EP1109887A1 - Production et utilisation de cellules dopaminergiques pour traiter des deficiences dopaminergiques - Google Patents

Production et utilisation de cellules dopaminergiques pour traiter des deficiences dopaminergiques

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EP1109887A1
EP1109887A1 EP98959412A EP98959412A EP1109887A1 EP 1109887 A1 EP1109887 A1 EP 1109887A1 EP 98959412 A EP98959412 A EP 98959412A EP 98959412 A EP98959412 A EP 98959412A EP 1109887 A1 EP1109887 A1 EP 1109887A1
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Prior art keywords
cells
neurons
hnt
cell
dopaminergic
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EP1109887A4 (fr
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Michael P. Mcgrogan
Gary L. Snable
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Layton Bioscience Inc
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Layton Bioscience Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/48—Regulators of apoptosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/24—Genital tract cells, non-germinal cells from gonads
    • C12N2502/246—Cells of the male genital tract, non-germinal testis cells

Definitions

  • the present invention is in the field of tissue engineering/transplantation and more particularly dopaminergic cells, their production and use in transplantation.
  • Dopaminergic neurons are those which synthesize and use dopamine (DA) as a neurotransmitter.
  • Dopaminergic neurons are found in a number of areas of the brain, including the nigrostriatal, mesolimbic, mesocortical and tubero-hypophysial systems.
  • the rate-limiting step in dopamine synthesis is catalysis of tyrosine by tyrosine hydroxylase (TH).
  • TH tyrosine hydroxylase
  • Dopamine is stored in synaptic vesicles.
  • Dopamine is released from presynaptic vesicles by exocytosis.
  • Dopamine acts on as many as five classes of receptors.
  • Dopamine is recycled by reuptake and/or degradation by monoamine oxidase B (MAO-B) (RK Murray, Ch. 64. The Biochemical Basis of Some Neuropsychiatric Disorders. In: Harper's Biochemistry, ed. by Murray, et al. 24th ed., Appleton & Lange, Stamford, CT, 1996, pp. 794-814).
  • MAO-B monoamine oxidase B
  • Parkinson's disease is a neurodegenerative disorder characterized by a loss of dopaminergic cells from the substantia nigra par compacta, resulting in decreased dopaminergic input to the striatum.
  • the hallmark motor symptoms include tremor, rigidity, bradykinesia, and instability.
  • Levodopa the gold standard pharmacological treatment to restore DA, is plagued by decreased efficacy and increased side effects over time.
  • Adjunct treatment with DA agonists is frequently necessary; however, recently approved DA agonists with greater receptor subtype specificity may provide only incremental clinical benefit.
  • COMP monoamine oxidase
  • Schizophrenia is often treated by neuroleptic drugs which decrease the amount of dopamine activity in mesolimbic dopaminergic neurons.
  • “Positive symptoms” e.g., hallucinations, delusions, playful behavior
  • “Negative symptoms” of schizophrenia e.g., social withdrawal, emotional blunting, and catatonia
  • prefrontal dopaminergic neurons may normally inhibit the activity of subcortical dopamine neurons, a lowering of dopamine in the prefrontal area could lead to the elevated dopaminergic activity in the subcortical neurons.
  • Progressive Supranuclear Palsy (Steele-Richardson-Olszewski Syndrome) is due to a loss of neurons and gliosis in the tectum and tegmentum of the midbrain, the subthalamic nuclei of Luys, the vestibular nuclei, and to some extent the ocular nuclei. Some symptoms are shared with Parkinson's disease, including rigidity of the neck and other trunk muscles and occasional sensitivity to L-dopa.
  • torsion dystonia is dramatically L-dopa-responsive. Starting in childhood, the dystonia first affects gait. Most individuals later develop parkinsonism. Some focal dystonias also are reported to be L-dopa responsive.
  • positron emission tomography has shown decreased uptake of dopamine derivatives in the putamen and caudate, probably reflecting a loss of nigrostriatal dopaminergic neurons.
  • Current treatment is symptomatic. The parkinsonian symptoms may be helped by L-dopa or other dopaminergic drugs, but later most patients become refractory to these drugs.
  • Depression is associated with heterogeneous dysregulations of the biogenic amines. Although norepinephrine and serotonin have been most implicated in the pathophysiology, dopamine also may play a role in depression. Dopamine may be reduced in depression and increased in mania. Drugs that reduce dopamine concentrations (e.g., reserpine) and diseases that reduce dopamine concentrations (e.g., Parkinson's disease) are associated with depressive symptoms. Also, drugs that increase dopamine concentrations (e.g., tyrosine, amphetamine and bupropion) reduce the symptoms of depression.
  • dopamine may be reduced in depression and increased in mania.
  • Drugs that reduce dopamine concentrations e.g., reserpine
  • diseases that reduce dopamine concentrations e.g., Parkinson's disease
  • drugs that increase dopamine concentrations e.g., tyrosine, amphetamine and bupropion reduce the symptoms of depression.
  • the search for a continuous, stable, regulated, site-specific source of DA delivery has turned to tissue transplantation, cell therapy and genetic engineering, with the ultimate goal of finding an effective treatment to halt or reverse disease progression.
  • Human fetal mesencephalic tissue transplants are the most extensively studied procedure. They have demonstrated therapeutic potential in animal models of PD and in Parkinson's disease patients. Fetal tissue transplants have been performed in the clinic for over a decade on more than 200 patients throughout the world with positive outcomes (Kordower JH, Goetz CG, Freeman TB, Olanow CW. Experimental Neurology 144:41-46, 1997). Grafts survive, form synaptic connections, and improve motor function in many patients. However, ethical, moral and technical constraints limit the widespread use of human fetal tissue. Future progress in the field of neural transplantation will depend largely on the development of alternative sources of cells.
  • Xenotransplantation the use of cells from different species, is a viable approach to circumventing the limitations associated with human fetal neural transplantation (Galpern WR, Burns LH, Deacon TW, Dinsmore J, Isacson O. Experimental Neurology 140:1-13, 1996).
