Jun., 1999 | Wille, Jr.
| |
| 6001654 | Dec., 1999 | Anderson et al.
| |
| Foreign Patent Documents |
| 93/01275 | Jan., 1993 | WO.
| |
| 94/09119 | Apr., 1994 | WO.
| |
| 94/10292 | May., 1994 | WO.
| |
| 94/16718 | Aug., 1994 | WO.
| |
| WO 9416718 | Aug., 1994 | WO.
| |
| 95/13364 | May., 1995 | WO.
| |
| WO 9512665 | May., 1995 | WO.
| |
| WO 9741208 | Nov., 1997 | WO.
| |
| WO 9956759 | Nov., 1999 | WO.
| |
Other References
Calof et al., Neuron, 3, 315, Jul. 1989.
Mayo et al., Int. J. Dev. Biol. 36, 255, 1992.
Fraichard A, et al. In vitro differentiation of embryonic stem cells and functional
neurons. Journal of Cell Science 108, 3181-3185 (1995).
Kaufman SJ, Replicating myoblasts express a muscle-specific phenotype. Proc.
Natl. Acad. Sci. USA vol. 85, pp. 9606-9610, Dec. 1988.
La Salle G. An Adenovirus vector for Gene Transfer into Neurons and Glia in the
brain. Science, vol. 259, pp. 988-990 (1993).
Mayo ML, Desmin expression during early mouse tongue morphogenesis. Int. J. Dev.
Biol. 36:255-263 (1992).
Schubert D, Ontogeny of electrically excitable cells in cultured olfactory epithelium.
Proc. Natl. Acad. Sic. USA, vol. 82, pp. 7782-7786, Nov. 1985.
Sosnowski JS, Chemical traumatization of adult mouse olfactory epithelium in
situ stimulates growth and differentiation of olfactory neurons in vitro. Brain
Res. Dec. 8, 1195;702(1-2):37-48.
Avoli et al. Pharmacology and Electrophysiology of a synchronous GABA-Mediated
Potential in the Human Neocortex. Neuroscience vol. 62 No. 3, pp. 655-666, 1994.
Bruckenstein et al. Morphological Differentiation of Embryonic Rat Sympathetic
Neurons in Tissue Culture. Developmental Biology vol. 128 pp. 324-336, 1988.
Burns S. et al., (1983) "A primate model of parkinsonism: selective destruction
of dopaminergic neurons in pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine."
Proc Natl Acad Sci (USA) 80:4546-4550.
Fahn S. (1992) "Fetal-tissue transplants in Parkinson'disease." New England Journal
of Medicine. 327:1589-1590.
Dunnett SB. et al., (1991) "Nigral transplants in primate models of parkinsonism.".
In: Lindvall O., Bjorkland A., Widner H., eds. Intracerebral transplantation in
movement disorders. Restorative Neurology 4:27-51.
Langston JW. et al., (1983) "Chronic parkinsonism in humans due to a product
of meperidine analog synthesis." Science 219:979-980.
Widner H. et al., (1993) "Bilateral fetal mesencephalic grafting in two patients
with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)."
New England Journal of Medicine 327:1556-1563.
Winkler C. et al., (1995) "EGF-responsive neural progenitor cells, survive, migrate
and differentiate after transplantation into the adult rat striatum." Society for
Neuroscience Abstracts 21:2029.
Gage FH. et al., (1995) "Survival and differentiation of adult neuronal progenitor
cells transplanted to the adult brain." Proc Natl Acad Sci (USA) 92:11870-11883.
Reynolds B. and Weiss S., (1992) Science 255:107.
Weiss S. et al., (1996) "Is there a neural stem cell in the mammalian forebrain?"
T.I.N.S. 19:9:1.
Peel AL. et al., (1995) "Co-localization of glial and neuronal markers in EGF-generated
cultures of pluripotent CNS stem cells." Society for Neuroscience Abstract 21:285.
Ruth S. Slack et al., (1996) "Adenovirus-mediated Gene Transfer of the Tumor
Supressor, p53, Induces Apoptosis in Postmitotic Neurons." The Journal of Cell
Biology, vol. 135, No. 4 1085-1096.
Le Gal La Salle et al., (1993) "An adenovirus vector for gene transfer into neurons
and glia in the brain." Science, 259: 988-990.
R.S. Slack et al., (1996) "Viral vectors for use in modulating gene expression
in neurons", Curr. Opin. Neurobiot, 6:576-583.
I. Lefkowitz et al., "Limiting Dilution Analysis of Cells in the Immune System."
Cambridge University Press, Cambridge, U.K. (1979).
C. G. Bellows et al., Dev. Biol. 133, 8 (1989).
A. Carlsson et al., Nature 180, 1200 (1957).
U. Ungerstedt et al., Brain Res. 24, 485 (1970).
A. Gloster et al., J. Neurosci, 14, 7319 (1994).
S. Bamji et al. Comp. Neurol. 374, 52 (1996).
E. Soriano et al., J. Histochem. Cytochem. 39, 255 (1991).
Ehringer, H. et al., "Verteilung von noradrenalin und dopamin (3-hydroxytyramin)
im gehirn des menschen und ihr verhalten bei erkrankungen des extrapyramidalen
systems", Kllin. Wschr. 38: 1236-1239 (1960).
Ourendnik et al., "Neural stem cells- a versatile tool for cell replacement and
gene therapy in the central nervous system", Clin. Genet. 56:267-278 (1999).
|
Primary Examiner: Murphy; Joseph
Attorney, Agent or Firm: Clark & Elbing LLP, Bieker-Brady; Kristina
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/024,590,
filed Aug. 26, 1996, and U.S. Provisional Application No. 60/024,456 filed Aug.
27, 1996 which are incorporated by reference herein in their entirety.
