JP2003512333A - Methods for inducing the in vivo proliferation and migration of transplanted precursor cells in the brain - Google Patents

Abstract

  (57) [Summary] The present invention provides methods for in vivo migration and in vivo proliferation of precursor cells transplanted into the brain. Also disclosed is the isolation, characterization, expansion, differentiation, and transplantation of mammalian neural stem cells. In one embodiment, the precursor cells are transplanted into a subject's brain at a first location and in vivo migration of the transplanted cells is induced by injecting a mitogenic growth factor into a second location in the brain. Thus, a method is provided for inducing migration of precursor cells transplanted into the brain in vivo. In some preferred embodiments, the first location is the striatum of the brain, and the second location where mitogenic growth factor is injected is the lateral ventricle of the brain.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to the isolation of human central nervous system stem cells and methods and media for expanding, differentiating and transplanting these cells.

BACKGROUND OF THE INVENTION During the development of the central nervous system (“CNS”), pluripotent progenitor cells (also known as neural stem cells) proliferate and consequently constitute the adult brain. It gives rise to transiently dividing precursor cells that differentiate into cell types. Stem cells (or other tissues) are classically defined as capable of self-renewing (ie, forming mostly stem cells), proliferating, and differentiating into multiple different phenotypic lineages. Has been done. In the case of neural stem cells, this includes neurons, astrocytes, oligodendrocytes. For example, Potten and Loeffler (Deve
Lopment, 110: 1001, 1990), "(a) proliferation,
Flexibility in (b) maintenance of self, (c) production of large numbers of differentiated functional offspring, (d) tissue regeneration after injury, and (e) use of these selections is possible,
Undifferentiated cells ".

These neural stem cells have been isolated from several mammalian species including mouse, rat, pig and human. For example, WO 93/01275, WO 94 /
09119, WO 94/10292, WO 94/16718 and Catt.
aneo et al., Mol. Brain Res. 42, 161-66 (1996)
), All of which are incorporated herein by reference.

Human CNS neural stem cells, like their rodent homologues, contain mitogenic factors, typically epidermal growth factor or epidermal growth factor and basic fibroblast growth factor. When maintained in serum-free culture medium, they grow in suspension culture and form cell aggregates known as "neurospheres". Human neural stem cells have been shown to have a double acceleration of approximately 30 days. For example, Cattaneo et al., Mol. Brain Res. , 42, 161-66
See page (1996). During removal of mitogens and supply of substrates,
The stem cells differentiate into neurons, astrocytes and oligodendrocytes. In the prior art, the majority of cells in the differentiated cell population were identified as astrocytes and very few neurons (<10%) were observed.

[0005] Of recent interest has been the adult central nervous system (CNS), which exhibits stem cell properties.
The ability of the cell population within to self-renew to produce the differentiated mature cell phenotype of the adult CNS. In vivo intracerebroventricular infusion of epidermal growth factor (EGF) causes proliferation of at least two different cell populations found in the periventricular region, both of which are constitutively dividing neural progenitor cells. And a relatively resting cell population with stem cell-like properties. For example, Craig et al., Journal
of Neuroscience 16, pp. 2649-2658 (1996).
checking ... When stimulated to divide by the presence of EGF, these endogenous stem / progenitor cells do not follow the normal migration pattern along the rostral migration pathway towards the olfactory bulb to regenerate neurons (eg, Lois et al., Scie
nce 264, 1145-1148 (1994); Luskin, Neur.
on 11, pp. 173-189 (1993)) rather migrate laterally to the striatum, cortex, and surrounding parenchyma of the septum, where they differentiate into glia and the adult CNS. It exists in an associated position with respect to the endogenous neuron. For example, Kuhn et al., The Journal of Neuroscience.
17, pp. 5820-5829 (1997).

Cell transplantation offers the potential to provide novel cellular components in response to adult mammalian injury as a means to modify the brain's response to injury or degeneration by the implantation of new neurons or glia. Many neurotrophic factors have been shown to affect progenitor cell proliferation, differentiation capacity, and survival in vitro (eg, Ahmed et al., Journal of Neuroscience).
15, pp. 5765-5778 (1995)), but the problems associated with the limited migration, proliferation, and differentiation of transplanted cells in vivo remain.

There remains a need to increase the growth rate of neural stem cell cultures. There also remains a need to increase the number of neurons in the differentiated cell population. There is a further need to improve the viability of neural stem cell grafts upon transplantation into the host, including the need to improve in vivo proliferation and directed migration of undifferentiated progenitor cells following transplantation into the brain. ,Remaining.

SUMMARY OF THE INVENTION The present invention provides methods for in vivo migration and proliferation of progenitor cells transplanted into the brain. In one embodiment, the precursor cells are first in the brain of the subject.
In vivo migration of transplanted progenitor cells by inducing in vivo migration of transplanted cells by injecting mitogenic growth factor into a second location in the brain and injecting into a second location in the brain A method is provided for. In some preferred embodiments, the first location is the striatum of the brain and the second location where the mitogenic growth factor is infused is the lateral ventricle of the brain. In another preferred embodiment, the injection of mitogenic growth factor is carried out at this second location (eg injection location).
But not the differentiation of these precursor cells.

In another embodiment, progenitor cells are transplanted to a location in the brain of a subject and in vivo proliferation of the transplanted cells is induced by injecting a mitogenic growth factor at or near the transplant location. A method for inducing in vivo proliferation of progenitor cells transplanted into the brain by is provided. In some preferred embodiments, the implant location is a striatum of the brain and the mitogenic growth factor is
Injected into the lateral ventricles of the brain. In another embodiment of the method of the present invention, the precursor cells are mammalian embryonic precursor cells, and the precursor cells are cultured in a medium containing mitogenic growth factor prior to transplantation. To be done.

The present invention further provides novel human central nervous system stem cells, as well as methods and media for expanding, differentiating and transplanting these cells. In one embodiment, the present invention provides novel human stem cells having a double acceleration of between 5 and 10 days, as well as defined growth media for extended expansion of human neural stem cells. In another embodiment, the invention provides a defined medium for the differentiation of human neural stem cells, such as enriching for neurons, oligodendrocytes, astrocytes or combinations thereof. The present invention also provides a large number of neurons previously unavailable,
And a differentiated cell population of human neural stem cells that provide astrocytes and oligodendrocytes. The invention also provides novel methods for transplanting neural stem cells that improve graft viability during transplantation in a host.

The method of the invention can be used in the preparation of a medicament for inducing in vivo proliferation and migration of transplanted progenitor cells in the brain.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the isolation, characterization, expansion, differentiation and transplantation of CNS neural stem cells. The invention further relates to inducing in vivo migration or proliferation of progenitor cells transplanted into the brain.

The neural stem cells described and claimed in this application can be grown in suspension or adherent cultures. When the neural stem cell of the present invention proliferates as a neurosphere,
Human nestin antibody can be used as a marker to identify undifferentiated cells. Proliferating cells show little GFAP and β-tubulin staining (
However, some staining may be present due to the diversity of cells within the sphere).

When differentiated, most cells lose their nestin-positive immunoreactivity. In particular,
Antibodies specific for various neuroproteins or glial proteins can be used to identify the phenotypic characteristics of differentiated cells. Neurons can be identified using neuron-specific enolase (“NSE”), nerve fibrils, antibodies to τ, β-tubulin, or other known neuronal markers. Astrocytes can be identified using antibodies against glial fiber acidic protein (“GFAP”), or other known astrocyte markers. Oligodendrocytes can be identified using antibodies against galactocerebroside, 04, myelin basic protein (“MBP”), or other known oligodendrocyte markers. Glial cells
It can generally be identified by staining with an antibody such as the M2 antibody or other known glial marker.

In one embodiment, the invention provides novel human CNS stem cells isolated from the forebrain. Four neural stem cell lines have been isolated from human forebrain, all of which exhibit neural stem cell properties; Prolonged growth in factors, and when differentiated, these cells contain a population of neurons, astrocytes and oligodendrocytes. These cells, in contrast to human neural stem cells derived from the diencephalon of the prior art,
It can be doubled every 5-10 days. The reported proliferation rate of human neural stem cells from diencephalon was a doubling of about every 30 days. Cattaneo et al., Mol. Brain Res. , 42, 161-66 (1996).

Any suitable tissue source can be used to derive the neural stem cells of the invention. Neural stem cells can be induced to proliferate and differentiate by culturing the cells in either suspension or adherent cultures. For example, US Pat.
, 750,376 and US Pat. No. 5,753,506 (both of which
See, herein, in its entirety by reference, and the prior art media described therein. Both allografts and autografts are intended for transplantation purposes.

The present invention also provides a novel growth medium for the expansion of neural stem cells. Provided herein are serum-free or serum-depleted culture media for short-term and long-term proliferation of neural stem cells.

A number of serum-free or serum-depleted culture media have been developed due to the unwanted effects of serum that can lead to inconsistent culture results. For example, WO 95/006
32, which is incorporated herein by reference, and the prior art media described therein.

Prior to the development of the novel medium described herein, neural stem cells were treated with epidermal growth factor (“EGF”) or analogs of EGF (eg, amphiregulin (amp).
hiregulin) or transforming growth factor α (“TGF-α”)
)) As a mitogen for growth. See, for example, WO 93/01275, WO 94/16718, both of which are incorporated herein by reference. In addition, basic fibroblast growth factor (“bFGF”), alone or in combination with EGF, was used to enhance long-term neural stem cell survival.

The improved medium according to the invention, which comprises leukocyte inhibitory factor (“LIF”), significantly and unexpectedly increases the proliferation rate of neural stem cells, especially human neural stem cells.

The proliferation rates of forebrain-derived stem cells described herein were compared in the presence and absence of LIF. Unexpectedly, LIF was found to dramatically increase the cell proliferation rate in almost all cases.

The medium according to the invention comprises the following components in effective amounts for cell viability and cell growth: (a) serum-free (containing 0-0.49% serum) or serum-depleted (0.5-). 5.
Standard culture medium, which is 0% serum), known as a "defined" culture medium such as: For example, Iscove's modified Dulbeccc.
o's medium (“IMDM”), RPMI, DMEM, Fischer's, α medium, Leibovitz's, L-15, NCTC, F-10, F-12, ME.
M and McCoy's; (b) a suitable carbohydrate source (eg glucose); (c) a buffer such as MOPS, HEPES or Tris (preferably HE).
PES); (d) hormone sources including insulin, transferrin, progesterone, selenium and putrescine; (e) at least one growth factor that stimulates proliferation of neural stem cells (eg, EG
F, bFGF, PDGF, NGF, and their analogs, derivatives and / or
Or a combination, preferably a combined EGF and bFGF); and (f) LIF.