  • a phase I clinical trial sponsored by Diacrin, Inc. is evaluating transplants of porcine cells harvested from the midbrains of pig fetuses.
  • Another technique, developed by Cytotherapeutics, Inc. uses encapsulated xenografts of rat PC12 cells that secrete dopamine.
  • a semipermeable polymer membrane allows diffusion of the small therapeutic molecules but prevents diffusion of the larger immunogenic molecules. Whether the release of dopamine from encapsulated sources will be sufficient to restore optimal DA levels in PD patients remains to be determined.
  • viral vectors herpes simplex virus, adeno virus, adeno-associated virus, or lentivirus.
  • viral vectors herpes simplex virus, adeno virus, adeno-associated virus, or lentivirus.
  • Genetically modified vectors encoding genes such as TH or glial-derived neurotrophic animal models of PD.
  • the extent of gene expression, long-term efficacy, and cytopathogenicity associated with viral vectors is unknown.
  • GDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • Lithium the primary treatment for mania and bipolar affective disorder, has been reported to significantly influence the activity of signaling systems.
  • PC 12 cells as a model system, Li and Jope (J Neurochem 65:2500-08, 1995) studied the NGF-induced expression of several signal transduction proteins, including subtypes of G proteins, protein kinase C and phospholipase C and its modulation by lithium. Their results demonstrated that lithium, at a therapeutic concentration (ImM), modulates the level of signal transduction proteins.
  • the ideal cell for a CNS transplant system should meet the following criteria: It should be of human CNS origin, capable of growth cessation and differentiation, clonal and defined, transfectable and selectable, immunologically inert, capable of long-term survival following implantation, non- tumorigenic, functional and integrated into the host brain, of consistent quality, and readily available.
  • Dopaminergic neuronal cells suitable for transplantation in dopaminergic deficiency are derived from progenitor cells as follows.
  • the progenitor cells are treated with retinoic acid for a time period sufficient to optimize expression of tyrosine hydroxylase.
  • the optimized neuronal cells being further treated with at least one lithium salt or a combination thereof.
  • the transplantation-ready cells are selected with trypsin and so harvested.
  • the resulting neuronal cells are highly purified and have a phenotype optimized for at least one neurodegenerative disease, such as Parkinson's Disease.
  • the progenitor cells also are cultured with Sertoli cells, astrocytes, or glial cells.
  • Figure 1 is a tyrosine hydroxylase Western blot of NT cells at different stages of maturation in the hNT-Neuron process. It was developed with anti-TH monoclonal antibody and biotin-strepavadin alkaline phosphatase system. The following lanes contain: 1) 500 pg TH rat standard, 2) 1x10° hNT-Neurons positive control, 3) 1x10° Replate-I Neurons, 4) 1x10° Replate-I Accessory cells, 5) lxlO 6 NT2 D1 Precursor cells. Lanes 6-8 contain lxlO 6 Replate-II neurons each harvested after 1 week (lane 6), 2 weeks (lane 7), and 3 weeks (lane 8) in extended culture.
  • Figure 2 is a tyrosine hydroxylase Western blot comparing different maturation conditions for the hNT-Neurons. It was developed with anti-TH monoclonal antibody and biotin-strepavadin alkaline phosphatase system. NT2/D1 cells were induced with RetA for 6 weeks and processed as Replate-I or Replate-II cultures in mitotic inhibitors for 1 week. Then the cultures were allowed to mature in conditioned media for 1 day (1 week replate), 1 week (2 week replate), or 2 weeks (3 weeks replate). Pure hNT-Neurons were harvested from the mature replate cultures and cell extracts corresponding to lxl 0 6 cells were loaded in the following lanes.
  • Lanes 1-3 show the results for the Extended Replate-I Neurons which were matured for 1 week (lane 1), 2 weeks (lane 2), and 3 weeks (lane 3). Lanes 5 and 6 have Replate-II Neurons which were matured for 1 week and 2 weeks, respectively. Lanes 4 and 7 contain lxlO 6 hNT- Neurons positive control.
  • Figure 3 is a TH Western Blot showing the time course of RetA Induction. It was developed with anti-TH monoclonal antibody and biotin-strepavadin alkaline phosphatase system. The NT2/D1 cells were induced with RetA for 4, 5, or 6 weeks, and after induction Replate-I cultures were maintained in mitotic inhibitors for either 1 week (Lanes 2-4) or a total of 2 weeks matured (Lanes 5-7). Purified hNT-Neurons were harvested from each sample and cell extracts corresponding to lxlO 6 cells were loaded in the following lanes. The 1 week Replate-I: 2) 4w-RetA, 3) 5w- RetA, 4) 6w-RetA.
  • Replate I The 2 weeks matured Replate I: 5) 4w-RetA, 6) 5w-RetA, 7) 6w-RetA. 8) hNT positive control, and rat TH standard was in lanes 1 (500pg) and 9 (5ng).
  • Figure 4 is a bar graph showing the effects of different doses of lithium chloride on TH expression in cultured hNT neurons.
  • Figures 5a -5d are photomicrographs of a representative culture of hNT cells treated with lithium chloride for four days and immunostained with antibodies to tyrosine hydroxylase.
  • Figures 5a and 5b show hNT cells cultured with 1.0 mM concentration of lithium chloride.
  • Figures 5c and 5d represent cultures of hNT cells treated with 3.0 mM concentration of lithium chloride.
  • Figures 6a through 6c are photomicrographs illustrating the effects of 4 weeks of RetA and 5 days of LiCl on hNT cells on frequency of TH-expressing cells (6a), TH and PI staining (6b) and bcl-2 expression.
  • Figure 7 is a bar graph showing the effects of different doses of lithium chloride on Bcl-2 expression in cultured hNT neurons.
  • Figures 8a and 8b are photomicrographs comparing cytochrome oxidase activity in 4- week RetA-treated control hNT cells (8a) and LiCl-treated hNT cells (8b).
  • Figures 9a, 9b, 9c, 9d, and 9e are photomicrographs of the distribution, morphological appearance and phenotype of hNT neurons 5days in control culture and lithium-treated cultures.