Claims
1. A composition consisting of an isolated population of neural stem cells of
a postnatal mammal and a carrier, wherein said neural stem cells form non-adherent
clusters in culture, are self renewing, proliferate in an EGF-independent manner,
express nestin, and differentiate, in the presence of serum, into neurons expressing
tyrosine hydroxylase, said stem cells produced by a method comprising the steps of:
(a) providing a culture of peripheral tissue containing sensory receptors from
said mammal;
(b) isolating neural stem cells from said peripheral tissue, based on the tendency
of said neural stem cells to aggregate and form non-adherent clusters in culture,
wherein said neural stem cells form non-adherent clusters in culture, are self
renewing, proliferate in an EGF-independent manner, express nestin, and differentiate,
in the presence of serum, into neurons expressing tyrosine hydroxylase.
2. A composition consisting of an isolated population of neural stem cells of
a postnatal mammal and a carrier, wherein said neural stem cells form non-adherent
clusters in culture, are self renewing, proliferate in an EGF-independent manner,
express nestin, and differentiate, in the presence of serum, into neurons expressing
tyrosine hydroxylase.
3. The composition of claim 1, wherein said peripheral tissue comprises olfactory epithelium.
4. The composition of claim 1, wherein said peripheral tissue comprises tongue.
5. The composition of claim 1, wherein said neural stem cells are transfected
with a heterologous gene.
6. The composition of claim 5, wherein said gene encodes a trophic factor.
7. The composition of claim 1, wherein said neural stem cells are human stem cells.
8. The composition of claim 1, formulated in a pharmaceutically acceptable carrier,
auxiliary or excipient.
9. The composition of claim 2, formulated in a pharmaceutically acceptable carrier,
auxiliary or excipient.
10. The composition of claim 2, wherein said neural stem cells are human stem cells.
11. The composition of claim 2, formulated in a pharmaceutically acceptable carrier,
auxiliary or excipient.
Description
FIELD OF THE INVENTION
The present invention relates to multipotential precursor cells isolated from
peripheral tissues containing sensory receptors such as the olfactory epithelium
and tongue. The invention also relates to cells differentiated from the precursor
cells. The invention includes pharmaceutical compositions containing precursor
cells. The invention also includes cells differentiated from precursor cells and
uses for those cells.
BACKGROUND OF THE INVENTION
There are a number of diseases of the central nervous system ("CNS") which
have a devastating effect on patients. These diseases are incurable and debilitating.
They include Alzheimer's disease, Huntington's disease, Parkinson's disease and
Multiple Sclerosis, to name a few.
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 striatum. Parkinson's disease occurs when
these dopaminergic neurons die. There are a number of methods to treat Parkinson's disease.
One method is to treat humans having parkinsonism with L-DOPA. Another method
is to transplant cells into the substantia nigra or striatum. Transplanted cells
replace endogenous cells that are lost as a consequence of damage. Transplanted
cells may also be used as vectors for the expression of therapeutic molecules.
Another method is to implant fetal brain grafts containing dopaminergic neurons.
This method is experimental (Widner et al., 1993; Callahan et al., 1992). An animal
model of Parkinson's disease is an MPTP-treated non-human primate. The animal models
have been transplanted with dopamine-rich embryonic neurons with some success (Dunnett
et al., 1991). (MPTP is a selective dopaminergic toxicant that produces parkinsonian
symptoms in humans and in primates after a one-hit lesion to the neurons in the
substantia nigra (Langston et al., 1983; Burns et al., 1983)).
Investigators studying other neurodegenerative diseases, such as Alzheimer's
disease and Huntington's disease, are exploring the possible usefulness of fetal-tissue
implants in the treatment of these diseases.
Current approaches to transplantation suffer from a number of serious limitations.
First, many investigators are utilizing non-neural cells such as fibroblasts or
transformed cell lines for transplantation. Second, the safety of transplantation
of immortalized cell sources into the human brain is a concern. These cells may
become unregulated and develop into tumors. Third, transplants of dopaminergic
neuron fetal tissue to Parkinson's disease patients have a number of difficulties:
- the fate of implanted dopaminergic neurons in patients with Parkinson's
disease is uncertain—whatever caused the loss of endogenous dopaminergic
neurons may also eventually injure the implanted ones,
- in many cases, implants provide only temporary relief as the symptoms
associated with the disease often return after a number of years,
- the patient may reject foreign fetal tissue,
- there are adverse reactions associated with immunosuppression (immunosuppression
is needed to try to help the patient accept the foreign fetal tissue, even though
the brain is, to some degree, immunologically privileged),
- a sufficient number of cells in the fetal tissue being implanted are
unable to survive during and after implantation,
- the implants may not be regulated by the host brain,
- other diseases or disorders may be transmitted to the patient via the implant,
- the cost and effort associated with implanting fetal tissue may not
be justified by the results, and
- there are objections to the ethics associated with implanting fetal tissue.
Many of these problems are encountered with transplants used to treat other
neurodegenerative diseases, disorders or abnormal physical states.
In some tissues, stem cells and progenitor cells are proposed as a source for
alternative treatments of disease or injury to tissues. The proposed treatments
involve transplants of healthy tissue or endogenous stimulation of stem cells or
progenitor cells to produce healthy tissue.
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 specialised tissues and organs of the developing fetus.
In adults, specialised stem cells in individual tissues are the source of new cells
which replace cells lost through cell death due to natural attrition, disease or
injury. No stem cell is common to all tissues in adults. Rather, the term "stem
cell" in adults describes different groups of cells in different tissues and organs
with common characteristics.
Stem cells are capable of producing either new stem cells or cells called progenitor
cells. A progenitor cell differentiates to produce the mature specialized cells
of mammalian organs. In contrast, stem cells never terminally differentiate (i.e.
they never differentiate into specialized tissue cells). Progenitor cells and stem
cells are referred to collectively as "precursor cells". This term is often used
when it is unclear whether a researcher is dealing with stem cells or progenitor
cells or a combination of both cells.
Progenitor cells may differentiate in a manner which is unipotential or
multipotential. A unipotential progenitor cell is one which can form only one particular
type of cell when it is terminally differentiated. A multipotential progenitor
cell has the potential to differentiate to form more than one type of tissue cell.