A standard culture medium typically contains various essential components required for cell survival, including, for example, inorganic salts, carbohydrates, hormones, essential amino acids, vitamins and the like. Preferably, DMEM or F-12 is used as the standard culture medium, most preferably a 50/50 mixture of DMEM and F-12. Both media are commercially available (DMEM-Gibco 12100-046; F-12-Gibco).
21700-075). Premixed formulations are also commercially available (N2-Gi
bco 17502-030). It is advantageous to provide additional glutamine (preferably about 2 mM). It is also advantageous to provide heparin in the culture medium. Preferably, the conditions for culturing should be as close to physiological as possible. The pH of the culture medium is typically between 6 and 8, preferably about 7, most preferably about 7.4. The cells are typically between 30 and 40 ° C., preferably 32.
It is incubated between ~ 38 ° C, most preferably between 35-37 ° C. The cells are preferably grown in 5% CO ₂ . The cells are preferably grown in suspension culture.

[0024] In one exemplary embodiment, the neural stem cell culture comprises the following components at the indicated concentrations:

[0025]

[Table 1] .

Serum albumin may also be present in the main culture medium—however, the medium is usually serum starved or serum free (preferably serum free), chemically well defined and highly purified. Certain serum components that have been (> 95%) included, such as serum albumin, may be included.

The human neural stem cells described herein can be cryopreserved according to conventional procedures. Preferably, about 1 to 10 million cells are cryogenically in a "frozen" medium consisting of growth medium (absence of mitogenic growth factor), 10% BSA (Sigma A3059) and 7.5% DMSO. Saved. The cells are centrifuged. Growth medium is aspirated and replaced with freezing medium. The cells are gently resuspended as spheres rather than as detached cells. The cells are slowly frozen, for example by placing the container at -80 ° C. The cells are thawed by swirling in a 37 ° C bath, resuspended in fresh growth medium and grown as usual.

In another embodiment, the invention provides a differentiated cell culture comprising a number of previously unobtainable neurons and astrocytes and oligodendrocytes. In the prior art, typically, cultures of differentiated human diencephalic-derived neural stem cells formed few neurons (ie, 5-10%).
Less than). In accordance with the present technology, neuronal concentrations of between 20% and 35% (and higher than otherwise) are routinely achieved in cultures of differentiated human forebrain-derived neural stem cells. This is a great advantage. This is because it allows the enrichment of neural populations prior to transplantation in a host of disease manifestations in which nerve function is impaired or lost.

Furthermore, according to the method of the present invention, a differentiated neural stem cell culture highly enriched in GABAergic neurons was achieved. GAB like this
A-agonist neuron-enriched cell cultures are associated with excitotoxic neurodegenerative disorders (eg,
It is particularly advantageous in the powerful treatment of Huntington's disease or epilepsy.

To identify the cell phenotype during either proliferation or differentiation of neural stem cells,
Various cell surface markers or intracellular markers can be used.

When the neural stem cells of the invention are proliferating as neurospheres, human nestin antibodies can be used as a marker to identify undifferentiated cells. Proliferating cells are GFA
Little to no P staining and β-tubulin staining (although some staining may be present due to the diversity of cells within the sphere).

When differentiated, most cells lose their nestin-positive immunoreactivity. In particular,
Antibodies specific for various neuroproteins or glial proteins can be used to identify the phenotypic characteristics of differentiated cells. Neurons can be identified using neuron-specific enolase (“NSE”), nerve fibrils, antibodies to τ, β-tubulin, or other known neuronal markers. Astrocytes can be identified using antibodies against glial fiber acidic protein (“GFAP”), or other known astrocyte markers. Oligodendrocytes can be identified using antibodies against galactocerebroside, 04, myelin basic protein (“MBP”), or other known oligodendrocyte markers.

By identifying the compounds characteristically produced by these phenotypes,
It is also possible to identify the phenotype of the cell. For example, it is possible to identify neurons by the acidity of neurotransmitters (eg, acetylcholine, dopamine, epinephrine, norepinephrine, etc.).

Specific neural phenotypes can be identified according to the specific products produced by these neurons. For example, GABAergic neurons can be identified by their production of glutamate decarboxylase ("GAD") or GABA.
Dopaminergic neurons have their dopa decarboxylase (“DDC”)
, Dopamine, or tyrosine hydroxylase (“TH”) production. Cholinergic neurons can be identified by their production of choline acetyl transferase (“ChAT”). Hippocampal neurons are NeuN
Can be identified by staining with. It will be appreciated that any suitable known marker for identifying a particular neural phenotype may be used.

The human neural stem cells described herein can be genetically engineered or genetically modified according to known techniques. The term "genetic modification" refers to a stable or transient modification of the genotype of a cell by the deliberate introduction of exogenous DNA. DNA may be synthetic or naturally derived, and may include genes, parts of genes, or other useful DNA sequences. The term "genetic modification" is not meant to include naturally occurring modifications (eg, modifications that occur through natural viral activity, natural genetic modification, etc.).

The gene of interest (ie, the gene encoding the biologically active molecule) can be inserted into the appropriate expression vector cloning site by using standard techniques. These techniques are well known to those of ordinary skill in the art. See, for example, WO 94/16718, incorporated herein by reference.

The expression vector containing the gene of interest can then be used to transfect the desired cell line. Standard transfection techniques such as calcium phosphate co-precipitation, DEAE-dextran transfection, electroporation, biolistic, or viral trunk infection can be used. Commercially available mammalian transfection kits can be purchased, for example, from Stratagene. Human adenovirus transfection is described by Berg et al., Exp. Cell Res. 1
It can be achieved as described on page 92 (1991). Similarly, lipofectamine-based transfection was performed in Cattaneo, Mol. Brain Res. 42, 161-66 (1996).

A wide variety of host / expression vector combinations may be used to express the gene encoding the biologically active molecule of interest. For suitable cell-based production expression vectors, see, eg, US Pat. No. 5,545,723 (incorporated herein by reference).

Increased expression of biologically active molecules can be achieved by increasing or amplifying the transgene copy number using amplification methods well known in the art. Examples of such an amplification method include DHFR amplification (for example, Kaufma).
n et al., U.S. Pat. No. 4,470,461), or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464, and European Application Publication EP 338,841). , All of which are incorporated herein by reference).

[0040] In another embodiment, the genetically modified neural stem cells are derived from transgenic animals.

If the neural stem cells are genetically modified for the production of biologically active substances, this substance is preferably useful for the treatment of CNS disorders. To this end, genetically modified neural stem cells can be generated that can secrete therapeutically effective biologically active molecules in a patient. Further consideration is the production of biologically active molecules that have a proliferative or trophic effect on the transplanted neural stem cells. Inducing the differentiation of cells into neuronal lineages is further considered. Thus, genetically modified neural stem cells provide cell-based delivery of biological agents with therapeutic value.

The neural stem cells described herein, and their differentiated progeny, can be immortalized or conditionally immortalized using known techniques. Conditional immortalization of stem cells is preferred, and most preferably conditional immortalization of these differentiated progeny. Among the conditional immortalization techniques, Tet conditional immortalization (WO96 / 31
242, incorporated herein by reference), and Mx-1 conditional immortalization (see WO 96/02646, incorporated herein by reference). .

The present invention also provides a method for differentiating neural stem cells to obtain a cell culture enriched in neurons to a degree previously unavailable. According to one protocol, proliferating neurospheres are depleted of growth factor mitogens and LIF, and sources of 1% serum, substrate and ionic charge (eg, polyornithine or extracellular). Differentiation is induced by the provision of a cover glass) covered with a matrix component. A suitable base medium for this differentiation protocol is otherwise the same as the growth medium, except for the growth factors mitogen and LIF. This differentiation protocol produces cell cultures enriched for neurons. According to this protocol, neuronal concentrations of between 20% and 35% were routinely achieved in differentiated human forebrain-derived neural stem cell cultures.

According to the second protocol, the proliferating neurospheres are depleted of growth factor mitogens, and sources of 1% serum, substrate and ionic charge (eg polyornithine or extracellular matrix). Cover glass covered with ingredients), as well as PD
Differentiation is induced by the provision of a mixture of growth factors including GF, CNTF, IGF-1, LIF, forskolin, T-3 and NT-3. A cocktail of growth factors can be added at the same time as the neurospheres are removed from the growth medium, or can be added to the growth medium and cells preincubated with the mixture prior to removal from the mitogen. This protocol produces cell cultures that are highly enriched for neurons and oligodendrocytes. According to this protocol, neuron concentrations of> 35% were routinely achieved in differentiated human forebrain-derived neural stem cell cultures.

The presence of bFGF in the growth medium, contrary to expectations, inhibits the differentiation potential of oligodendrocytes. bFGF is trophic to oligodendrocyte precursor cell lines. Oligodendrocytes are induced under differentiating conditions when passaged without bFGF containing EGF and LIF in the culture medium.

The human stem cells of the present invention have numerous uses, including for drug screening, diagnostics, genomics and translation. Stem cells can be induced to differentiate into a neural cell type of choice using the appropriate medium described in this invention. The drug to be tested can be added prior to differentiation to test for developmental inhibition, or post-differentiation to monitor neuronal type-specific responses.

The cells of the invention may be administered to a patient according to conventional techniques, eg, US Pat. No. 5,082.
, 670 and 5,618,531, each of which is incorporated herein by reference, or in the CNS, as described herein, or at any other suitable body part. Can be transplanted.

[0048] In one embodiment, human stem cells are transplanted directly into the CNS. Sites of parenchyma and sites within the sheath are considered. It will be appreciated that the exact location in the CNS will vary according to the disease state.

Cells to be transplanted can be labeled with bromodeoxyuridine (BrdU) prior to transplantation. As observed in various experiments, cells that are dually stained for neuronal marker and BrdU in various grafts show differentiation of BrdU-stained stem cells into appropriate differentiated neuronal cell types (see Example 9). See). Transplantation of human forebrain-derived neural stem cells into the hippocampus produced neurons that predominantly stain NeuN but are GABA-negative. NeuN antibodies are known to stain hippocampal neurons. GABAergic neurons were formed when these same cell lines were transplanted into the striatum. Therefore, the transplanted cells respond to environmental cues in both the adult and neonatal brains.

According to one aspect of the invention, there is provided herein a methodology for improving the viability of transplanted human neural stem cells. In particular, graft viability is improved when compared to transplantation of dissociated single cell suspensions, where transplanted neural stem cells may aggregate or form neurospheres prior to transplantation. It Preferably, small neurospheres with a diameter of about 10-500 μm, preferably 40-50 μm, are implanted. Alternatively, about 5-100, preferably 5-20 per sphere.
Spheres containing cells of About 10,000 to 1,000,000 per 1 μl
Of cells, preferably a density of 25,000 to 500,000 cells per μl is preferred for transplantation.