  • (9c) and (9d) are low-magnification phase contrast photomicrographs of hNT neurons treated with l.OmM (9c) and 3.0mM (9d) concentration of lithium chloride.
  • Figures 10a, 10b, 10c, lOd, and lOe are photomicrographs that show the effect of lithium chloride on TH expression in hNT neurons cultured for 5 days.
  • Figures 1 la, 1 lb, l ie, 1 Id, and 1 le are photomicrographs of representative control and lithium-treated hNT neurons cultured for 5 days and immunostained for tyrosine hydroxylase.
  • Ha control culture of hNT neurons reveals few TH-positive cells.
  • (1 lb) and (l ie) show hNT cells cultured with l.OmM (1 lb) and 3.0 mM (l ie) lithium chloride
  • (l id) and (l ie) show the representative morphology of TH-positive hNT cells treated with 1.0 mM (l id) and 3.0 mM (l ie) of lithium chloride.
  • Figure 12 is a table which shows the effect of lithium chloride on soma size (12a) and neurite growth (12b) of hNT neurons cultured for 5 days. * denotes significant difference (p ⁇ 0.01) compared to control.
  • Figure 13 represents a process for LiCl Induction of DA neurons.
  • the levels of TH expressed are shown in Figure 13 (DA-Neuron Co-Culture).
  • Lane 1 is 500 pg TH, lane 2 if fresh DA-Neurons bulk harvested.
  • Lane 3 shows the results after 1-week co-culture with polylysine, TM-4 cells are in lane 4 and rat glial cells are in lane 5.
  • Figure 14 represents the production of LiCl Induced DA-Neurons.
  • the enhanced expression of TH in the 4-week DA-Neurons was found to be optimal in small-scale cultures after replating the neurons in serum free media containing 1 mM LiCl for 5 to 7 days.
  • Figure 14 shows the results for 500 pg TH (Fig. 14 lane 1); control DA neurons in DF-5/Inh, 7 days (Fig. 14 lane 2); DA neurons in DF-5/Inh/ ImM LiCl, 7 days (Fig. 14 lane 3); DA neurons in DF- 5/Inh/ ImM LiCl, 3 days (Fig.
  • hNT-Neurons The six criteria for transplantable cells summarized above are met by hNT-Neurons.
  • hNT-neurons surprisingly were able to be optimized for stable TH production similar to that seen with primary mesencephalic cells.
  • TH is vital because it performs the rate-limiting step in production of dopamine.
  • These optimized PD-hNT neurons have improved dopaminergic properties arising from manipulating the hNT-neuron natural capabilities. These procedures eliminated the need to transfect the cells with exogenous gene constructs.
  • hNT-Neurons have the potential to overcome many of the limitations associated with human fetal tissue transplantation, including poor graft survival (5-10%), high tissue variability, and low degree of host re-innervation.
  • hNT-Neurons demonstrate excellent graft survival and behavioral improvements in animal models of CNS disorders. There are preliminary data suggesting that hNT-Neurons may have immunosuppressive properties. Thus, long-term, systemic immunosuppression may not be necessary in humans.
  • hNT-Neurons are human cells derived from the human teratocarcinoma NT2/D1 cell line through induction with RetA treatment (Andrews, PW, Damjanov J, Simon D, Banting G, Carlin C, Dracopoli NC, Fogh J. Lab Invest 50:147-162, 1984).
  • NT2/D1 cells which share many characteristics of neuroepithelial precursor cells, undergo significant changes resulting in the loss of neuroepithelial markers and the appearance of neuronal markers (Pleasure SJ, Page C, Lee VM. J Neurosci 12:1802-1815, 1992; Lee VM, McGrogan M, Lernhardt W, Huvar A.
  • a dopaminergic deficiency is a condition in which there is a shortage of dopamine.
  • the dopaminergic deficiency may have a variety of causes, including, but not limited to, underproduction by dopaminergic neurons, deficit of dopaminergic neurons, or insensitivity of dopaminergic neurons to dopamine. Examples of such conditions include, but are not limited to, Parkinson's disease, schizophrenia, progressive supranuclear palsy (Steele-Richardson- Olszewski Syndrome), and a Dopa-responsive form of torsion dystonia.
  • a beneficial effect is an observable improvement over the baseline clinically observable signs and symptoms.
  • a beneficial effect can include improvements in graft survival, improvements in one or more of the signs and symptoms associated with a dopaminergic deficiency, such as movement or mood.
  • N2/D1 precursor cells refers to a special cell line available from
  • NT2 D1 cells are unique among other teratocarcinoma cell lines because these cells act like progenitor cells whose progeny are restricted to the neuronal lineage (Andrews, ibid.)
  • LBS-NeuronsTM human neuronal cells refers to the special neuronal cell line disclosed in U.S. Patent No. 5,175,103 to Lee et al. Briefly, NT2/D1 precursor cells are induced to differentiate into neurons by administration of 10 (M RetA which is replenished three times weekly for 6 weeks, after which the cells are replated with special manipulations to become more than 99% pure hNT-Neurons. These are the cells which are used in the subsequent experiments. Alternately, for human use, there is a cell line manufactured without antibiotics (used in the research grade LBS-Neurons and under good manufacturing practices (GMP) which is termed LBS NEURONS human neuronal cells (Layton Bioscience, Inc.).
  • “Inducing agent” includes, but is not limited to compound which have the same effect in causing NT2 D1 precursor cells to differentiate into hNT neurons, one example of which is retinoic acid.
  • an inducing agent includes not only retinoic acid in any of it isomers and trans/cis forms, but also similarly active compounds.
  • Immunosuppressant as used herein is a substance which prevents or attenuates immunologic phenomena.
  • immunologic phenomena include inflammation, autoimmunity, GVHD and graft rejection.
  • current immunosuppressants include but are not limited to cyclosporine A, cyclophosphamide, prednisone and tacrolimus (FK506).
  • an immunosuppressant can be administered at the time of the transplant.
  • One regimen calls for administering the immunosuppressant for two days before and on the day of transplantation.