Which type of cell it ultimately becomes depends on conditions in the local environment
such as the presence or absence of particular peptide growth factors, cell—cell
communication, amino acids and steroids. For example, it has been determined that
the hematopoietic stem cells of the bone marrow produce all of the mature lymphocytes
and erythrocytes present in fetuses and adult mammals. There are several well-studied
progenitor cells produced by these stem cells, including three unipotential and
one multipotential tissue cell. The multipotential progenitor cell may divide to
form one of several types of differentiated cells depending on circumstances such
as which hormones or factors act upon it and cell—cell contact.
Weiss et al, 1996, summarises the five defining characteristics of stem cells
as the ability to:
- Proliferate: Stem cells are capable of dividing to produce daughter cells.
- Exhibit self-maintenance or renewal over the lifetime of the organism:
Stem cells are capable of reproducing by dividing symmetrically or asymmetrically
to produce new stem cells. Symmetric division occurs where one stem cell divides
into two daughter stem cells. Asymmetric division occurs where one stem cell forms
one new stem cell and one progenitor cell. Symmetric division is a source of renewal
of stem cells. This permits stem cells to maintain a consistent level of stem cells
in an embryo or adult mammal.
- Generate large number of progeny: Stem cells may produce a large number
of progeny through the transient amplification of a population of progenitor cells.
- Retain their multilineage potential over time: Stem cells are the ultimate
source of differentiated tissue cells, so they retain their ability to produce
multiple types of progenitor cells, which will in turn develop into specialized
tissue cells.
- Generate new cells in response to injury or disease: This is essential
in tissues which have a high turnover rate or which are more likely to be subject
to injury or disease, such as the epithelium or blood cells.
Thus, the key features of stem cells are that they are multipotential cells
which are capable of long-term self-renewal over the lifetime of a mammal.
There has been much effort to isolate stem cells and determine which peptide
growth factors, hormones and other metabolites influence stem cell renewal and
production of progenitor cells, which conditions control and influence the differentiation
of progenitor cells into specialized tissue cells, and which conditions cause a
multipotential progenitor cell to develop into a particular type of cell.
Stem cells or progenitor cells may be used as substrates for producing healthy
tissue where a disease, disorder or abnormal physical state has destroyed or damaged
normal tissue. For example, stem cells and progenitor cells may be used as a target
for in vivo stimulation with growth factors or they may be used as a source of
cells for transplantation. The stem cells or progenitor cells may be transplanted
or they may be induced to produce healthy differentiated cells for transplant.
In several tissues, stem cells have been isolated and characterised in an attempt
to develop new therapies to repair or replace damaged tissues. For example, neural
stem cells have been isolated from the mammalian brain (Reynolds and Weiss, Science
255:107 (1992)) and these cells were shown to be multipotential and able to differentiate
into neurons, astrocytes and oligodendrocytes. WO 93/01275, WO 94/16718, WO 94/10292
and WO 94/09119 describe uses for these cells.
WO 95/13364 reports the delivery of growth factors to the ventricles of the CNS
in order to stimulate neural stem cells to proliferate and produce neural progenitor
cells which will develop into neurons, oligodendrocytes or astrocytes. This procedure
has many complications which must be addressed before it may be used clinically.
Differentiating the target neural stem cells or neural progenitor cells into a
desired type of tissue which is functional is one complication. Another complication
is choosing a growth factor which does not cause side effects in other areas of
the brain.
These publications are limited to isolating or using adult stem cells from
the brain (in particular, the tissue around the brain ventricles, the ventricle
ependyma, which is the remnant of the embryonic brain germinal zone). Although
these publications suggest that progenitor cells may be isolated from the adult
peripheral nervous system ("PNS"), the publications define the PNS as the system
which originates from the neural crest. There is no reported isolation of a stem
cell from the PNS which does not originate from the neural crest.
There are no clinical treatments involving transplants of neural stem cells
or neural progenitor cells isolated from the brain nor are there clinical treatments
using differentiated cells produced from the neural stem cells or neural progenitor
stem cells isolated from the brain. There are also no clinical treatments to endogenously
stimulate the neural stem cells or neural progenitor cells of the brain in vivo
to produce differentiated cells. Even if there were clinical procedures to transplant
fetal neural stem cells or neural progenitor cells from the brain, or to transplant
cells differentiated from these stem cells or progenitor cells (e.g. dopaminergic
neurons into Parkinson's disease patients), this would not overcome the many problems
of transplants from one human to another. As mentioned above, the only current,
accessible human source for these neural stem cells and neural progenitor cells
is aborted human fetuses, raising serious ethical concerns. Heterologous transplants
are also very risky and complicated because of problems with graft rejection, immunosuppression,
and the potential for donor grafts transferring diseases or disorders to a recipient.
Encapsulation of cells in microspheres has the potential to decrease the likelihood
of graft rejection, but this effect is lost if the integrity of the microsphere
is disrupted. There is a clear need for safer tissue grafts which can be transplanted
to a recipient without being rejected.
The safest type of tissue graft would be 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 would harm the site
where the healthy neurons originate. Neural stem cells or neural precursor cells
that can be differentiated into dopaminergic neurons may be available at the damaged
site or at other sites from which they may be transplanted, but the CNS, particularly
the brain, is physically difficult to access. It would be impractical or impossible
to 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 or progenitor cells that could be differentiated into CNS cell types,
such as dopaminergic neurons, to provide a source of cells for autologous transplants.
It would be useful if neural stem cells or progenitor cells could be identified
and isolated outside the CNS and outside the PNS which originates from the neural
crest. Medical treatments could then be developed using those neural stem cells,
neural progenitor cells or cells differentiated from those cells. It is clear that
despite the work that has been done to attempt to treat neurodegenerative diseases
by tissue transplant, a need still exists for a pharmaceutical composition in which
(1) the composition is accepted by the patient, thus avoiding the difficulties
associated with immunosuppression, (2) the composition is safe and effective, thus
justifying the cost and effort associated with treatment, (3) the composition provides
long term relief of the symptoms associated with the disease, (4) the composition
is efficacious during and after transplantation and (5) there are no objections
to the ethics of the composition's use.