Cells can also be encapsulated and used to deliver biologically active molecules according to known encapsulation techniques. As this encapsulation technique, microencapsulation (for example, US Pat. No. 4,352,883; US Pat.
No. 3,888; and 5,084,350, incorporated herein by reference) (b) Macroencapsulation (eg, US Pat. No. 5,284).
, 761; 5,158,881; 4,976,859; and 4,968,733, and published PCT patent application WO 92/191.
95, WO 95/05452, each incorporated herein by reference).

When human neural stem cells are encapsulated, US Pat. No. 5,284,761
No. 5,158,881; No. 4,976,859; No. 4,968,
733; 5,800,828; and published PCT patent application WO9.
The macroencapsulation described in 5/05452, each incorporated herein by reference, is preferred. The number of cells in the device can vary; preferably each device contains between 10 ^{3 and} 10 ⁹ cells, most preferably 10 ⁵ to 10 ⁷ cells. Multiple macroencapsulation devices can be implanted in a patient; preferably between 1 and 10 devices.

In addition, “naked” transplantation of human stem cells in combination with a capsule-like device is also contemplated, where the capsule-like device is a biologically active molecule that is therapeutically effective in a patient. , Or a biologically active molecule that has a proliferative or trophic effect on the transplanted neural stem cells, or a biologically active molecule that induces the differentiation of neural stem cells towards a specific phenotypic strain. Secrete.

The present invention further provides methods of inducing migration and proliferation of progenitor cells transplanted into the brain in vivo. In one embodiment, in vivo migration of progenitor cells transplanted to a first location in the brain of a subject is induced by injection of EGF at a second location in the brain. In some preferred embodiments, the first location is in the striatum of the brain and the second location infused with EGF is the lateral ventricle of the brain. In another preferred embodiment, EGF injection induces migration towards a second location (eg, the location of injection) but not differentiation of progenitor cells.

In another embodiment, the in vivo proliferation of progenitor cells transplanted at the location of the brain of the subject is induced by injecting EGF at or near the location of the implantation. In some preferred embodiments, the locus of implantation is in the striatum of the brain and EGF is injected into the lateral ventricles of the brain. In another embodiment of the method of the present invention, the progenitor cells are mammalian embryonic progenitor cells, and the progenitor cells are cultured in medium containing EGF prior to transplantation.

Any EGF-responsive neural stem cell suitable for treatment of a given neurological disease state may be utilized. For example, EGF-responsive stem cells can be excised from the striatal discs of, for example, a transgenic embryonic mammal (eg, mouse). Progenitor cells
It can be cultured and expanded as described above. These cells can be cultured in growth medium containing EGF and centrifuged and the desired final concentration (typically 250,000).
Prepared for transplantation by collecting small "spheres" of cells (typically about 15-30 cells), as described above, by resuspending in 00 cells / μl). Progenitor cells can also be encapsulated for transplantation, as described above.

Transplantation of cells into the brain of a subject is performed by stereotactic surgery under anesthesia. Multiple stacks of cell sphere suspensions (eg, 500,000 cells per stack) can be made in the striatum of the brain. After implantation, an injection cannula can be placed in the ventricles (eg, lateral ventricles) for EGF injection and fixed using dental cement. A minipump can be used to infuse EGF (eg, dissolved in serum / gentamicin / saline solution) over several days. The total dose of EGF required to induce migration and proliferation of transplanted cells will vary somewhat from subject to subject, but for example, about 400 ng / day of EGF can be infused. Dividing cells can be labeled for study by BrdU, for example by intraperitoneal injection of BrdU following cell transplantation. Alternatively, the encapsulated EGF-producing cells can be transplanted into the ventricles adjacent to the progenitor cell transplant.

EGF-responsive neural progenitor cells may respond to EGF after transplantation in vivo. Cells transplanted into the striatum of adult rats can proliferate and migrate to the source of intraventricular EGF, and this response is maintained over several days of EGF infusion. Some of these newly generated cells subsequently differentiate into glial cells and express the astrocyte marker GFAP. Subventricular region (sub-ventri)
Newly developed BrdU positive cells in the color zone (SVZ) can be found at a maximal distance of 1 mm labial to the infusion cannula and are further apart in labial migrating flow on the pathway to the olfactory bulb. Absent. In addition, some cells are maintained at the site of proliferation, forming small nodules of SVZ, which project into the lateral ventricles.

The transplanted progenitor cells show an active response to EGF in vivo,
Cell proliferation and directed migration are directed away from the graft core towards the EGF source. The EGF protein may diffuse through and across the striatum in order to exert an effect on the transplanted cells, which after implantation in vivo E
Maintains responsiveness to GF. Accordingly, the present invention provides nerve growth factors (eg, E
Intraventricular delivery of GF) is provided as a promising system for manipulating cells after transplantation. Injection of EGF in vivo provides a means to manipulate post-transplant progenitor cells for at least a short period of time to direct these cells to specific differentiation or directed migration, or to increase their survival. This technique plays an important role in overcoming the problems associated with the restricted migration and differentiation of transplanted cells, and thus the ability of transplanted neurons to re-invade host tissue in the paradigm of neural transplantation. Can be increased.

No morphological differences are observed between transplanted cells exposed to EGF in vivo and transplanted cells that received only vehicle injection. Extensive glial differentiation is seen in all implants as evidenced by the M2 positive profile, but neuronal differentiation is observed using both the early neuronal markers Hu and β-III-tubulin. Not done. Therefore, it is unlikely that EGF exerts its effect on different types of cells within the mixed population found in these precursor cell cultures, both on the progenitor cells themselves and on the more differentiated glial progenitor cells. Is likely.

The cells and methods of the invention may be useful in treating various neurodegenerative diseases and other disorders. It is contemplated that the cell replaces diseased, damaged or lost tissue in the host. Alternatively, the transplanted tissue may increase the function of the endogenously affected host tissue. The transplanted neural stem cells can also be genetically modified to provide therapeutically effective biologically active molecules.

Excitotoxicity includes various pathological conditions including epilepsy, seizures, ischemia, and neurodegenerative diseases such as Huntington's disease, Parkinson's disease and Alzheimer's disease.
Related to. Therefore, neural stem cells may provide one means of preventing or replacing the behavioral abnormalities of cell loss and associated disorders. Neural stem cells can replace cerebellar neurons lost in cerebellar ataxia, and clinical development can be readily measured by methods known in the medical arts.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by a ruthlessly progressive movement disorder with catastrophic psychiatric and cognitive deterioration. HD is associated with consistent and severe atrophy of the neostriatum, which is associated with a marked loss of GABAergic medium-sized spinous protrusion neurons (major striatal output neurons). Related. Excitotoxic intrastriatal injections such as quinolinic acid (QA) mimic the pattern of selective neuronal fragility seen in HD. QA
The lesions result in motor and cognitive deficits, which are among the major symptoms seen in HD. Therefore, intrastriatal injection of QA has been a useful model for HD and aimed to prevent, ameliorate, or reverse HD-related neuroanatomical and behavioral changes. , And may serve to evaluate new therapeutic strategies. Since GABAergic neurons are characteristically lost in Huntington's disease, the treatment of patients with Huntington's disease can be achieved by transplantation of cell cultures enriched in GABAergic neurons induced by the methods of the invention. Can be achieved.

Epilepsy is also associated with excitotoxicity. Therefore, GAB induced by the present invention
A-acting neurons are intended for transplantation into patients suffering from epilepsy.

The cells of the invention can be administered to various demyelinating and myelinating disorders (eg, Perizeus-
It may be used in the treatment of Merzbacher's disease, multiple sclerosis, various leukoatrophy, post-traumatic demyelination, and cerebrovascular (CVS) disorders), and various neuritis and neuropathy (especially ocular). The present invention contemplates the use of oligodendrocytes or oligodendrocyte precursors or cell cultures enriched for progenitor cells, which cultures have been prepared and transplanted according to the invention, Promotes remyelination of demyelinated areas in the host.

The cells of the present invention can also be used in the treatment of various acute and chronic pains, and for certain nerve regeneration applications such as spinal cord injury. The present invention also contemplates the use of human stem cells for use in ocular photoreceptor sparing or development.

In another aspect of the invention, local delivery of neurotrophic factors (eg, EGF) to newly transplanted cells according to the invention provides a means of in vivo regulation and undifferentiated precursors. Re-control of host tissue and associated behavioral recovery to guide cells into division, migration or differentiation into specific phenotypes and increase graft survival,
For enhancing the effectiveness of transplantation as a potential restorative treatment for neurodegenerative diseases,
It may provide a controlled means.

The cells and methods of the invention are intended for use in a mammalian host, recipient, patient, subject or individual (preferably primate, most preferably human).

All references cited herein are incorporated herein by reference. The following examples are provided for illustrative purposes only and are not intended to be limiting.

[0070]   (Example)   (Example 1: Medium for growing neural stem cells)   Growth medium was prepared with the indicated concentrations of components:

[0071]

[Table 2] .

Example 2 Isolation of Human CNS Native Stem Cells Sample tissues from human fetal forebrain were collected and dissected in Waden and were kindly provided by Huddinje Sjukhus. Blood samples from donors were sent for virus testing. Dissection was performed in saline and selected tissues were placed directly into growth medium (described in Example 1). Tissues were stored at 4 ° C until dissected. The tissue was dissociated using a standard glass homogenizer in the absence of any digestive enzymes. The dissociated cells were counted and seeded in flasks containing growth medium. After 5-7 days, 100 contents of the flask.
Centrifuge for 2 minutes at 0 rpm. The supernatant was aspirated and the pellet resuspended in 200 μl growth medium. The cell culture was treated with P200 to destroy the clusters.
Milled using Pipetman approximately 100 times. Cells for 75,000-100,
Re-seeded to growth medium at 000 cells / ml. Cells were passaged every 6-21 days depending on mitogen and seeding density. Typically, these cells are
BrdU (index of cell proliferation) is incorporated. T75 flask culture (initial volume 2
For 0 ml), cells are "fed" three times weekly by adding 5 ml of growth medium. In a preferred embodiment, a Nunc flask is used for the culture.