  • Vehicle is a biologically compatible solution, such as 0.9% normal saline and the like, which is used to suspend and inject the dopaminergic cells into mammals. This definition also includes any gel which firms at body temperature and is biodegradable.
  • sample is meant to refer to one or more treated cells.
  • a sample contains a plurality of cells.
  • a sample of treated cells is implanted into either a non-human mammal or a human.
  • lithium is meant generally a lithium salt, wherein the anion includes but is not limited to chloride, bromide, carbonate, citrate, or other biologically compatible monovalent anion.
  • lithium chloride LiCl
  • Therapeutic agent as used herein means the transplanted cells themselves or chemical entities secreted by these cells. Examples of chemical entities secreted by the cells include, but are not limited to dopamine, other neurotransmitters, proteins and hormones.
  • hNT-Neurons The production of hNT-Neurons is an 8-10 week process. All cell culture work is performed in T-flasks. The neurons are induced from NT-2/D1 cells following exposure to growth media containing 10 ⁇ M RetA for 4-6 weeks. Cells are harvested using trypsin and replated at reduced density. Replate I cultures are maintained in growth media for 2 days and then selectively harvested with trypsin to give an enriched neuron population and replated (replate II) into flasks. Both extended replate I and replate II cultures are treated with mitotic inhibitors for 7 days. The cells are maintained in neuron-conditioned media and allowed to mature in culture. Neurons constitute approximately 10% of the cell population; 90% are non- neuronal accessory cells.
  • Neurons are post-mitotic and no longer capable of dividing, whereas the accessory cells are mitotically inhibited by the addition of cytosine arabinoside (Ara-C) and fluorodeoxyuridine (FUdR) to culture medium. Neurons, which are loosely attached to the surface of the cell bed, are selectively harvested by brief trypsin treatment. Harvest results in a purified bulk product of >95% neurons.
  • cytosine arabinoside Ara-C
  • FdR fluorodeoxyuridine
  • RT-PCR Reverse transcript PCR
  • the expected TH band was present in the sample of immature hNT-Neurons which were harvested after 6 weeks of RetA, as well as in the sample of mature purified hNT-Neurons that were aged in culture for 5 weeks after mitotic inhibitor treatment.
  • the TH band was not detected for either the uninduced NT2 precursor cells or the 24hr RetA-induced cultures. These results are consistent with other observations that TH is not expressed in the precursor cells or the NT2 cells early in the induction period (personal communication, Virginia Lee). TH begins to appear only after several weeks of RetA induction as the neurons develop, and continues during the differentiation of hNT-Neurons. Similar results were obtained for the PCR analysis of the dopamine Dl and D2 receptor expression.
  • Protein samples and cell extracts were denatured in SDS sample buffer containing ⁇ - mercaptoethanol and electrophoresed on a Laemmli SDS polyacrylamide gel which separates the proteins by size. The proteins were then transferred to a nitrocellulose membrane by electro- blotting and the specific protein of interest was immunodetected by reacting to a primary antibody which is developed using the appropriate secondary antibody enzyme conjugate system.
  • Samples were prepared by harvesting cells with trypsin EDTA solution, or by scraping, and resuspending in media. An aliquot of cells was taken for viable cell count, and total viable cells were determined. The remaining cells were centrifuged and washed, and the resulting cell pellet were quick-frozen and stored at -80°C. Samples were thawed and lysed in cold RIPA buffer at a concentration of 10 6 cells in lO ⁇ l of buffer, an equal volume of 2X SDS reducing sample buffer added, and heated in boiling water bath for 5 min and then placed on ice.
  • the TH protein standard (STI, Catalog No. P-20233) at 500pg/ sample and the prestained protein molecular weight marker samples were prepared in SDS reducing sample buffer. The gel was electrophoresed until protein markers were well separated and bromophenyl blue dye had run off the bottom of the gel.
  • the protein samples were transferred from the gel and immobilized on a nitrocellulose membrane by electroblotting in a Western transfer chamber. The Western blot was blocked overnight in PBS containing 2% dried milk and incubated with anti-TH monoclonal (Boehringer-Mannheim clone 2/40/15) in PBS-Tween containing 1.0% BSA.
  • the blot was incubated at room temperature with biotinylated goat anti-mouse secondary antibody (1 : 1000) and then with Strepavidin-Alkaline phosphatase conjugate (1 :2000). The blot was washed in PBS-Tween and developed using the insoluble alkaline phosphatase substrate BCIP NTB (Sigma). The TH specific bands are visualized within l-2min. and appeared in the 55 to 60Kd size range. The sensitivity of the assay was determined to be approximately 50pg/ lane using serial dilutions of the TH-protein standard (STI) ranging from 5ng to 20pg.
  • STI TH-protein standard
  • TH levels confirmed and quantified by Western blot assay.
  • Western blot assay In order to confirm and quantitate the levels of TH expressed in different samples, a Western blot assay was developed. Several monoclonal antibodies were evaluated for detection of TH on blots containing human cell extracts and rat TH standard (STI). The anti-TH monoclonal 2/40/15 (Boehringer Mannheim) was found to detect reproducibly 50 to 100 pg of TH/lane. Initial estimates of the amount of TH protein expressed were determined using the Western blot assay and purified rat TH (STI) as standard. Analyzed samples of hNT-Neurons contained a range of cells from 2x10 to 2xl0 5 per lane.
  • the intensity of the TH-specific band was compared to samples containing 500 pg to 50 pg of the TH standard. The results of this analysis indicated that approximately 200-500 pg of TH protein are present in extracts containing 10 6 hNT-Neurons.
  • a similar analysis was performed using human mesencephalic samples ranging from 10 6 to 10 5 per lane. Equivalent levels (200-500 pg/10 6 cells) were detected in the hNT-Neurons compared to the human mesencephalic tissue.
  • the commercial process of producing hNT-Neurons involves inducing the NT2/D1 precursor cells with RetA for 6 weeks.
  • the resulting culture contains 10%o neurons and 90% non-neuronal accessory cells, which are next mitotically inhibited.