Thus, there is a clear need to develop neural stem cell cultures or neural
progenitor cell cultures from accessible tissues of the PNS which can act as a
source of cells that are transplantable to the CNS, PNS, spinal cord or other tissues
in vivo in order to replace damaged tissue.
SUMMARY OF THE INVENTION
This invention relates to the isolation of "precursor cells" (which may be neural
stem cells or neural progenitor cells or a combination of both types of cells)
from peripheral tissue with sensory receptors, specifically olfactory epithelium
and tongue, of the PNS. The olfactory epithelium is part of the PNS, but does not
originate from the neural crest. Rather, it is of placodal origin. Hence, peripheral
sensory neurons of the olfactory epithelium are developmentally distinct from the
neurons of the neural crest derived PNS. Olfactory precursor cells have been isolated,
determined to be multipotential and capable of generating CNS cell types. Thus,
they are a useful source of tissue for autologous or heterologous transplant to
the CNS, PNS, spinal cord and other damaged tissues.
The invention also includes isolated and purified precursor cells of a mammal
from peripheral tissue containing sensory receptors, wherein the precursor cells
are selected from a group consisting of neural stem cells, neural progenitor cells
and a combination of neural stems cells and neural progenitor cells. The cells
can be isolated from tongue.
The inventors have isolated precursor cells from the olfactory epithelium of
mammals (juvenile and adult mice, adult rat and humans). The precursor cells of
the olfactory epithelium possess the two key characterising features of stem cells:
they are mutipotential and are self-renewing. They can be passaged and differentiated
into cell types of the CNS, including astrocytes, oligodendrocytes, and dopaminergic
neurons. Precursor cells isolated from the olfactory epithelium of neonatal mice
express the immunological marker of neural stem and progenitor cells, nestin. These
cells are not restricted to assuming an olfactory phenotype, but instead can differentiate
into astrocytes, oligodendrocytes, and dopaminergic neurons. This shows that the
olfactory epithelium is a useful source of dopaminergic neurons for homotypic grafts
into Parkinson's Disease patients. The precursor cells of the olfactory epithelium
may also be used for autologous or homologous transplants to treat other neurodegenerative
diseases, disorders or abnormal physical states.
Precursor cells were also isolated from tongue and these may also be used
for autologous or homologous transplants to treat neurotrauma or neurodegenerative
diseases, disorders or abnormal physical states.
The stem cells or progenitor cells can be taken from an individual suffering
from a neurodegenerative disease and then differentiated into neurons, astrocytes,
oligodedrocytes for implantation into the nervous system of the individual. In
a preferred mode of the invention, cells may be transplanted into the CNS, PNS,
spinal cord or other damaged tissues.
Thus, this invention overcomes the needs outlined above in that the precursor
cells of this invention (1) are accepted by the patient because they can be taken
from the patient's own olfactory epithelium or tongue, (2) are safe in that the
patient is not receiving cells or tissue from another source, (3) are effective
in that the cells are of neural tissue origin and can be differentiated into neurons,
astrocytes and oligodendrocytes for implantation and the cells survive during and
after implantation, (4) offer the potential to provide long term relief of the
symptoms associated with neurodegenerative diseases, and (5) would not raise objections
to the ethics of their use.
Therefore, this invention relates to isolated and purified precursor cells
of peripheral tissues with sensory receptors, such as the olfactory epithelium
of a mammal (juvenile or adult). Under appropriate conditions, the precursor cells
can differentiate into neurons, astrocytes or oligodendrocytes. The precursor cells
may be transfected with a heterologous gene encoding, for example, a trophic factor.
The precursor cells may then be implanted into the CNS, PNS, spinal cord or other
damaged tissues of a patient and the heterologous gene expressed.
This invention also relates to neurons, astrocytes and oligodendrocytes differentiated
from the precursor cells of this invention.
The invention also includes a pharmaceutical composition for use in implant therapy.
The composition includes the precursor cells of this invention or neurons, astrocytes
or oligodendrocytes differentiated from the precursor cells of this invention,
in a pharmaceutically acceptable carrier, auxiliary or excipient. The composition
may include one or more types of cells selected from a group consisting of precursor
cells, neurons, oligodendrocytes and astrocytes.
A method of treating an individual suffering from a neurodegenerative disease
is
included within this invention. The method includes implanting the precursor cells
of this invention, or the neurons, astrocytes or oligodendrocytes derived from
the precursor cells of this invention, into the CNS, PNS, spinal cord or other
damaged tissues of the individual. Another method consists of treating an individual
suffering from a neurodegenerative disease by administering the pharmaceutical
composition of this invention to the individual.
This invention also includes a method for isolating and purifying precursor
cells from the olfactory epithelium of a mammal. The method includes (1) taking
a sample of the olfactory epithelium from the mammal, (2) dissociating the sample
into single cells, (3) placing the cells in culture, (4) isolating the cells which
survive in culture. These isolated cells may be differentiated into neurons, astrocytes
or oligodendrocytes. The precursor cells which survive in culture are spherical
aggregates. The step of placing the cells in culture includes placing the cells
in a tissue culture incubator in an appropriate medium. We isolate precursor cells
from the tongue and other peripheral tissues with sensory receptors using a similar technique.
In this method, the mammal may be a human who is suffering from a neurodegenerative
disease, disorder (such as neurotrauma) or abnormal physical state. The method
may further include implanting the precursor cells or the neurons, astrocytes or
oligodendrocytes differentiated from the neural stem cells, into the CNS, PNS,
spinal cord or other damaged tissues of the human. In another case, the mammal
is a human and is not suffering from a neurodegenerative disease or neurotrauma.