Nestin Staining for Proliferating Neurospheres Cells were stained for nestin (a unit of measurement of proliferating neurospheres) as follows. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells were washed twice with 0.1 M PBS (pH 7.4) for 5 minutes. 100 cells
Permeated with% EtOH for 2 minutes. The cells were then washed twice with 0.1 M PBS for 5 minutes. The cell preparation was treated with 0.1 M PBS (pH 7.
4) and 5% normal goat serum ("NGS") diluted in 1% Triton X-100 (Sigma X-100) and blocked for 1 hour at room temperature with gentle agitation. Cells were diluted in 1% NGS and 1% Triton X-100 with a primary antibody against human nestin (Dr. Lars Wahlger).
Rabbit polyclonal antibody from G., Karolinska, Sweden,
Used at 1: 500) and incubated for 2 hours at room temperature. Then
The preparation was washed twice with 0.1 M PBS for 5 minutes. Cells were diluted in 1% NGS and 1% Triton X-100, secondary antibody (GAM / FITC pool used at 1: 128, Sigma F-0257; GAR / TRIT).
C was used at 1:80, Sigma T-5268) and incubated in the dark for 30 minutes at room temperature. The preparation is washed twice with 0.1 M PBS for 5 minutes in the dark. The preparation was placed in a monitoring medium (Vectashield Mount).
ing Medium, Vector Labs. ． , H-1000) and mounted on table-top slides and stored at 4 ° C.

FIG. 1 shows proliferating spheres of human forebrain-derived neural stem cells.
sphere) (herein referred to as the "neurosphere"). Proliferation of four strains of human forebrain-derived neural stem cells was evaluated in growth medium as described above with or without LIF. As illustrated in Figure 2, LIF significantly increased cell growth rate in 3 out of 4 strains (6.5 Fbr, 9 Fbr and 10.5 Fbr).
). The effect of LIF was most pronounced after about 60 days in vitro.

The effect of bFGF on the rate of proliferation of human forebrain-derived neural stem cells was also evaluated. As FIG. 3 illustrates, stem cell proliferation was significantly enhanced in the presence of bFGF.

Example 3 Differentiation of Human Neural Stem Cells In a first differentiation protocol, proliferating neurospheres were treated with the removal of growth factors mitogen and LIF, and 1% serum, substrate and ionic charge. Differentiation was achieved by supplying a source (eg, a cover glass covered with polyornithine).

The staining protocol for neurons, astrocytes and oligodendrocytes was as follows: (β-tubulin staining for neurons) Cells were incubated with 4% paraformaldehyde at room temperature. It was fixed for 20 minutes. Cells
The plate was washed twice with 0.1 M PBS (pH 7.4) for 5 minutes. Cells with 100% Et
Soak for 2 minutes with OH. The cells were then washed twice with 0.1 M PBS for 5 minutes. Cell preparation was diluted in 0.1 M PBS, pH 7.4, 5
Blocked in% normal goat serum ("NGS") for 1 hour at room temperature. Cells
Primary antibody against β-tubulin (Sigma T diluted in 1% NGS).
-8660, mouse monoclonal; used at 1: 1,000) for 2 hours at room temperature. The preparation was then washed twice with 0.1 M PBS for 5 minutes. Cells were diluted in 1% NGS with a secondary antibody (GAM / FITC).
Pool used at 1: 128, Sigma F-0257; GAR / TR
Incubated for 30 minutes at room temperature in the dark with ITC used at 1:80, Sigma T-5268). The preparation is washed twice with 0.1 M PBS for 5 minutes in the dark. The preparation was placed in a monitoring medium (Vectashield Mou).
nting Medium, Vector Labs. ． , H-1000) and mounted on table-top slides and stored at 4 ° C.

In some examples, cells were also stained with DAPI (nuclear stain) as follows. Coverslips prepared as above were treated with DAPI solution (1: 1000 diluted in 100% MeOH, Boehringer Mannheim, # 23.
6 276). The coverslips are incubated at 37 ° C. for 15 minutes in DAPI solution.

(O4 Staining on Oligodendrocytes) Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells
The plate was washed 3 times for 5 minutes with 0.1 M PBS (pH 7.4). Add the cell preparation to 0.1
Blocking was performed in 5% normal goat serum ("NGS") diluted in MPBS (pH 7.4) for 1 hour at room temperature. Cells were diluted in 1% NGS with primary antibody to O4 (Boehringer Mannheim, # 1518 92).
5, mouse monoclonal; used 1:25) for 2 hours at room temperature. The preparation was then washed twice with 0.1 M PBS for 5 minutes.
Cells were incubated with secondary antibody and further treated as described above for β-tubulin.

(GFAP Staining on Astrocytes) Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Cells
The plate was washed twice with 0.1 M PBS (pH 7.4) for 5 minutes. Cells with 100% Et
Soak for 2 minutes with OH. The cells were then washed twice with 0.1 M PBS for 5 minutes. Cell preparation was diluted in 0.1 M PBS, pH 7.4, 5
Blocked in% normal goat serum ("NGS") for 1 hour at room temperature. Cells
Incubated with primary antibody against GAFP (DAKO Z334, rabbit polyclonal; used at 1: 500) diluted in 1% NGS for 2 hours at room temperature. The preparation was then washed twice with 0.1 M PBS for 5 minutes. Cells were incubated with secondary antibody and further treated as described above for β-tubulin.

This differentiation protocol resulted in neuron-enriched cell cultures as follows:

[0082]

[Table 3] .

The ability of the single cell lines to differentiate when the cultures were aging (ie at different passages) was also evaluated using the differentiation protocol described above. The data is as follows:

[0084]

[Table 4] . These data suggest that cells follow a reproducible differentiation pattern independent of passage number or culture age.

Example 4 Differentiation of Human Native Stem Cells In a second differentiation protocol, proliferating neurospheres were depleted of growth factors mitogen and LIF, and 1% serum, substrate (eg, cover). Glass or extracellular matrix components), source of ionic charges (eg poly-ortinin)
, And 10 ng / ml PDGF A / B, 10 ng / ml CNTF,
10 ng / ml IGF-1, 10 μM forskolin, 30 ng / ml T
Differentiation was achieved by feeding a mixture of growth factors containing 3, 10 ng / ml LIF and 1 ng / ml NT-3. This differentiation protocol resulted in cell cultures that were highly enriched for neurons (ie, greater than 35% of differentiated cell cultures) and oligodendrocytes.

Example 5: Differentiation of Human Neural Stem Cells In a third differentiation protocol, cell suspensions were first cultured in a mixture of hbFGF, EGF and LIF, then 20 ng / mL hEGF (GIBC).
O) and 10 ng / mL human leukemia inhibitory factor (LIF) (R & D System
em) was plated onto a modified growth medium containing but not hbFGF. The cells were initially grown significantly slower than cultures that also contained hbFGF (see Figure 3). Nonetheless, the cells continued to grow and were passaged 22 times. Stem cells are removed from the growth medium and a mixture of growth factors (hPD
GF A / B, hCNTF, hGF-1, forskolin, T3 and hNT-
Differentiation was achieved by plating on poly-ornithine coated coverslips in differentiation medium supplemented with 3). Surprisingly, GalC immunoreactivity was found in these differentiated cultures at levels well above the number of 04-positive cells found in the growth factor induction protocol described in Example 4.

Thus, this protocol resulted in differentiated cell cultures enriched for oligodendrocytes. Neurons were found only occasionally, had a simple process, and appeared to be very immature.

Example 6 Genetic Modification The human neural stem cell-derived glioblast cell line described herein is transformed into WO9.
Conditionally immortalized using the Mx-1 system described in 6/02646.
In the Mx-1 system, the Mx-1 promoter drives the expression of SV40 large T antigen. The Mx-1 promoter is driven by interferon. When induced, large T is expressed and resting cells proliferate.

Human glioblasts were derived from human forebrain neural stem cells as follows. Proliferating human neurospheres were removed from the growth medium and plated on polyornithine plastic in mitogenic factors EGF, bFGF and LIF, and a mixture of 0.5% FBS and N2 (24 well plate). . 0.5 ml N2 medium and 1% FBS was added. The cells were incubated overnight. The cells are then
p31 using the nvitrogen lipid kit (lipids 4 and 6)
8 (a plasmid containing the Mx-1 promoter operably linked to the SV40 large T antigen). The transfection solution is optiM
EM medium contained 6 μl / ml lipid and 4 μl / ml DNA. Cells
Incubated in transfection solution for 5 hours. The transfection solution was removed and cells were placed in N2 and 1% FBS and 500 U / ml A / D interferon. Cells were fed twice a week. After 10 weeks, cells were assayed for large T antigen expression. The cells at this time showed robust T antigen staining. As shown in FIG. 4, the cell number was higher in the presence of interferon than in the absence of interferon.

Large T antigen was monitored using immunocytochemistry as follows. Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The cells are
It was washed twice with 1 M PBS, pH 7.4 for 5 minutes. Cells with 100% EtOH2
Permeabilized in minutes. The cells were then washed twice with 0.1 M PBS for 5 minutes. The sub-preparation was diluted with 0.1 M PBS, pH 7.4, and diluted with 5% normal goat serum ("
NGS ") at room temperature for 1 hour. Cells were incubated for 2 hours at room temperature with primary antibody against large T antigen (used 1:10) diluted in 1% NGS. Antibodies to large T antigen were prepared by culturing PAB149 cells and obtaining conditioned medium. The preparation is then added to 0.1M PBS for 5 minutes.
Washed twice per minute. Cells were diluted with 1% NGS as a secondary antibody (Vector L
laboratories Vectastain Elite ABC mou
Incubation with a 1: 500 biotinylated goat anti-mouse from se IgG kit, PK-6102) for 30 minutes at room temperature. The preparation is 0.1M
Wash twice for 5 minutes with PBS. The preparation is 1: 1 in 0.1 M PBS, pH 7.4.
Incubate for 30 minutes at room temperature in 500 diluted ABC reagent. 0 cells
． Wash twice for 5 minutes in 1M PBS, pH 7.4, then 0.1M Tris
Wash twice for 5 minutes at pH 7.6. Cells are incubated in DAB (nickel-enhanced) for 5 minutes at room temperature. DAB solution was removed and cells were washed with dH ₂ O.
Wash 3-5 times with. Cells are treated with 50% glycerol / 50% 0.1M PBS.
, Stored at pH 7.4.

Example 7 Encapsulation The following procedure may be used when encapsulating human neural stem cells: Hollow fibers are polyethersulfone (PES) with an outer diameter of 720 μm and a wall thickness of 100 μm. (AKZO-Nobel Wuppertal)
, Germany). These fibers are described in US Pat. Nos. 4,976,859 and 4,968,733 (incorporated herein by reference).
Fibers may be selected for their molecular weight cutoff. In a preferred embodiment, a 280 kd MWCO PES # 5 membrane is used. In another preferred embodiment, a MWCO PES # 8 membrane of about 90 kd is used.