  • the hNT-Neurons are selectively harvested, leaving the accessory cells behind.
  • the levels of TH expressed in hNT- Neurons, NT-accessory cells, and NT2 precursor cells were compared by Western blot ( Figure 1). Only the sample of hNT-Neurons (lane 3) gave the expected TH band that migrated at 55-60 kd, and no TH-specific bands were detected in the accessory cell sample (lane 4) or NT2 precursor cell sample (lane 5).
  • neuronal precursor cell lines were evaluated for their potential to produce neurons with dopaminergic properties.
  • the 10 sister clones to the NT2/D1 (p51) production cell line were obtained from the Wistar Institute and analyzed for growth and production of neurons.
  • the four best growing clones were evaluated for production and yield of neurons which were further tested for the presence of TH by Western analysis. Only the neurons from the NT2 B9 and D1(P30) clones expressed TH at reproducibly detectable levels on Western blots and also were determined using the HPLC assay to produce HVA at levels similar to the hNT- Neurons.
  • hNT-Neurons A study examined the effects of in vitro maturation of hNT-Neurons on TH levels. After the hNT-Neurons have developed during the RetA induction, cultures are routinely replated and treated with mitotic inhibitors in the process of purifying the neurons (see Example 1). The hNT-Neurons were maintained in culture and allowed to mature either as a replate-I or as an enriched replate-II culture. To optimize the effects of neuron expression of TH, neurons were purified after culturing under different replate conditions and the levels of TH compared in the Western Blot assay.
  • Samples were prepared from purified hNT-Neurons that had been treated with inhibitors for 7 days as replate-I or replate-II cultures, and then maintained in growth media for a total of 1, 2, or 3 weeks of maturation. Extracts of the purified neurons were analyzed by Western Blot. The level of TH expression decreased dramatically with maturation in culture, and it was no longer detectable after 2 weeks in replate-II or after 3 weeks in replate-I. The levels of TH found in the replate-I neurons not only were significantly higher, but also were expressed for a longer period. Control Western Blots were developed for each assay using an anti-Tau monoclonal to confirm that equivalent numbers of hNT-Neurons were loaded, and the samples were not degraded.
  • hNT-Neurons To optimize production of hNT-Neurons for expression of TH and dopaminergic properties, a time course of RetA induction was performed and the TH levels in purified replate-I neurons from different RetA inductions were analyzed. The NT2 precursor cells were induced with RetA to differentiate into hNT-Neurons.
  • the cultures were replated and treated with mitotic inhibitors for 7 days.
  • the cells were refed with growth media and the neurons harvested either after 1 day (1 week replate-I) or after an additional 7 days ( 2 weeks replate-I).
  • the extracts were prepared for denaturing SDS-PAGE and samples containing 10 6 cells/lane were transferred to Western blots for analysis.
  • a small-scale dose range study analyzed whether therapeutic (0.5 - 1.0 mM) and clinically toxic (2.0 - 6.0 mM) concentrations of LiCl enhanced TH expression in cells treated for 6 weeks with retinoic acid and then LiCl.
  • the control as well as lithium-treated cultures (0.5, 1.0 and 3.0 mM) was scored for TH immunoreactivity, which was expressed as the number of TH-positive (TH+) cells/well.
  • TH immunohistochemistry the cells were rinsed in 0.1 M phosphate buffered saline (PBS), fixed in 4% paraformaldehyde and then again rinsed in PBS.
  • PBS phosphate buffered saline
  • the cells were incubated in blocking serum (10% normal horse serum, in 0.02%> Triton X-100 in 0.1 M PBS, pH 7.4) for one hour and then incubated overnight in monoclonal antibody against TH (1:4000, LNCSTAR, Stillwater, MN).
  • blocking serum 10% normal horse serum, in 0.02%> Triton X-100 in 0.1 M PBS, pH 7.4
  • monoclonal antibody against TH (1:4000, LNCSTAR, Stillwater, MN).
  • secondary antibody biotinylated horse antimouse (1:300, Vector, Burlingame, CA) was applied for one hour.
  • the antibody complex was developed using avidin-biotin complex (ABC-Elite kit; Vector) and the developed product was visualized by using DAB (Pierce, Rockford, IL).
  • the numbers of labeled and unlabeled cells were assessed in a blind- coded manner using a 20X objective and a photographic frame. The frame was placed in 16 predetermined sites per well. This test was performed in duplicate.
  • TH+ neurons represented only 1% of the entire population of hNT cells. After treatment with 0.5 and 2.0 mM LiCl, almost 5%o cells were TH+. The TH+ cells constituted nearly 7% of hNT cells treated with 1.0 mM of lithium. At higher concentrations (3.0 and 6.0 mM lithium), TH+ cells decreased to fewer than 2%. The treated cells are shown in Figures 5A through 5D. Arrows in Figure 5a point to individual TH+ neurons. In Figure 5b some cells reveal extremely long TH+ processes (arrowheads). Figures 5c and 5d show cultures of hNT cells treated with 3.0 mM of lithium.
  • hNT-neurons were tested after 4-week induction with RetA after mitotic inhibitor treatment.
  • "Fresh Cells” had undergone Replate II treatment (see above).
  • Prior to freezing, other cells underwent Replate I treatment.
  • Control and lithium-treated cells fresh or frozen
  • the TH-immunostained slides were counterstained with propidium iodide to identify dead cells.
  • Total numbers of TH+ and propidium iodide-positive (PI+) neurons were counted in control and lithium-treated cultures from standardized fields at 20X magnification, as described above.
  • FIG. 6a is a control culture of hNT neurons immunostained with antibodies to TH which had a significantly higher number of TH+ cells in comparison to controls treated for six weeks with RetA (See Fig. 4).
  • Fig. 6c is a control culture of hNT neurons immmunostained with antibodies to bcl-2 to demonstrate the co- localization of an anti-apoptotic gene with TH expression. Incubation of hNT cells with LiCl increased the size of the TH neurons as well as the length and number of processes.