Then, the method includes implanting the precursor cells or the neurons, astrocytes
or oligodendrocytes differentiated from the precursor cells, into a second human
who is suffering from the neurodegenerative disease or neurotrauma. The neurodegenerative
disease may be one selected from a group consisting of Parkinson's disease, Alzheimer's
disease, Huntington's disease and Multiple Sclerosis, while types of neurotrauma
include stroke and spinal cord injury.
This invention also includes a kit for the treatment of a disease, disorder
or abnormal physical state. The kit includes one or more types of cells including
the precursor cells of this invention, or the neurons differentiated from these
precursor cells, the astrocytes differentiated from these precursor cells and the
neurons, astroycytes and oligodendrocytes differentiated from these precursor cells.
The invention also provides precursor cell cultures which may be used in toxicity
testing, drug development testing or studies of genes and proteins. Precursor cell
cultures may also be induced to produce healthy differentiated cells which may
be used for toxicity testing or drug development testing. Toxicity testing is done
by culturing precursor cells or cells differentiated from precursor cells in a
suitable medium and introducing a substance, such as a pharmaceutical or chemical,
to the culture. The precursor cells or differentiated cells are examined to determine
if the substance has had an adverse effect on the culture. Drug development testing
may be done by developing derivative cell lines, for example a pathogenic cell
line, which may be used to test the efficacy of new drugs. Affinity assays for
new drugs may also be developed from the precursor cells, differentiated cells
or cell lines derived from the precursor cells or differentiated cells. The methods
of performing toxicity testing and drug development testing are well known to those
skilled in the art.
Precursor cells also provide a culture system from which genes, proteins
and other metabolites involved in cell development can be isolated and identified.
The composition of stem cells may be compared with that of progenitor cells and
differentiated cells in order to determine the mechanisms and compounds which stimulate
production of stem cells, progenitor cells or differentiated cells. Methods of
isolating proteins and genes from cells are well known to those skilled in the art.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should 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, since 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 FIGURES
The invention will now be described in relation to the figures:
FIG. 1.
a) Bright field photograph of a small group of cells; 4 days in vitro ("DIV").
Scale bar=40 μm.
b) Bright field photograph of 3 floating olfballs; 12 DIV. Scale bar=200 μm.
c) Bright field photograph of 3 olfballs in the process of fusing; 12 DIV.
Scale bar=200 μm.
d) Nestin staining of an olfball. 6 DIV and one day after plating down.
Scale bar=30 μm.
FIG. 2.
a) GFAP staining of differentiated olfballs. 16 days after plating down.
Scale bar=50 μm.
b) GFAP staining of differentiated cells derived from olfballs which had
been passaged twice. 16 days after plating down. Scale bar=50 μm.
c) GC staining of differentiated olfballs. 16 days after plating down. Scale bar=50/u.
d) Bright field of same field as shown in c). Scale bar=50 μm.
e) GC staining of differentiated olfballs derived from olfballs which had
been passaged twice. 16 days after plating down. Scale bar=200 μm.
FIG. 3.
a) NF-160 staining of differentiated olfballs. 16 days after plating down.
Scale bar=50 μm.
b) Bright field of same field as shown in c). Scale bar=50 μm.
c) LacZ staining of differentiated olfballs derived from T∝1:nlacZ
mice (Gloster et al., 1994) that express a neuron-specific E. coli β-galactosidase
marker gene. 16 days after plating down. Scale bar=50 μm.
d) TH staining of differentiated olfballs. 16 days after plating down. Scale
bar=50 μm.
e) TH staining of differentiated olfballs derived from olfballs which had
been passaged twice. 16 days after plating down. Scale bar=50 μm.
f) Bright field of same field as shown in e). Scale bar 50 μm.
g) βIII tubulin staining of differentiated olfballs. 16 days after
plating down. Scale bar=100 μm.
h) NeuN staining of differentiated olfballs. 16 days after plating down.
Scale bar=50 μm.
FIG. 4
a) Bright field photograph of a small floating adult derived olfball; 8
DIV. Scale bar=50 μm
b) Bright field photograph of a larger adult derived floating olfball; 15
DIV. Scale bar=50 μm
c) nestin staining of a differentiated cell derived from an adult olfballs;
16 days after plating down. Scale bar=25 μm
FIG. 5
Limiting dilution curve. Cells were plated at 700 to 7000 cells per well,
cultured for 14 days in vitro, and then examined for the presence of olfballs.
The fraction of wells without olfballs was plotted against the number of cells
plated. Based upon the Poisson distribution, the probability of a well not having
an olfball at the 0.37 level (1/e) indicates that 1 of every 9000 cells plated
has the capacity to generate an olfball. The correlation value of the line is r=-0.992.
FIG. 6
Demonstration that the olfballs can be genetically modified. Olfballs were
plated on polylysine in the presence of 2% FBS. CMV-β-galactosidase adenovirus
was added at an MOI (Multiplicity of infection) of 25. Three days later the cultures
were X-gal stained (standard histochemical technique to reveal cells expressing
β-galactosidase), and 90% of cells were found to be expressing β-galactosidase.
FIG. 7
Generation of TH-positive neurons upon transplantation of olfballs into
the adult rat striatum. The striatum of adult rats was unilaterally denervated
using 6-hydroxydopamine to eliminate dopaminergic fibers, and neonatal olfballs
were transplanted into the striatum of the same animals. (a) With transplants of
olfballs from Tα1:nlacZ mice, β-galactosidase positive nuclei (arrows)
are detected along the graft tract. (b) A complex TH-positive neuron (arrow) with
multiple processes (arrowheads). (c) A cluster of morphologically simple TH-positive
cells that are double-labelled with BrdU. Note the black speckled appearance of
the BrdU-labelling (arrow). (d) A TH-positive neuron (arrowhead) with a single
process whose nucleus is double-labelled with BrdU (arrow). In this case, the BrdU
staining fills the entire nucleus. Scale bar: a=100 μm, b,c=25 μm,
d=5 μm.