Devices typically include: 1) Semipermeable poly (ether sulfone) hollow fiber membranes made by AKZO Nobel Faser AG; 2) Hub membrane segments 3) Light cured methacrylate (LCM) resin tips. And 4) Silicone rope. The semipermeable membranes used typically have the following characteristics: internal diameter 500 ± 30 μm wall thickness 100 ± 15 μm force at fracture 100 ± 15 cN elongation at fracture 44 ± 10% hydraulic permeability 63 ± 8 (ml / Min m ² mmHg) nMWCO (Dextran) 280 ± 20 kd The components of the device are commercially available. LCM adhesive is Ablestik La
available from laboratories (Newark, DE) (Luxtr
ak Adhesives LCM23 and LCM24). The rope material is Spe
ciality Silicone Fabricators (Robles, C
It is available from A). The size of the rope is 0.79mm OD × 0.43mm I
D × length 202 mm. The morphology of the device is as follows: The inner surface has permselective skin. The wall has an open cell foam structure. The outer surface has an open structure with pores up to 1.5 μm which occupy 30 ± 5% of the outer surface.

The fiber material is first cut into 5 cm long segments and the distal end of each segment is sealed with a photopolymerized acrylic adhesive (LCM-25, ICI). After sterilization with ethylene oxide and degassing, the fiber segments were placed between 10 ⁴ to 10 ⁷ cells in either a liquid medium or a hydrogel matrix (eg collagen solution (Zyderm®), alginate, agarose or chitosan). Of suspension is loaded through a Hamilton syringe and an attached inlet through a 25 gauge needle. Seal the proximal end of the capsule with the same acrylic adhesive. The volume of the device intended in human studies is approximately 15-18 μl
Is.

Silicone Rope (Specialty Silicone Fabrication)
n, Taunton, MA) (ID: 690 μm; OD: 1.25 mm) is placed on the proximal end of the fiber to allow easy manipulation and recovery of the device.

Example 8 Neural Stem Cell Transplantation Human neural stem cells were transplanted into rat brain and transplanted in graft viability, integration, phenotypic consequences of transplanted cells and in animals in disability. Behavioral changes associated with cells were evaluated.

Transplantation was performed according to standard techniques. Adult rats were anesthetized with sodium pentobarbitol (45 mg /
(kg, intraperitoneal), and aligned with a Kopf stereotaxic instrument. A midline incision was made in the scalp and punctured for injection of cells. Rats received grafts of undifferentiated human neural stem cells into the left striatum, unmodified using a glass capillary attached to a 10 μl Hamilton syringe. Each animal has a total of about 2 in a total volume of 2 μl.
Received 50,000-500,000 cells. Cells were transplanted 1-2 days after passage and this cell suspension was constructed with undifferentiated stem cell clusters of 5-20 cells. Following implantation, the skin was sutured closed.

[0097]   Animals were behaviorally tested and then sacrificed for histological analysis.

Example 9 Intraventricular EGF Delivery by Transplantation of Neural Stem Cells Approximately 300,000 neural stem cells as small neurospheres were applied to adult rat striatum near the lateral ventricles using standard techniques. Transplanted. EGF during the same surgical session
Osmotic minipumps releasing either (400 ng / day) or vehicle were also implanted intrastriatum. Rats were treated with EG at a flow rate of 0.5 μL / hour for 7 days.
F, resulting in a total delivery of 2.8 μg of EGF to the lateral ventricle of each animal. A subset of transplanted rats was further immunosuppressed by intraperitoneal cyclosporine injection (10 mg / kg / day). During the last 16 hours of pump infusion, animals received BrdU infusions (120 mg / kg) every 3 hours.

One week after transplantation, the animals were perfused with 4% paraformaldehyde and serial sections were cut at 30 μm thickness on a frozen microtome. Brain sections were stained for astrocytes, oligodendrocytes, neurons, and undifferentiated precursor cell markers. Minimal migration was shown in adult CNS in the absence of EGF.
Good survival of 7 day old grafts was found in rats that received EGF as shown by M2 immunoreactivity, and grafts in EGF-treated animals were much larger than those in animals treated with vehicle alone. there were. In addition, host cell proliferation was observed for EGF treatment. Animals that received BrdU injection prior to sacrifice
It showed an increased number of dividing cells in the treated ventricles, but not in adjacent ventricles.

Example 10: Treatment of syringomyelia Primary fetal transplantation was used to occlude the cavity formed around spinal cord injury in a patient. The neural stem cells described in the present invention are suitable for replacement. This is because only structural function is required of the cell. Transplant neural stem cells into the bone marrow of a patient to prevent cavity formation. Preferably, the results are measured by MRI imaging. Clinical trial protocols have been written and can be readily modified to include the neural stem cells described.

Example 11: Progeny of human neural stem cells grown in vitro (progen)
Treatment of Neurodegenerative Diseases Using t) Cells were obtained from ventral midbrain tissue from an 8 week old human fetus following conventional aspiration abortion. The tissue was collected into a sterile collection device. A 2 × 4 × 1 mm piece of tissue was dissected and separated as in Example 2. The neural stem cells were then expanded. Neural stem cell progeny were used for nerve transplantation into blood groups consistent with hosts with neurodegenerative disease. Surgery was performed using a BRW computed tomography (CT) stereotactic guide. Intravenous administration of midazolam to patients resulted in local anesthetic spiementhea (
suppiemencea). The patient underwent CT scanning to establish the coordinates of the area receiving the implant. The infusion cannula consists of a 17 gauge stainless steel outer cannula with a 19 gauge inner stylet. It was inserted into the brain at the correct coordinates and then removed and replaced with a 19 gauge infusion cannula preloaded with 30 μl of tissue suspension. The cells are slowly infused at a rate of 3 μl / min while cannulating. Multiple stereotactic needle passes were made into the area of interest, approximately 4 mm apart. Patients were tested on CT scans for post-operative bleeding or edema. Neurological assessments will be performed at various post-operative intervals and PET scans will be performed to determine the metabolic activity of the graft cells.

Example 12 Genetic Modification of Neural Stem Cell Progeny Using Calcium Phosphate Transfection Neural stem cell progeny are grown as described in Example 2. The cells are then transfected using the calcium phosphate transfection technique. For standard calcium phosphate transfection, cells are mechanically separated into single cell suspensions and plated onto tissue culture treated dishes at 50% confluence (50,000-75,000 cells / cm ² ). , And attach overnight.

The modified calcium phosphate transfection procedure is performed as follows: T
DNA (15-25 μg) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA, pH 7.5) diluted to 440 μl with E and 60 μl 2M CaCl ₂ (pH up to 5.8 with 1M HEPES buffer). To DNA / TE
Add to buffer. 500 μl total of 2 × HeBS (HEPES buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na ₂ HPO ₄ ,
12 mM dextrose, 40 mM HEPES buffered powder, pH 6.92) is added dropwise to this mixture. The mixture is left at room temperature for 20 minutes. 1 x H cells
Wash briefly with eBS, and add 1 ml of calcium phosphate-precipitated DNA solution to each plate, and incubate cells for 20 minutes at 37 ° C. Following this incubation, complete medium was added 10ml into cells, and plates incubator _{(37 ℃, 9.5% CO 2} ) placing an additional 3-6 hours in. DN
A and media are removed by aspiration at the end of the incubation period and cells are washed 3 times with complete growth media and then returned to the incubator.

Example 13: Genetic Modification of Neural Stem Cell Progeny The cells grown as in Example 2 are transfected with an expression vector containing a gene for the FGF-2 receptor or NGF receptor. Vector DNA containing this gene was added to 0.1 × TE (1 mM Tris pH 8.0, 0.1
Dilute to a concentration of 40 μg / ml in mM EDTA). 22 μl of DNA was added to 250 μl of 2 × HBS (in a disposable, sterile 5 ml plastic tube).
280 mM NaCl, 10 mM KCl, 1.5 mM Na ₂ HPO ₄ 2H ₂ O, 12 mM dextrose, 50 mM HEPES). 31 μl of 2M CaCl ₂ is added slowly and the mixture is incubated for 30 minutes at room temperature. During this 30 minute incubation, cells are centrifuged at 800 g for 5 minutes at 4 ° C. Resuspend cells in 20 volumes of ice cold PBS, and 1
Divide into aliquots of 10 ⁷ cells and centrifuge again. 1 aliquot of each cell
Resuspend in ml of DNA-CaCl ₂ suspension and incubate for 20 minutes at room temperature. The cells are then diluted with growth medium and incubated for 6-24 hours at 37 ° C. with 5% -7% CO ₂ . Cells are recentrifuged, washed with PBS and returned to 10 ml of growth medium for 48 hours.

This transfected neural stem cell progeny is either transplanted into a human patient using the procedure described in Example 8 or Example 11 or used for drug screening procedures as described in the Examples below. To do.

Example 14 Screening for Drugs or Other Biological Agents for Effects on Pluripotent Neural Stem Cells and Progeny of Neural Stem Cells A. Effects of BDNF on Neuronal and Glial Cell Differentiation and Survival ) Progenitor cells were grown as described in Example 2 and differentiated as described in Example 4. BDNF was added at a concentration of 10 ng / ml when the cells were plated. In vitro days 3, 7, 14 and 21 (DIV)
Secondly, cells were processed for indirect immunocytochemistry. BrdU labeling was used to monitor neural stem cell expansion. The effect of BDNF, oligodendrocytes and astrocytes on neurons is found in neurons (MAP-
2, NSE, NF), an antigen recognizing an antigen found in oligodendrocytes (O4, GalC, MBP) or an antigen recognizing an antigen found in astrocytes (GFAP) Assayed by examining the culture. Cell survival was determined by counting the number of immunoreactive cells at each time point and morphological observations were made. BDNF had a significant increase in neuronal differentiation and survival relative to the number observed under control conditions. The number of astrocytes and oligodendrocytes did not change significantly from control values.

B. Effect of BDNF on Differentiation of Neuronal Phenotypes Cells treated with BDNF according to the method described in Part A are antibodies that recognize neurotransmitters or enzymes involved in the synthesis of neurotransmitters. It investigated using. These included TH, ChAT, substance P, GABA, sonatostatin and glutamic acid. Control-treated culture conditions and BDN
In the F-treated culture conditions, the neurons showed substance P and GABA.
Was tested positive for the presence of Also, an increase in number, neurons grown in BDNF showed a dramatic increase in neurite outgrowth and branching when compared to control cases.

C. Identification of Growth Factor Responsive Cells Cells were differentiated as described in Example 4 and about 100 ng / ml BDNF was added at 1 DIV. The cells were fixed and treated for double-labeled immunocytochemistry 1 h, 3 h, 6 h, 12 h and 24 h after the addition of BDNF. Antibodies that recognize neurons (MAP-2,
NSE, NF), antibodies that recognize oligodendrocytes (O4, GAlC, MBP)
Alternatively, an antibody that recognizes astrocytes (GFAP) was used in combination with an antibody that recognizes c-fos and / or other immediate early genes. Exposure to BDNF resulted in a selective increase in the expression of c-fos in neuronal cells.