  • the hNT cells were seeded in lO ⁇ g/mL poly-L-lysine-coated 8-well chamber slides at a concentration of 100,000 hNT neurons/cm 2 in a DMEM medium supplemented with 10% FBS and 50 g/mL gentamicin (Sigma). Plated cells were maintained in a humidified CO incubator (5% CO 2 , 90% air at 37 "c . After 24 hours, the media was changed to DMEM:F12 containing 0.1%) ITS (Sigma), gentamicin and LiCl (Sigma) at 0, 1.0 and 3.0 mM concentrations. The cells were incubated for an additional four days. Then, trypan blue was added to the medium. In three random fields from control and both lithium- treated cultures, the numbers of viable and dead cells were counted.
  • bcl-2 has been shown to protect a variety of cell types from programmed cell death, it is often considered an inhibitor of apoptosis (Sentman et al. 1991). Lithium-treated cells for the involvement of bcl-2 which could help protect hNT neurons.
  • bcl-2 Lithium-treated cells for the involvement of bcl-2 which could help protect hNT neurons.
  • Immunostaining was performed as described for TH (above), except that the monoclonal antibody to bcl-2 (Ab-1, 1:50, Calbiochem, Oncogene Research Products, Cambridge MA) was used.
  • Figure 7 summarizes the effects of different doses of LiCl on bcl-2 immunostaining. These values were expressed as the number of bcl-2+ cells/well and clearly indicated significantly enhanced expression of anti-apoptotic agent in LiCl-treated hNT cells.
  • TH tyrosine hydroxylase
  • the hNT neurons (Layton Bioscience, Inc., Atherton, CA), previously treated for 6 weeks with retinoic acid, were stored at - 80 C prior to use. Cells were thawed rapidly at 37 C and transferred into a 15-cc tube containing DMEM (Gibco, BRL, Grand Island, NY) and 10%) fetal bovine serum (FBS; Gibco, BRL). After centrifugation (1000 rpm/7 min), the cells were resuspended in 1 ml of the above fresh media and the cell number assessed with a trypan blue exclusion method. Cells were plated at a concentration of 100,000/cm 2 on eight-well poly- L-lysine (10 g/ml, Sigma, St.
  • GAP-43 monoclonal primary antibody against growth-associated protein (GAP-43, 1 : 8000; Chemicon, Temecula, CA), GAP -43 is a neuronal marker highly expressed during neuronal differentiation and neurite outgrowth (Skene JHP, Ann Rev Neurosci 12: 127-156, 1989; Strittmatter SM, Vartanian T, Fishman MC, J Neurobiol 23: 507-520, 1992). In our study it was used to demonstrate the morphological appearance of cultured hNT neurons.
  • the antibody complex was developed using an avidin-biotin kit (ABC-Elite kit; Vector) and the developed product visualized by using 3,3 - diaminobenzidine (DAB; Pierce, Rockford, IL).
  • TH-positive hNT neurons could be detected throughout the culture dish (Fig. 10).
  • TH-positive hNT neurons revealed oval or spindle-shaped soma with short and smooth neurites preferentially emerging from basal and apical parts of the soma.
  • TH-positive neurons did not display processes at all (Fig. 10A).
  • the TH-positive cell bodies were larger and their neurites substantially longer with numerous varicosities (Fig. 10D, 10E).
  • TH-positive neurons represent only 1% of the entire population of hNT cells.
  • TH expression was slightly lower in hNT cells after treatment with 2.0 mM concentration of lithium. In the last two concentrations (3.0 and 6.0 mM) the TH-positive neurons represented about 2% of the entire population of hNT cells (Fig. 11).
  • FIG. 11 A Photomicrographs of representative control and lithium-treated hNT neurons cultured for 5 days and immunostained for tyrosine hydroxylase are shown in Fig. 11 A, control culture of hNT neurons reveals few TH-positive cells (arrow). The bar represents 50 m .
  • FIG. 1 IB and 1 IC show hNT cells cultured with 1.OmM (Fig. 1 IB) and 3.0 mM (Fig.1 IC) lithium chloride; arrows point to individual TH-positive neurons. In lithium-exposed cultures more TH-positive cells with longer TH-immunoreactive processes (arrowheads) were present.
  • Figures 1 ID and HE show the representative morphology of TH-positive hNT cells treated with l.OmM (Fig.
  • lithium an effective psychotherapeutic agent induced the expression of TH in hNT cells.
  • the increased TH expression can be due to increased TH synthesis and/or increased Th activity, both of which are mediated by signal transduction pathways including protein kinase C (Zigmond RE, Schwarzschild MA, Rittenhouse AR, Ann Rev Neurosci 12:415-461, 1989).
  • Example 9.D Effect of Lithium on Size and Neurite Outgrowth of hNT Neurons
  • TH soma size
  • ⁇ m neurite outgrowth
  • the second parameter characterizing the effect of lithium on the development of TH- positive hNT cells was neurite outgrowth.
  • Neurite outgrowth in NaCl and KCl-treated 5 days in cultures did not significantly differ from control values (Fig. 12B).
  • Soma size and neurite outgrowth were also measured in hNT cells maintained in culture for 10 days and treated for 5 days with the most effective dose of lithium (1.0 mM).
  • the mean soma size of TH-positive hNT cells was significantly larger (102.8 + 2.5 ⁇ m 2 ) (p ⁇ 0.01) than in 5 days in cultures, but did not differ significantly from the mean value of the lithium-treated group (103.2 + 2.7 ⁇ m 2 ).
  • the mean length of neurite processes in controls was 24.8 + 2.4 ⁇ m which was not different from younger (5DIV) control cultures but significantly different from 10 day lithium-treated group (55.5 + 5.1 ⁇ m).
  • numerous TH-positive cells revealed multiple branching processes with varicosities.
  • Example 9.E Effect of Lithium on Viability of hNT Neurons The effect of lithium chloride on the viability of hNT neurons was evaluated in living, trypan blue stained cultures. Our preliminary results (Zigova et al. Abst. Neuroscience 24:
  • the effect of lithium on survival of hNT neurons was also evaluated from cultures fixed and immunostained for neuronal marker, growth-associated protein GAP-43.