DETAILED DESCRIPTION OF THE INVENTION
The inventors have isolated multipotential precursor cells from the olfactory
epithelium of mammals juvenile and adult mice, adult rat and humans). The isolated
cells proliferate in culture, so that large numbers of precursor cells can be generated.
In culture, these cells form floating spheres which are named "olfballs". These
cells can be induced to differentiate into neurons, astrocytes, and oligodendrocytes
by altering the culture conditions. The precursor cells can generate differentiated
cells for use in autologous transplants for the treatment of certain neurodegenerative
disorders or neurotrauma. For example, precursor cells may be differentiated into
dopaminergic neurons and implanted in the substantia nigra or striatum of Parkinson's
disease patients. They can also be used to generate oligodendrocytes for use in
autologous transplants for multiple sclerosis. The precursor cells are easily accessible
by biopsy from the olfactory epithelium, so they are a ready source of cells for
autologous transplants. Finally, they could be used as autologous cellular vectors
to introduce growth factors into the diseased or traumatized CNS, PNS, spinal cord
and other damaged tissues.
The olfballs display some similarities to forebrain stem cells, but also possess
some distinctive differences. In particular, (i) when olfballs differentiate in
the presence of serum, almost half of the differentiated cells express neuronal
markers, whereas differentiated forebrain stem cell neurospheres generate only
a small percentage of neurons, (ii) significant numbers of dopaminergic neurons
are found in all differentiated cultures of olfballs, whereas they are never found
in cultures of forebrain stem cell neurospheres differentiated in serum, and (iii)
many of the undifferentiated progenitor cells that are found in olfball cultures
express glutamic acid-decarboxylase (GAD), a neurotransmitter enzyme that is expressed
transiently in many neuroepithelial cells in vivo; in contrast, the only GAD-positive
cells that derive from forebrain stem cell neurosphere cultures are neurons.
The precursor cells of this invention may be used to prepare pharmaceutical compositions
which can be administered to humans or animals. Dosages to be administered depend
on patient needs, on the desired effect and on the chosen route of administration.
The invention also relates to the use of the cells of this invention to introduce
growth factors into the diseased, damaged or physically abnormal CNS, PNS, spinal
cord or other damaged tissues. The precursor cells act as a vector to transport
a recombinant molecule, for example, or to transport a sense or antisense sequence
of a nucleic acid molecule. In the case of a recombinant molecule, the molecule
would contain suitable transcriptional or translational regulatory elements.
Suitable regulatory elements may be derived from a variety of sources, and
they may be readily selected by one with ordinary skill in the art. If one were
to upregulate the expression of the gene, one would insert the sense sequence and
the appropriate promoter into the vehicle. If one were to downregulate the expression
of the gene, one would insert the antisense sequence and the appropriate promoter
into the vehicle. These techniques are known to those skilled in the art.
Examples of regulatory elements include: a transcriptional promoter and
enhancer or RNA polymerase binding sequence, 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 precursor
cells or the cells differentiated from the precursor 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 coprecipitation
and incorporation of DNA into liposomes. The genetically altered cells may be encapsulated
in microspheres and implanted in the CNS, PNS, spinal cord and other damaged tissues.
The following examples describe (i) the derivation of olfballs from postnatal
mouse and adult mouse tissue, (ii) the derivation of olfballs from rat and human
tissue, (iii) the use of olfballs to generate endogenous CNS cell types in the
transplanted adult mouse brain, (iv) methods for genetically manipulating olfballs
for use as therapeutic vectors, (vi) isolation of precursor cells from other peripheral
tissues with sensory receptors such as tongue We characterize and use these cells
using procedures similar to those used with olfballs. These studies provide us
with novel tools for the treatment of the traumatized or diseased adult nervous system.
EXAMPLE 1
Isolating Multipotential Precursor Cells from Postnatal Olfactory Epithelium
of Mice
Postnatal mice were stunned with a blow to the head and then decapitated.
The heads were sagitally sectioned with a razor blade. The olfactory epithelium
of about 6 postnatal (P 1-9) mouse pups were stripped from the conchae, nasal septum,
and vomeronasal organs using watch-maker forceps. This tissue was placed into 3
mls of media (DMEM/F-12 1:3 (Hyclone media) supplemented with 2% B-27 (Gibco),
20 ng/ml EGF (Collaborative Research), 0.1% fungizone, 0.5 ml/100 ml penicillin/streptomycin
(Gibco). After epithelium from the postnatal pups was collected, the epithelium
was teezed apart with watch maker forceps, releasing a large number of single cells.
The media was transferred to a 15 ml tube, and 7 ml more media was added. The cells
were dissociated into single cells, by titration with 10 ml plastic pipette (Falcon),
and passed through a 60 micron filter (Gibco). Typically dissociated cells from
the olfactory epithelium from 6 pups was plated into 2 50 ml tissue culture flasks
(Falcon). The dissociated cells were then placed in 50 ml flasks in a 37°
C., 5% CO2 tissue culture incubator. Two days later most cells in the cultures
were dead or dying. However, a small number of large phase bright cells were present,
most of which attach to the flask bottom. Over the next 2-6 days these cells divided
and produced spherical aggregates which became larger over time. On day 4 (FIG.
1A) there were approximately 500 clusters of dividing cells per pup used in the
original isolation (n=2 independent isolations). Most of these cellular aggregations
lifted from the flask surface over the next few days (FIG. 1B). These floating
spheres (olfballs) continued to grow and fused together to become macroscopic (FIG.
1C), reaching 100 microns in diameter if left for 10 days days in vitro. After
14 days in vitro, the diameter of the spheres was approximately 1 mm.
If EGF was not added to the media, small clusters of dividing cells were still
seen by day 4, and some of these cells developed into olfballs, suggesting that
the cells were producing trophic factors themselves in quantities which in some
cases was sufficient for their proliferation.