D. BDNF for expression of markers and regulators during proliferation and differentiation
Effect of BDNF-treated cells according to the method described in Part A, modulators,
Processed for analysis of FGF-R1 or other marker expression.

E. Effect of Chlorpromazine on Proliferation, Differentiation and Survival of Stem Cell Progeny Produced by Growth Factors Chlorpromazine, a drug widely used in the treatment of psychosis, replaces BDNF in Examples 14A through 14D above. 10 ng / ml to 10
Used at concentrations ranging from 00 ng / ml. The effect of drugs at various concentrations on stem cell proliferation and differentiation and survival of stem cell progeny is monitored. Changes in gene expression and electrophysiology characteristics of differentiated neurons are determined.

Example 15 Induction of In Vivo Proliferation and Migration of Transplanted Progenitor Cells in the Brain EGF-Responsive Mouse Progenitor Cells Remain Responsive to Indoorly Administered EGF After They Are Implanted In Vivo To determine if there is a EGF generated from transgenic mice carrying the β-galactosidase enzyme (lacZ) gene under the control of a promoter for myelin basic protein (MBP).
Embryonic cells grown in a culture medium containing were transplanted into the medial striatum of adult rats. EGF
Was administered for 7 days after transplantation to assess its effect on proliferation, migration and differentiation of transplanted cells.

Cell Source EGF-responsive stem cells were generated from transgenic mice containing an insert for the galactosidase enzyme (MPB-lacZ) under the control of the MPB promoter. Striatal discs were excised from el4.5-e15.5 mouse embryos as previously described. See: Reynolds et al. , Journa
l of Neuroscience 12, pp. 4565-4574 (19
92). Tissue sections were broken into single cell suspensions by mechanical trituration with a flame-polished Pasteur pipette, and cells were resuspended in the following growth medium: N2
, Defined DMEM: F12-based GIBCO medium: 0.6
% Glucose, 25 μg / ml insulin, 100 μg / ml transferrin, 20 nM progesterone, 60 pM putrescine, 30 nM selenium chloride, 2 nM
Glutamine, 3 mM sodium bicarbonate, 5 mM HEPES and 20 ng /
ml human recombinant epidermal growth factor (EGF, R & D Systems). The cells grew as free-floating clusters or "spheres" and were passaged every 7 days by breaking into single cell suspensions.

(Preparation of cells for transplantation) After 5 weeks of culture, cells were prepared for transplantation. ³ H-thymidine, 2.5 μCi
/ Ml was added to the cultures 1 and 3 days after the last passage. At day 5 post passage, small spheres, typically 15-30 cells, were collected by centrifugation and 250
Resuspended to a final concentration of 1,000 cells / μl. Viability was checked using trypan blue exclusion and approximately 90% viable cells were found within the sphere.

Surgery Adult female Sprague-Dawley rats (weighing approximately 220 g) were used in this study. Animals were maintained in a controlled temperature and wet environment using a 12 hour light / dark cycle and were allowed food and water ad libitum during the experiment. Three experimental groups were included in this study: EGF injection with immunosuppression (n = 8), EGF injection without immunosuppression (n = 6) and vehicle injection with immunosuppression (n =).
6). Animals undergoing immunosuppression received cyclosporine 1 daily at the beginning of the day of transplantation.
Received an intraperitoneal injection of 0 mg / kg.

Stereotactic surgery was performed under deep anesthesia (3 ml / kg body weight, intraperitoneal). Each rat received six 0.3 μl sphere suspensions. This equated to approximately 500,000 cells and was done with the following coordinates: AP =
+0.4, L = -2.0, V = -4.5, -4.0, -3.5; AP = + 0.0
, L = -2.0, V = -4.5, -4.0, -3.5.

Immediately after implantation, a steel injection cannula was placed in place in the room (coordinates: A
P = + 0.2, L = -1.2, V = -3.5), and fixed with dental cement. The extracranial end of the cannula is attached to the minipump device (Alzet, 1007
D, infusion rate 0.5: 1 / hour) and placed on the back under the skin of the neck. Injections were performed over 7 days with either 400 ng / day EGF in 0.1% rat serum and 0.01% gentamicin in 0.9% saline, or a control vehicle without EGF. This gives a total of 3 during the study.
． Delivery of 2 μg EGF was performed.

BrdU Labeling of Dividing Cells In Situ Seven days after transplantation, each rat was replicated with 120 mg / kg BrdU in sterile saline every 3 hours starting 16 hours prior to perfusion. An intraperitoneal injection was made.

Histology One hour after the last injection, the rats were terminally anesthetized with an excess dose of chloral hydrate and transcardially with 0.1 M phosphate buffered saline (PBS). Reflux, followed by reflux with 250 ml of 4% paraformaldehyde in PBS for 5 minutes. Brains were removed and soaked in 4% paraformaldehyde overnight, then rinsed and transferred to a 25% sucrose solution in PBS.

The brain was cut with a freezing microtome to a thickness of 3 μm. Fluorescent immunohistochemistry was performed on serial sections for different marker combinations. Free-floating sections were pre-incubated for 1 hour in a block solution of calcium phosphate buffered saline (KPBS) containing 5% normal donkey serum (NDS) and 5% normal rabbit serum (NRS). This solution was then replaced with primary antibody made in blocking solution for 36 hours at 4 ° C. For M2, Triton was not included in the procedure. The antibodies used in this study were: M2 (Ca
1:50, a mouse-specific glial marker presented by Dr. rl Lagenhauer; BrdU (Calbiochem) 1: 100, or (Beck).
ton Dickinson 1:25; glial fiber acidic protein (GFAP)
, Dakopatts) 1: 500; VIM (Dakopatts) 1:25;
Nestin (gift from Dr. Ron McKay) 1:50; (β-tubulin-III (Sigma, St Louis, MO) 1: 400; Hu (SG)
1: 2000 from Dr. Oldman). After three rinses in KPBS, the sections were cut into appropriate secondary antibodies (donkey anti-mouse secondary antibody 1 conjugated with FITC 1).
: 200 (Jackson); Cy3 conjugated donkey anti-mouse secondary antibody 1:
200 (Jackson); donkey anti-rat secondary antibody conjugated to FITC 1:
200 (Jackson); Cy5 conjugated donkey anti-rat secondary antibody 1: 2
00 (Jackson); donkey anti-rabbit secondary antibody conjugated with FITC 1: 2
00 (Jackson); biotinylated rabbit anti-rat 1: 200 (DAKO))
With KPBS with 2% appropriate normal serum for 2 hours at room temperature,
Incubated in the dark. After 3 additional rinses and when using biotinylated secondary antibody, the final incubation was at room temperature in the dark for 2 hours in streptavidin conjugated with Cy3 in -KPBS. The section
Mounted on chrome alum-coated glass slides, air dried for 5 minutes, and mounted coverslips with PVA / DABCO mounts.

An additional series of sections were stained for BrdU, this time using diaminobenzidine (DAB) as the chromogen. These were mounted, defatted, and soaked in Kodak 50 emulsion for 6 weeks to assess thymidine labeling. The sections were then counterstained with cresyl violet, then dehydrated and coverslipped with DPX. Expression of the lacZ transgene was expressed using β-galactosidase (βgal, 1: 500, 5′3′Inc) using a similar immunohistochemistry protocol.
． ) Was used.

Analysis Fluorescent sections were observed with a Bio-Rad MRC1024UV confocal scanning microscope, allowing precise definition of each antibody. The double-labeled cells were
This was confirmed by collecting 2 μm serial sections.

Graft core volume was measured using M2 positivity. A series of 1: 8 sections was taken through each implant and the area of densely stained graft cores was outlined in each section and the area was calculated using an image analysis system. Then
Areas are standard ANOVA test (Statview software). Was converted to volume for comparison using.

Results All animals had grafts that survived well as observed with M2 immunoreactivity and ³ H thymidine labeling. There was a clear effect of EGF injection on graft cells both in increasing migration towards the source of EGF and in its proliferation. Therefore, transplanted mouse progenitor cells were able to respond to EGF in vivo in a manner similar to how they respond in culture conditions.

1. Host Response to EGF Infusion Continuous infusion of BrdU into host animals for 16 hours prior to perfusion revealed good labeling of the endogenous population of cells located in the SVZ adjacent to the lateral ventricles. became. On the opposite side of the cannula configuration, some BrdU positive nuclei were observed scattered in the monolayer adjacent to the chamber wall (Fig. 5A). These were found in both vehicle and EGF treated animals. Brd under the striatal ipsilateral ventricle, lying in the lateral wall, in animals pumped vehicle alone
There was a slight increase in the number of U-positive nuclei. The cells were observed in layers 4-5. Here, with some scattered nuclei up to 1 mm lateral to the chamber wall (Fig. 5B).
, D). However, in animals receiving EGF injection, a significant number of BrdU-positive nuclei were observed in the adjacent 12-15 cell layer of the chamber. Here, many more positive cells were scattered in the striatum lateral to the injection site (Fig. 1C, E, L). The host response to EGF infusion with increased numbers of BrdU positive cells converges to the lateral ventricular wall and is SV up to 1 mm rostral and caudal for cannula placement.
Observed at Z. In addition, SVZ nodes appeared, which protruded into the room space. These were filled with BrdU-positive nuclei (see, eg, FIG. 5E top).

The astrocyte marker 3FAP was used to identify host glial responses to EGF injection. GFAP in vehicle-injected animals that also received grafts
Reactive astrocytes were observed around the graft core, mixed with the M2-positive profile (Figure 8D) in BGR-injected animals, GFAP positivity was more concentrated, and individual cells were associated with the graft. Scattered in the area between the lateral chambers and observed around and inside the graft core itself (FIG. 5E). By high power microscopy, numerous GFAP-positive cells were also labeled with BrdU both in the graft core (FIG. 5F) and in the area of the striatum between the graft and the lateral chamber (FIG. 5G). Became clear. This means that these cells are the last one before injection.
Shows splitting at 6 hours.

Vimentin (VIM) positivity, present in the host striatum, was used to distinguish both SVZ immature cells and reactive immature astrocytes. In vehicle-injected animals, VIM staining of immature cells was restricted to SVZ and immature cells scattered around the graft core (Fig. 5H). VIM-positive SVZ appeared thicker in EGF-injected animals (FIG. 5I). This was an indicator of an increase in the number of cells and was accompanied by the extension of radial VIM-positive projections protruding from SVI toward the adjacent striatum (Fig. 5K). Just further away from the SVZ (200-400 μm),
Individual immature VIM-positive glia were observed (Fig. 5J).