  • the mean number of GAP-43- positive cells per field was calculated from 4 wells per condition.
  • the immunostaining was selected as further confirmation of hNT' s neuronal phenotype and to facilitate the neuronal counts.
  • Morphologically, GAP-43 positive hNT neurons usually exhibited round or oval perikarya and neurites including growth cones (Fig. 9). In Fig.
  • FIG. 9B higher magnification shows that virtually all cultured hNT cells are immunoreactive for GAP43, and thus neuronal phenotype.
  • GAP-43 -positive hNT cells usually have oval-shaped cell bodies with one or two processes, some of which demonstrate growth cones. The bar represents 50/tfm .
  • hNT neurons Independent of survival time or lithium dose used, hNT neurons were aggregated into tightly or loosely packed clusters frequently interconnected with each other.
  • FIG. 9C, 9D Low- magnification phase contrast photomicrographs of hNT neurons treated with 1.0 mM (Fig. 9C) and 3.0 (Fig. 9D) concentration of lithium chloride.
  • GAP-43 immunostaining facilitated the neuronal counts on these cultures, whose aggregates hampered the cell counting if unstained.
  • the number of viable neurons varied between 110-140/ field that was similar to cultures receiving 1.0 and 3.0 mM lithium chloride.
  • the individual counts per field ranged from 110-150 and in the group treated with 3. OmM lithium chloride these varied between 100-140 / field. These counts were not statistically significantly different from the control values.
  • NT-2/D1 Precursor cells were induced with 10 ⁇ M RetA for four weeks. The cultures were replated and treated with mitotic inhibitors for seven days. Purified DA-Neurons were selectively harvested. Approximately 2 X 10 6 neurons were plated in flasks that contained one of the following feeder cultures of mouse 3T3 cells, TM-4 cells (a Sertoli cell line), NT-2 Accessory cells, primary human astrocytes or primary rat glial cells. These co-cultures were maintained for seven days, after which the mature neurons were differentially harvested for the feeder cultures. Extracts were prepared from about 0.5 x 10 6 Cells, which were reduced, denatured and analyzed for TH using a Western blot technique.
  • TH levels are shown in Figure 13 (DA-Neuron Co-Culture).
  • Lane 1 is a control of co-cultured 500 pg of TH
  • lane 2 shows TH from fresh DA-Neurons bulk harvested.
  • TH from DA cells 1-week with polylysine/Laminin (lane 3)
  • TM-4 cells (lane 4)
  • rat glial cells (lane 5).
  • figure 13 is that TH levels decreased dramatically from that found in the fresh DA-neurons (lane 2) in cultures plated on polylysine alone, or on rat glial cells (lane 5). Similar results were found for most of cells with the exception of the TM-4 cells (lane 4).
  • the level of TH expressed in DA-Neurons co-cultured for 1 week with the TM-4 cells was comparable to fresh neurons (lane 2).
  • the stabilization of TH levels in the TM-4 co-cultured neurons appears to be aided by cell-cell interactions, since TM-4-conditioned media had no effect.
  • cytokines e.g., FGF, TFG and LIF
  • neurotrophic factors e.g., FGF, TFG and LIF
  • BDNF, GDNF, NT4/5) on the development of their dopaminergic properties in addition to TH levels are also investigated.
  • the DA/hNT-neurons from the best plating and enhancement protocols are then analyzed for production of DA using the HVA assay.
  • the optimal process/treatment regimen is then scaled up for production of DA hNT neurons for testing in animal studies.
  • the enhanced expression of TH in the 4-week DA-Neurons was found to be optimal in small scale cultures after replating the neurons in serum-free media containing 1 mM LiCl for 5 to 7 days.
  • the neurons analyzed in these initial studies were purified and frozen prior to plating on polylysine for testing, conditions where TH levels decrease without treatment. It would be ideal to incorporate the LiCl induction into the DA-Neuron process to obtain 4-week RetA induced DA-Neurons that are also treated with LiCl.
  • a series of LiCl treatments were designated to evaluate conditions for optimal TH expression.
  • the purpose of this experiment was to determine on 4-week RetA cultures the effect of LiCl treatment during the Replate I mitotic inhibition step of the process, which occurs just before harvest of the DA-Neurons.
  • Replate cultures were treated with 1 mM LiCl in the presence of inhibitors (FudR & AraC) for the standard 7 days of replate or during the last three of the 7 days (prior to harvest).
  • the 3-day LiCl treatment was also evaluated without mitotic inhibitors and in serum-free media (+ITS).
  • After treatment the neurons were selectively harvested, processed and TH levels analyzed using Western Blots.
  • the 7-day-LiCl Neurons contain about 50%> higher levels of TH than the 7-day inhibitor only control (compare Fig. 14 Lanes 2 and 3).
  • the 3-day-LiCl neurons also expressed levels of TH comparable to the control, with the uninhibited sample (lane 5) expressing somewhat more TH.
  • the neurons harvested from serum-free media expressed significantly less TH (Fig. 14 D/F+ITS, lanes 6 and 7). Serum-free neurons may not have developed as well and were contaminated with accessory cells.
  • Figure 14 shows the results for 500 pg TH (lane 1); control DA neurons in DF-5/Inh, 7 days (lane 2); DA neurons in DF-5/Inh/ ImM LiCl, 7 days (lane 3); DA neurons in DF-5/Inh/ ImM LiCl, 3 days (lane 4); DA neurons in DF-5/lmM LiCl, 3 days (lane 5); DA neurons in DF- 5/ ImM LiCl, 3 days (lane 6); and DA neurons in DF/ 1% ITS/ ImM LiCl, 3 days (lane 7).
  • DA neurons treated with LiCl (lanes 3, 4 and 5) seemed to express more TH than did the DA neurons maintained in DF-5/Inh alone (lane 2).
  • different cells can be adapted for increased TH and dopaminergic production.