The cells in these dividing clusters expressed a marker for neural progenitor
cells and neural stem cells, the intermediate filament protein nestin; at six days,
olfballs were transferred to polylysine coated 35 mm dishes overnight in media
containing 2% fetal bovine serum to facilitate the cells adhering to the substratum,
and were processed for indirect nestin immunohistochemisty. Filamentous antibody
staining was observed in almost all the cells in the clusters (FIG. 1D).
These nestin positive cells could also be passaged. Six days after isolation,
the media (5 ml) was removed from the flasks. This media contained many olfballs
that had lifted from the flask surface. The media containing olfballs was titturated
with a fire polished pipette, thereby dissociating many of the cell clusters into
single cells, and placed in a larger flask with an additional 15 ml of fresh media
(total volume now 20 ml). After a further 6 days one quarter of the media was removed,
the olfballs were again triturated, and put into a new flasks with 15 ml fresh
media and EGF. These cells have been successfully passaged four times.
EXAMPLE 2
Differentiating Precursor Cells Into Neurons Astrocytes and Oligodendrocytes
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 isolation were plated down onto polylysine coated 35 mm culture dishes
(Falcon) and 4 multiwell culture dishes (NUNC), in DMEM/F12 media containing 2%
fetal bovine serum (Hyclone) and 2% B-27 (no EGF). Media was changed every 3-4
days. Over the next 6-19 days cells migrated out of the olfballs onto the dish
surface. Some of these cells had the morphology of neurons, astrocytes, or oligodendrocytes.
We determined the phenotype of these cells using marker antibodies to glial fibrillary
acid protein (GFAP) (FIGS. 2A, B) for astrocytes, antibodies to neurofilament 160
(NF-160) (FIG. 3A), β III tubulin (FIG. 3G), NeuN (FIG. 3H) for neurons,
and antibodies to galactocerebroside (GC) (FIGS. 2C-E) for oligodendrocytes. Antibodies
to tyrosine hydroxylase (TH) were used to identify dopaminergic, noradrenergic,
and adrenergic neurons (FIGS. 3D-F). Dopamine β-dehydrogenase (DBH) was also
used for noradrenergic and adrenergic neurons.
Immunohistochemical procedures. With the exception of GC immunohistochemistry,
culture dishes were washed twice with TBS (Tris Buffered Saline; 10 mM Tris, 150
mM NaCl, pH 8), then fixed with 4% paraformaldehyde, rinsed in three times with
TBS, blocked with TBS plus 2% goat serum (Jackson ImmunoResearch), and 0.1% Triton-X
(Sigma) for 30 min, then incubated with primary antibody in TBS plus 2% goat serum,
rinsed 3 times with TBS, incubated in secondary antibody in TBS plus 2% goat serum,
rinsed 3 times and then viewed under a Zeiss Axiovert 100 florescence inverted
microscope. The antibodies to GFAP (Boehringer Mannheim), βIII tubulin (Sigma
and a gift from Dr. D. Brown, U. Ottawa), NeuN (Dr. R. Mullen), NF-160 (American
Tissue Culture Collection) were monoclonals used at concentrations of 1:200; 1:25;
1:10, and 1:1 respectively. Antibodies to nestin (a gift from Dr. Ron MacKay (Nation
Institute of Health), TH (Eugenetech), and DBH (Eugenetech) were rabbit polyclonals
used at concentrations of 1:1000, 1:200, and 1:200 respectively. Secondary antibodies
were 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 FITC goat anti-mouse (Jackson ImmunoResearch). GC immunohistochemistry,
living cultures were incubated with a DMEM media; HEPES; 5% HS (heat inactivated
horse serum), and 1:10 GC antibody (BRD1; a gift of Dr. B. Juurlink U. Sask.) for
30 min at 37° C., rinsed 3 times with the media/HEPES/HS, fixed with 4% paraformaldehyde
for 15 min, rinsed 3 times in TBS, incubated in Cy3 conjugated goat anti-mouse
antibody (1:1500) for 2 hr., finally rinsed 3 times in TBS. Cultures processed
for immunohistochemistry without primary antibodies revealed no staining.
Astrocytes, neurons, and oligodendrocytes were found. We also cultured
olfballs from transgenic mice which express β-galactosidase off of the neuron
specific promoter Tα1 α-tubulin, which allowed us to use staining with
the ligand X-gal antibodies for β-galactosidase as an additional neuronal
marker (FIG. 3B, 3C).
Since the differentiated cells abutted each other and were piled up on top
of each other in the center where the olfball originally attached, it was not possible
to count the number 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 lacZ positive. A lower number of cells were NF-160 positive, β
III tubulin, TH, GAD or GC positive. Therefore the olfballs could differentiate
into neurons, astrocytes and oligodendrocytes. While a few of the βIII tubulin
positive cells had complex morphologies (FIG. 3A), most were simpler, possessing
only a few neurites. The TH positive cells were the most morphologically complex
cells in the cultures, with numerous neurites extending from the cell body (FIGS.
3D, E). These TH positive cells are probably 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 derived nestin positive olfball cells could be differentiated
into cell types never found in the olfactory epithelium-oligodendrocytes, astrocytes,
GABAergic neurons, and dopaminergic neurons. The coexpression of astrocytic and
neuronal markers has been reported for differentiated cells derived from EGF-generated
brain-derived progenitor cells (Peel et al., 1995). While most cells were either
lacZ or GFAP positive, there were a few cells which were both lacZ and GFAP positive,
however none of the TH positive cells were also GFAP positive. Therefore while
cells may transiently express both neuronal and glial markers during their differentiation
program, fully differentiated morphologically complex neurons express only neuronal markers.
Like the original olfballs, the passaged olfballs could also be differentiated
into neurons, astrocytes, and oligodendrocytes. Olfballs which had been passaged
twice were plated down on polylysine coated dishes. The olfballs cells migrated
out and spread out over the dish's surface, and after 16 days were immuno-positive
for GC (FIG. 2E), GFAP, βIII tubulin, NeuN, lacZ, and TH. The proportion
of cells positive for the various markers was similar to that seen in the differentiated
cultures from the original cultures.