The antibody nestin was used as a marker for immature progenitor cells. See: Lendahl et al. , Cell 60, pp. 585-595 (
1990). Nestin immunoreactivity showed a similar distribution to vimentin. In vehicle-injected animals, nestin-positive cells are restricted to SVZ, while E
This region was thicker in the GF-injected ones. This is an indicator of cell division (Fig. 5L). Furthermore, in animals that received EGF, multiple nestin-positive profiles were observed in the area between the lateral ventricles and the graft.
High power microscopes were revealed with double labeling. BrdU / Nestin positive cells are within this region (FIG. 5M).

2. Graft Response to EGF Injection All animals had well-surviving grafts revealed with the mouse-specific astrocyte marker M2 (FIG. 5). Most of the implants were in the striatum just proximal to the lateral ventricles. However, in two animals some of the implants were found to be misplaced in the chamber itself and attached to the chamber wall. All E
In GF-injected animals, the dense M2-positive cores of the graft appeared to be within a similar range of volume. Graft volume did not change between cyclosporine-treated and non-cyclosporine-treated animals injected with EGF. This is
It shows that non-immunosuppressed animals did not show any form of transplant rejection during their survival and that administration of cyclosporine did not alter the efficacy of EGF on the transplanted cells. Therefore, non-immunosuppressed animals were
Included in EGF injected group.

Cell migration towards the lateral ventricles: In vehicle-injected animals (FIGS. 5B, D, H and FIG. 6A), the implants were characteristically dense and outside the implant core. Is associated with very small M2 positive staining. M2 positive profiles were observed protruding from the implant in all directions to a limited extent in the surrounding parenchyma (Fig. 5B, D, H).

In EGF-injected animals, M2 outside the implant on the side facing the lateral ventricles.
There was a characteristic pattern of positive staining (Figure 5C, E, I, L and Figure 6B).
There was a significant increase in the number of profiles stained with M2, and these were found throughout the parenchyma up to the chamber wall itself. In some animals, SVZ
There was an increase in M2 positivity in and many M2 positive profiles were clustered in this area. Furthermore, many M2-positive profiles in the region between the graft and the SVZ were found to be directed towards the lateral ventricles (Fig. 51, L). On the distal side of the chamber, very little M2-positive staining was observed outside the graft core.

Expression of the M2 marker was observed up to 1 mm rostral and caudal to the implant (FIG. 6). In more caudal sections from EGF-injected animals, a profile was observed in the striatal medulla white matter duct (SM in Figure 6B), which runs parallel to the axon profile.

A series of sections initially stained for BrdU were soaked in emulsion for 6 weeks to investigate ³ H thymidine expression in transplanted cells. In all animals autoradiographic particles were observed throughout the graft core (Figure 7). Immediately around the implant, dispersed cells could be identified containing many silver particles around the nucleus. In vehicle-injected animals, these were only observed in the area immediately surrounding the graft and not in the zone between the transplanted cells and the lateral ventricles (Fig. 7B). However, in EGF-injected animals, dispersed ³ H thymidine-positive cells were seen throughout the parenchyma on the side of the graft adjacent to the chamber, up to the SVZ (FIG. 7A, arrow).

B. Proliferation of Transplanted Cells To assess whether the transplant population divides in response to EGF, Brd.
The U / 3 ^H-thymidine double-labeled sections were assessed for co-localization of these two markers. Most of the BrdU-labeled cells in the zone between the transplant deposit and the lateral ventricles did not contain significant numbers of silver particles above background levels. However, dispersed BrdU ^{/ 3} H-thymidine double labeling cells are sometimes
Observed (arrow head in Figure 7A). Furthermore, fluorescence immunohistochemistry shows that there was an increase in the number of BrdU positive cells found within the M2 positive regions in EGF-injected animals. BrdU / M2 double labeled cells could be found in the graft core (Fig. 5F) and in the region between the graft and lateral ventricles (Fig. 5G, M). Not all BrdU labeled cells were double stained with M2, but a certain proportion of these were positive for GFAP, as described above, and the lumber was labeled with neither M2 nor GFAP. Was not done. In vehicle-injected animals, BrdU-positive cells often show GFAP.
Or they were dispersed with M2 positive profile and only a few cells were double labeled for either marker.

Further evidence for the growth of transplanted cells within the EGF-injected group was obtained from analysis of graft volume. Each graft volume was calculated by measuring the dense M2 positive graft cores through a series of sections, eliminating the scattered M2 positive profile in the EGF-injected group. By this analysis,
Graft core volumes were shown to be similar in animals receiving EGF or vehicle injections compared to controls (vehicle injection 0.81 ± 0.2;
EGF injection = 1.15 ± 0.57). This indicates that M distributed outside the core (ie, in the region adjacent to the lateral ventricles in the EGF-injected group).
The increase in the 2-positive profile indicates that it was not solely due to the migration of cells from the graft core, but was also partially due to the proliferation of transplanted cells.

C. Graft Morphology No apparent differences in the morphology of the transplanted cells were observed when compared to vehicle or EGF injected animals. Immunohistochemistry in all animals showed a number of M2 positive profiles, indicating a large number of cells with glial morphology (FIG. 5). There was an overlap of GFAP and M2 positive staining.
With mixed profiles in these regions (FIGS. 5D-G). Overlapping GFAP and M2 profiles were observed, but a closer analysis of the Z series through tissue sections did not show co-localized expression of two of these markers. This was also the case with M2 and VIM. M2 positive profiles were often observed mixed with VIM positive glial, but not double labeled cells. Therefore, these populations of M2-positive but GFAP- or VEM-negative cells are presumed to be immature or non-reactive glial cells that express neither GFAP nor VIM. The graft was also stained with the mouse-specific marker M6, which stains both neurons and a subset of astrocytes. For example, see: Campbell et al. , Neuron 15, pp. 1259-1273 (1995). There was a complete overlap of M6 expression with M2 positive regions. An M6 positive profile with neuronal morphology was observed.

The cells used in this study were derived from transgenic mice with the β-galactosidase enzyme (lacZ) under the control of the myelin basic protein (MBP) promoter. The section was cut with β-galactosidase (βgal).
And the expression of the gene was examined. In all animals, there was very low βgal expression within the graft core regardless of the injection medium. Where positive staining was seen at this site, its expression was punctuate.
), Which gave the cells a spherical immature appearance (FIG. 8K). Good expression was seen throughout the cells and primary processes when positive βgal staining was observed away from the graft core. The cells found in the gray material had a relatively immature morphology, with either monopolar or bipolar extensions (FIG. 8C). In one case where cells were seen in the needle-like tube to the level of the corpus callosum, these cells had longer processes extending in the same direction as the host's axonal profile (Fig. 8B) and were immature. It had a rare oligodendrocyte morphology.

Grafts were also analyzed for the expression of the early neuronal markers Hu (4) and β-III-tubulin, in each case in combination with the M2 antibody.
Cells positive for any of these antibodies were not found in the graft area itself or in the area between the graft core and lateral ventricles (data not shown).

The above results suggest that EGF-responsive mouse neural progenitor cells may respond to EGF after in vivo transplantation. Cells transplanted into the adult rat striatum
It migrates towards the F source and this response is maintained for 7 days after EGF infusion.

As previously observed (Craig et al., Supra; Kuhn et al., Supra), injection of EGF into the lateral ventricles resulted in SVZ progenitor cell division and surrounding striatum. Stimulate the migration of that cell to. Current studies show that at 7 days after the start of EGF infusion, some of these newly generated cells differentiate into glial cells and express the astrocyte marker GFAP. Newly generated BrdU positive cells within the SVZ are found at a maximum distance of 1 mm rostral towards the infusion cannula and not further apart in rostral migratory flow in the pathway to the olfactory bulb. It was In addition, some cells remained at the site of proliferation and formed small nodules of SVZ protruding into the lateral ventricles. This means that EGF injection is SVZ
Interfering with active migration of progenitor cells towards their olfactory bulb in its normal pathway (Ku
hn et al. Threadgill, et al. , Science
ce 269, pp. 230-234 (1995)) and earlier reports of promoting differentiation to glial but not neural phenotype.

Transplanted mouse progenitor cells showed an in vivo active response to EGF.
Here, it was accompanied by proliferation and directed migration of cells away from the graft core towards the EGF source. Two conclusions that can be drawn from these results are that EGF protein can penetrate and disperse in striatal parenchyma and exert an effect on transplanted cells, and that murine cells are associated with in vivo transplantation. Retaining responsiveness to EGF even later. In vivo neurotrophic factors (see, eg, Ahmed et al., Supra; Kirschenbaum et a.
l. , Cerebral Cortex 6, pp. 576-589 (1994
The addition of)) may provide a means to manipulate progenitor cells after transplantation for at least a short time to direct them to specific differentiation or to increase their directed migration or their survival. This technique may play an important role in overcoming the challenges associated with the restricted migration and differentiation of transplanted cells, and thus enhances the ability of transplanted neurons to neurally stimulate host tissue in the neural transplant paradigm. You can There appears to be a threshold level of EGF that is required to influence the migration and differentiation of TransphiMed progenitor cells. Studies in combination with encapsulated EGF-secreting cells placed in chambers adjacent to EGF-positive progenitor cell implants had no effect on the migration capacity of these cells (unpublished observation). The amount of EGF differentiated by this cell line was 1 / 100th of the cannula injection and had no effect on endogenous progenitor cells. This suggests that this level is insufficient to elicit a response.

No morphological differences were observed between transplanted cells exposed to EGF and vehicle-injected cells in vivo. Extensive glial differentiation was seen in all grafts evidenced by the M2-positive profile, but neural differentiation was observed with both the early neural markers Hu and β-III-S tubulin. There wasn't. Thus, both on the progenitor cells themselves and on the more differentiated glial precursors, EGF appears to exert its effect on different types of cells in the mixed population found in these progenitor cell cultures.

Evidence for EGF to act on more mature glial-restricted progenitor cells, although EGF is de-regulated both in vitro and in vivo,
Derived from previous studies shown to support progenitor cells that are not committed to neurons 18 (Kilpatrick et al., J. Neuroscience).
ce 15, pp. 3653-3661 (1995); Kuhn et al.
, Above). Indeed, both GFAP and M2, the antibodies used to identify glia in this study, are known to label more mature glial cells, where progenitor spheres are labeled with both markers. Negative for (see, eg, Winkler et al., Neuroscience 1).
1, pp. 99-116 (1998)). Once these progenitor cells are induced to differentiate in vitro, cells that adopt the glial phenotype express GFAP and / or M2. In culture, M2 expression co-localizes with GFAP, but not all M2-positive cells are also GFAP-positive. This population of M2-positive / GFAP-negative cells can carry the transplanted cells. This is Br
Double labeled with dU and M2, but does not express GFAP. Indeed, our previous studies have shown that the expression of GFAP and m2 is not co-localized in mouse progenitor cells after in vivo transplantation (Winkler et al.,.
(See above).