  • the initial step is analyzing the cells' TH activity; examples of additional cell types are listed below. Subsequently, additional experiments as discussed above are undertaken to change plating, maturation and co-culturing techniques and thus optimize each cell type's TH activity.
  • U.S. Patent No. 5,639,618 discloses a stable line of lineage-specific neuronal stem cells.
  • the stem cells are constructed from blastocyst-derived ES cells transfected with a reporter construct under the control of the Otx regulatory region, Otx being an early marker for neurogenesis.
  • the reporter construct is used to segregate the neuronal stem cells by FACS isolation or other method. The segregated cells are then plated and permitted to terminally differentiate.
  • U.S. Patent No. 5,618,531 discloses a method for increasing the viability of cells, which are administered to the brain or spinal cord. The method is accomplished by attaching the cells to a support matrix and implanting the support matrix into the brain.
  • U.S. Patent No. 5,135,956 discloses using long-chain (23 to 29 carbons) fatty alcohols and prodrugs esters to cause extension of neurites in vivo and facilitate healing of traumatic injury to both the central and peripheral nervous systems by facilitating reconnection and reestablishment of function, decreasing ischemia and neuronal death, and reducing Wallerian degeneration after injury.
  • the most widely used model to evaluate the potential of neural transplantation to relieve the symptoms of PD is the rotational model of nigrostriatal function in rats (Koutouzis TK, Emerich DF, Borlongan CV, Freman TB, Cahill DW, Sanberg PR. Critical Reviews in Neurobiology 8: 125-162, 1994).
  • the toxin 6-OHDA is stereotaxically injected into the substantia nigra to destroy dopaminergic neurons and to reduce TH activity on one side, creating a unilateral DA receptor supersensitivity.
  • Lesioned animals develop stereotypical rotational behavior contralateral to the lesioned side in response to apomorphine, and ipsilateral to amphetamine that stimulates DA release.
  • Transplantation was done by stereotaxic injection into the striatum. Transplants consisted of hNT-Neurons alone, hNT-Neurons and Sertoli cells together, or hNT-Neurons co-cultured with Sertoli cells for 72 hours prior to transplantation. Doses of approximately 10 6 viable cells of either cell type were used. Functional recovery was measured by fewer post-transplantation turns in response to apomorphine.
  • Non-human higher mammal study bridges the gap between rodent and human. This study demonstrates efficacy, long-term effects, scale-up to doses close to humans, general and specific safety, and valid neurosurgical procedures in preparation for clinical trials for the indication of PD.
  • Non-human primates may be unilaterally lesioned in the caudate and putamen with l-methyl-4-phenyl-l, 2,3, 6-tetrahydropyri dine (MPTP) to produce hemiparkinsonian-like symptoms that provide a good model for evaluation of interventions in PD. Following the lesion, animals spontaneously circle in the direction of the lesion, but in response to apomorphine, they reverse and circle contralateral to the side of the lesion.
  • MPTP 2,3, 6-tetrahydropyri dine
  • Associated motor and behavioral symptoms include generalized slowing, rigidity and tremors characteristic of PD.
  • optimized DA/hNT neurons are administered to at least one part of the lesioned area.
  • Possible administration protocols call for delivery of the neurons in 1-20 tracks, preferably 1-10 tracks, more preferably 1-6 tracks and most preferably about 4 tracks. It is understood that one or more doses of cells is administered in each track, once as the needle is initially placed and optionally again or repeatedly as the needle is partially withdrawn.
  • the amounts of cells to be delivered in each track varies from about 5 x 10 4 to 5 x 10 6 cells per dose, preferably 1 x 10 5 to 2 x 10 6 cells per dose, most preferably about 5 x 10 5 to 1.5 x 10 6 per dose, and most preferably 10 6 per dose; however, these dose ranges are to be modified based on the identification of the optimal dose in rodents and the appropriate scale-up factor for higher mammals.
  • the cells or appropriate control(s) are administered to caudate and putamen of MPTP-lesioned animals. Animals undergo repeated behavioral testing at suitable intervals before being sacrificed for histological analyses. Histologic examination will be done for tumor formation, brain infection, and graft rejection.
  • hNT-Neurons While not wishing to be bound by any theory, one possible explanation for the effectiveness of hNT-Neurons in multiple brain disorders is that the morphology and differentiation of the TH-induced, transplanted hNT-Neurons may be determined by the microenvironments of the CNS region of the host into which they are introduced.
  • the ability of hNT-Neurons to migrate following transplantation was investigated (Zigova and Luskin, in preparation) by transplanting fluorescent-labeled neurons into the subventricular zone (SVZa) of newborn rat pups. The cells migrated tangentially along a stereotypical and extended pathway to the olfactory bulb, and then turn radially into one of the overlying neuronal cellular layers.
  • Fluorescence microscopy and immunohistochemistry revealed aggregates of labeled cells at the site of implantation in the SVZa, as well as along the migratory pathway leading to the olfactory bulb, and in the subependymal zone in the middle of the olfactory bulb. None of the labeled cells deviated from the pathway. Some of the transplanted labeled neurons exhibited a neuronal morphology with distinct processes and stained with the antibody TuJl, a neuron- specific marker, suggesting that the environment of the SVZa and the pathway leading to the bulb provided cues guiding the differentiation of hNT-Neurons.

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Abstract

Des cellules neuronales différenciées convenant pour une transplantation chez des sujets à déficience en dopamine sont dérivées de cellules souches. Les cellules souches sont traitées par au moins un agent inducteur, tel que l'acide rétinoïque, pendant une durée suffisante pour optimiser l'expression de la tyrosine hydroxylase. Les cellules destinées à la transplantation sont traitées éventuellement par un sel de lithium pour accroître la production et la survie de bcl-2. Facultativement, les cellules souches peuvent être co-cultivées avec des cellules Sertoli, astrocytes, gliales, des cellules accessoires ou une combinaison de celles-ci. Les cellules prêtes à la transplantation sont isolées et récoltées. Les cellules neuronales résultantes sont hautement purifiées et présentent un phénotype optimisé pour une déficience dopaminergique, telle que la maladie de Parkinson.
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