EXAMPLE 3
Isolating Multipotential Precursor Cells from Olfactory Epithelium of Adult Mice
and Adult Rats
Similar proliferating cells were also isolated from adult mouse and rat olfactory
epithelium and vomeronasal organs. We developed techniques for reproducibly culturing,
passaging, and differentiating the adult olfballs, on the basis of our experience
with their juvenile equivalents. As part of this aim, we (i) characterized the
growth factor and media requirements for the adult cells to proliferate in culture,
and (ii) characterized the growth factor and substrate requirements for the differentiation
of oligodendrocytes and dopaminergic neurons from both adult and juvenile olfballs.
We were informed in these studies by similar work on EGF- and FGF-dependent stems
cells from the CNS, since olfballs likely respond to at least some of the same
growth factors. The adult isolation procedures were essentially the same as for
the postnatal olfballs (described in examples 1 and 2).
Adult mice and rats were anaesthetized with injected with an overdose of somnitol,
and then decapitated. The olfactory and vomoeronasal organ epithelia were stripped
from the conchae and nasal septum and incubated in F12/DMEM culture media for 1
or 2 days after their dissection and prior to the rest of the isolation procedure
(B). After this incubation, the epithelia was dissociated in an identical manner
as the postnatal epithelia. Two days after the isolation almost all 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
seen in the postnatal cultures (FIGS. 4A, B). These also grew to give rise to the
large floating clusters which were routinely seen in the postnatal cultures. After
6 divisions some of these clusters began to differentiate and spread out over the
flask's surface, while some other clusters which had floated reattached to the
surface and then differentiated. (These cells multiplied to produce the small balls
or cells, but did not grow to form the large balls of cells like the postnatal
cultures). We passaged these cells using the same procedure as that described above
with respect to the cells isolated from postnatal olfactory epithelium.
These proliferating cells from the adult were also nestin positive. 10 days
after their initial isolation the cells were transferred to polylysine coated dishes
with 2% fetal bovine serum (FBS). Two hours later the cells were processed for
nestin immunohistochemistry (FIG. 4C).
After the cellular clusters of this Example had been generated they could be
differentiated into neurons and oligodendrocytes. Clusters from cultures 7 day
after isolation were plated down onto polylysine coated 35 mm culture dishes and
4 multiwell culture dishes, in media containing 2% fetal bovine serum and 2% B-27
(no EGF). Over the next month cells migrated out of the olfballs onto the dish
surface. We determined the phenotype of these cells using marker antibodies to
glial fibrillary acid protein (GFAP) for astrocytes, antibodies to βIII tubulin
for neurons, antibodies to TH for dopaminergic neurons and antibodies to galactocerebroside
(GC) for oligodendrocytes.
Neurons, and oligodendrocytes were found, although the number of these cells
was much lower than the number obtained from the neonate. The phenotype of these
adult derived differentiated cells was assessed using indirect immunohistochemistry.
The cells isolated from the adult were differentiated into βIII tubulin positive
cells (neurons), tyrosine hydroxylase positive cells (dopaminergic neurons), galactocerobroside
positive cells (oligodendrocytes). No astrocytes (GFAP positive) cells were found.
Therefore the adult derived olfballs could differentiate into neurons and oligodendrocytes.
EXAMPLE 4
Precursor Cells Differentiate Into Neurons When Transplanted Into Adult Brain
The major potential therapeutic use for olfballs is autologous transplantation
into the injured or degenerating CNS, PNS, spinal cord and other damaged tissues,
either to replace lost cell types and/or as vectors for expression of therapeutic
molecules. Transplantation experiments determine the fate of transplanted olfactory-derived
precursor cells. The precursor cells can differentiate into neurons when transplanted
into the adult brain. To this end, we transplant mouse derived precursor cells
into brains of immunosuppressed adult rats and identify which of the transplanted
cells differentiate into neurons, using double labelling with the mouse specific
and neuron specific antibodies (such as those which recognize neuron specific enolase
and neuron specific βIII β-tubulin). A similar approach has proved
successful in the study of transplanted brain-derived stem cells (Winkler, Hammang,
and Bjorklund, 1996).
In order for these stem cells to be useful for transplantation to treat neurodegenerative
diseases it is necessary to induce the differentiation of the appropriate neuronal
phenotype, such as dopaminergic neurons in the case of Parkinson's disease. Therefore,
initially we determine if the precursor stem cells transplanted into lesioned and
unlesioned striatum and substantia nigra, differentiate into dopaminergic neurons
in response to signals from their new environment, as they do when they differentiate
in vitro. Brain sections are double labelled with a mouse specific antibody and
antibodies to tyrosine hydroxylase to reveal dopaminergic neurons derived from
the transplanted cells. Transplants into neonatal rat brains show that a more immature
host environment is able to induce dopaminergic differentiation.
We transplanted olfballs into the denervated and intact striatum of adult rats.
Specifically, we unilaterally destroyed the dopaminergic innervation of the adult
striatum by a local infusion of 6-hydroxydopamine, under conditions where noradrenergic
neurons are spared. Several weeks following this lesion paradigm, olfballs were
transplanted into both the intact and lesioned striatum, and one week later, the
fate of the transplanted olfballs was determined immunocytochemically. These studies
demonstrated that transplanted olfballs can differentiate into tyrosine-hydroxylase-positive
neurons in vivo, as they can in vitro. Given that the primary deficit in Parkinson's
disease is a loss of dopaminergic innervation of the striatum due to neuronal loss,
these preliminary studies raise the exciting possibility that olfballs provide
an autologous source of dopaminergic neurons with which to treat this disease.
We characterize the neuronal and glial cell types that are generated by olfballs
transplanted into the adult striatum. In order to definitively identify the progeny
of the transplanted olfballs, we (i) derive olfballs from transgenic mice expressing
β-galactosidase from either the Tα1 α-tubulin and/or