Previous studies have shown that mouse EGF-responsive progenitor cells are pluripotent in vitro; they preferentially show glial expression after transplantation into either developing or adult rat brain. Differentiate into molds. For example, see: Hamman
g et al. , Experimental Neurology 147,
pp. 84-95 (1997); Winkler et al. , Molecu
lar and Cellular Neuroscience 11, pp.
99-116 (1998). In this study, double staining with BrdU and M2 showed newly generated mouse glial cells in EGF-injected animals when compared to the vehicle-injected group, which indicated that EGF Stimulated the differentiation of transplanted progenitor cells directed to the glial phenotype. Thus, it appears that EGF injection stimulated cell differentiation and migration, but did not stimulate differentiation of transplanted cells in aesthetics in a manner similar to its action in vitro.

Many BrdU-positive cells within the transplanted region expressed neither M2 nor GFAP. These cells can belong to one of two populations of host or transplanted progenitor cells. Both of these cells have a less differentiated, immature phenotype. It is possible that EGF may also play a role in maintaining the transplanted donor cells in a progenitor-like state, similar to their role in culture. See, for example: Reynolds et al. , D
developmental Biology 175, pp. 1-13 (199
6).

A third population of cells found in the region adjacent to the graft and lateral ventricles is Br.
It could be doubly labeled with dU and GFAP but not with M2. This population appears to represent newly dividing glial cells derived from the host SVZ.
This is as we previously observed that all GFAP-positive mouse progenitor cells co-express M2 after their differentiation in vitro. See, for example: Winkler et al. (1998), supra.
Further evidence for this is Br found in the region between the graft and the lateral ventricles.
It may be derived from dU / Nestin double labeled cells. These cells could be derived from either mouse or host progenitor cells that had divided in response to EGF injection.

The cells used in this transplantation study were obtained from transgenic mice. Therefore, they are transgene lac under the control of the MBP promoter.
Had Z. Expression of β-galactosidase as a sign of oligodendrocyte formation in vivo indicates a small number of transplanted cells with premature oligodendrocyte morphology, predominantly within the white matter ducts (eg, corpus callosum). The small number of lacZ-positive cells found in the grafts suggest that the majority of the cells differentiate into astrocytes rather than oligodendrocytes, as previously seen. For example, see: Wi
nkler et al. (1998), supra; Winkler et al.
, Society for Neuroscience Abstracts (
1995).

However, the presence of lacZ-positive cells in rat brain indicates that the transgene can still be expressed under appropriate conditions after xenotransplantation, and these cells serve as tools to allow the introduction of the relevant gene into the brain. Is shown to support the efficacy of using. It remains to be investigated whether more differentiated oligodendrocytes are observed after longer survival times.

These results indicate that nerve growth factor injection can stimulate mouse progenitor cells in vivo after transplantation into adult rat brain. This local delivery of neurotrophic factors to newly transplanted cells provides a novel means of regulation in vivo, leading undifferentiated progenitor cells to proliferate, migrate or differentiate into specific phenotypes, And further, a controlled means of increasing graft survival, enhancing neuralization of host tissues and restoration of associated behavior to enhance transplant efficacy as a possible retrograde treatment for neurodegenerative diseases. provide.

[Brief description of drawings]

FIG. 1 shows proliferating 9FBr human neural stem cells derived from human forebrain tissue (6th generation).
Shows the display of the sphere.

FIG. 2 Panel A of (a) EGF, FGF and leukocyte inhibitory factor (“LIF”).
Shows growth curves for a human neural stem cell line designated as 6.5Fbr cultured in defined medium (shown as black diamonds) containing (b) as well as (b) the same medium without LIF (shown as white diamonds); Panel B shows (a) EGF, FGF and LI
Panels show growth curves for a defined medium containing F (shown as black diamonds), and (b) a human neural stem cell line designated 9Fbr cultured in the same medium without LIF (shown as white diamonds); panel. C is (a) EGF, FGF and LIF
Shows growth curves for a human neural stem cell line designated 9.5Fbr cultured in defined medium containing B. (shown as black diamonds), and (b) in the same medium without LIF (shown as white diamonds); Panel D was cultured in (a) defined medium containing EGF, FGF and leukocyte inhibitory factor (“LIF”) (shown as black diamonds) and (b) the same medium without LIF (shown as white diamonds). 10.
3 shows a growth curve for a human neural stem cell line designated 5Fbr.

FIG. 3 shows (a) defined medium containing EGF and basic fibroblast growth factor (“bFGF”) (shown as open diamonds), and (b) EG without bFGF.
3 shows growth curves for a human neural stem cell line designated 9Fbr cultured in defined medium with F (shown as black diamonds).

FIG. 4 shows a graph of cell number vs. days in medium for Mx-1 conditionally immortalized human glioblastoma cell line derived human neural stem cell line. Open squares indicate growth in the presence of interferon; filled diamonds indicate growth in the absence of interferon.

FIG. 5 shows an image of rat brain after precursor cell transplantation. All transplanted cells are identified by the antigen M2 (red). Panels AC show low power images, the median striatum is labeled with M2 (red) and BrdU (green), A) to E
Contralateral side of GF-injected animals, B) Transplant center of vehicle-injected animals, and C) Transplant center of EGF-injected animals (LV = lateral ventricle). Panel D ~
G is M) (red) and GF of D) vehicle injected animals, EG) EGF injected animals.
Co-labeling with AP (green) and BrdU (blue) is shown, F) high power in the transplant center, G) high power in the region between the transplant and the lateral ventricles. . Arrowhead arrows indicate doubly labeled BrdU / M2 cells, and arrows doubly labeled BrdU / GFAP cells. Panels H-K show dual labeling of H) vehicle injection and I-K) EGF injection with M2 (red) and vimentin (VIM; green), J) transplant center and lateral ventricle. High output in the region between and K) SV
With increased expression of VIM in Z. Panel L shows triple labeling of M2 (red), nestin (green), and BrdU (blue) of EGF-injected animals, with high-power images of the region between the M) transplant center and lateral ventricles. . The arrowhead is BrdUI.
Double labeled cells of M2 are shown, and arrows indicate co-localization of BrdU and nestin. Scale bar in M, A to C = 300 μm; D, E, H, I = 4
00 μm; F, G, J, M = 15 μm; K, L = 200 μm.

FIG. 6 shows a series of 1 in A) vehicle-injected animals and B) EGF-injected animals.
: Camera lucida drawing of 8 coronal sections showing the distribution of M2 positive profiles across the implant and adjacent parenchyma.
CC: corpus callosum; Str: striatum; LV: lateral ventricle; SM: stria medu
llaris). Arrows indicate the level of cannula placement and associated damage to the cortex.

FIG. 7 shows an image of the distribution of ³ H-thymidine labeled cells (silver granules) and BrdU labeled cells in the region between the transplant center and lateral ventricles (A) EGF injected animals. And B) Vehicle-injected animals). Scattered ³ H-thymidine positive cells are indicated by arrows and occasional BrdU / ³ H-thymidine double labeled cells are indicated by arrowhead arrows (A insertion). It should be noted that B lacks ³ H-thymidine labeled cells. The scale bar is B = 80 μm.

FIG. 8 shows an image of β-galactosidase (βgal) labeling of a representative implant. A) Only immature βgal-positive cells were observed in the transplant center. B and C) Occasional cells were found to be interspersed within the striatum (B) or corpus callosum (C) and had immature oligodendrocyte identity. T: transplant; Ctx:
Cortex; CC: corpus callosum; Str: striatum. Scale bar: A = 50 μm; and C (
For B and C) = 20 μm.

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Claims (12)

[Claims] 1. For transplantation into the brain of at least about 500,000 mitogenic growth factor responsive precursor cells capable of differentiating into neurons, oligodendrocytes, or astrocytes. The system of claim 1, wherein the cells are: (a) transplanted at a first location in the brain of a living host subject; (b) after transplantation at a second location in the brain of the host subject. In vivo from the first location to another anatomical site for integration within the nervous system of the host subject after injection of a mitogenic growth factor that does not induce differentiation of the precursor cells at the location. (C) after transplantation, integrated in situ into parenchyma at a topographical site in the host subject after transplantation; and (d) after integration, neurons, oligodendrocytes, and astrocytes. In situ on cells selected from the group consisting of Differentiated; wherein said transplanted precursor cells retain their in vivo responsiveness to organic fiber mitogenic growth factors, system. 2. The system of claim 1, wherein the precursor cells comprise mammalian embryonic precursor cells. 3. The system of claim 1, wherein the first location is in the striatum of the brain and the second location is in the lateral ventricle of the brain. 4. The in vivo movement occurs towards the second direction,
The system of claim 1. 5. At least about 500,000 mitogenic growth factor responsive progenitor cells capable of differentiating into neurons, oligodendrocytes, or astrocytes transplanted into the brain, A system for inducing in vivo proliferation, wherein the cells are: (a) transplanted into a location of one of the brains of a living host subject; (b) the brain optionally after transplantation. Moving in vivo from the location to another anatomical site after injection of a mitogenic growth factor that does not induce differentiation of the precursor cells at or near the location of; ) A system that proliferates in situ, wherein said transplanted progenitor cells retain their in vivo responsiveness to said mitogenic growth factor. 6. The system of claim 5, wherein the precursor cells comprise mammalian embryonic precursor cells. 7. The system of claim 5, wherein the location is in the striatum of the brain. 8. The injection of the mitogenic growth factor is a lateral ventricle of the brain,
The system according to claim 5. 9. The system of claim 5, wherein the mitogenic growth factor is EGF. 10. The system of claim 5, wherein the progenitor cells are cultured in a medium containing the mitogenic growth factor prior to transplantation. 11. To induce the in vivo migration of at least 500,000 progenitor cells that are capable of differentiating into neurons, oligodendrocytes, or astrocytes transplanted to a first location in the brain. Use of a mitogenic growth factor in the preparation of a medicament according to claim 1, wherein the mitogenic growth factor is in a location in the brain different from the transplanted progenitor cells and different from the first location. Use to induce in vivo migration of said progenitor cells towards said injection site when injected. 12. To induce in vivo proliferation of at least 500,000 precursor cells capable of differentiating into neurons, oligodendrocytes, or astrocytes transplanted to a first location in the brain. Use of a mitogenic growth factor in the preparation of a medicament according to claim 1, wherein said mitogenic growth factor induces in vivo growth of said cell population when injected into different locations of said brain.