KR100880948B1 - A method of Obtaining matured dopaminergic neuron from neural precursor cells - Google Patents

Abstract

The present invention relates to a method for differentiating neural precursor cells into functional and morphologically mature dopaminergic neurons using a Nurr1 family transcription factor and a composition used therein. Dopaminergic neurons differentiated and proliferated from neural progenitor cells according to the present invention are used in cell replacement therapy and gene therapy therapy for the treatment of diseases of the neurological system such as Parkinson's disease, Alzheimer's disease, stroke, ischemia, etc. It can be widely used as a material for drug effect assays or various studies.

Neuronal Progenitor Cells, Neuronal Differentiation, Nurr1 Transcription Factors, Bcl-XL, SHH, Mash1

Description

A method of obtaining matured dopaminergic neuron from neural precursor cells}

1 is a process schematic diagram of the present invention.

FIG. 2 is a graph showing neural differentiation of Nurr1-induced DA cells in non- passaged (P0) and passaged (P1) cultures, where A is a graph of the mean fiber length in GFP + cells and B is the mean of TH + cells Contrast graph for fiber length, C is graph for TH + TuJ1 / TH + MAP2 + of total TH + cells.

3 is a graph depicting improved morphological differentiation of Nurr1-DA cells by SHH, where A is Nurr1-derived derived from unpassed (P0), passaged P1 monocellular (PID) and passaged P1 clusters. A graph comparing the fiber length of TH + cells to TH + cells, B is a graph showing the effect of SHH signaling on the differentiation of Nurr1-TH cells, the fiber length of TH + cells on cyclopamine treatment in P0 culture. A graph showing improved morphological differentiation of Nurr1-TH cells in P1, with total fiber of TH + cells for SHH (-), SHH (+), Nurr1-SHH (NH). This is a graph of length.

FIG. 4 is a graph demonstrating the effect of Bcl-XL on improved morphological differentiation of Nurr1-DA cells from precursors in late developmental phase, where A is transduced with Nurr1 proliferated neuronal precursors for 2 and 5 days. A graph comparing the fiber length of induced TH + cells, B is a graph comparing the fiber length of Nurr1-TH cells in PO culture from cortical precursors isolated from various natal days (E12-E18), and C is Bcl-XL Graph showing enhanced neurite production of Nurr1-TH cells by co-expression of vs. TH + fiber length on day 5 of differentiation for Nurr1-IRES-LacZ (control) and Nurr1-IRES-Bcl-XL (NB) to be.

FIG. 5 is a graph depicting the expression of MAP2 in Nurr1-induced TH cells, differentiated in culture transduced with Nurr1-LacZ (control) and Nurr1-SHH-Bcl-XL (NHB) and Nurr1-Mash1 (NM). It is a graph showing the result of TH / MAP2 immune cell staining performed on the day.

6 is a graph of biochemical and physiological analysis of the function of the transduced cells, A is a graph measured after application of 24 hours of medium or KCl for 15 minutes, and B in N, NHB, and NM-transduced cultures. (n = 5) A graph comparing the results of DAT-mediated DA uptake from total uptake, CE is a graph showing the biochemical properties of NHB-transduced cultures, and FH is the electrophysiological properties of NM-transduced cultures Is a graph.

7 is an in vitro cell survival graph, where A is% TH + cells in total DAPI + cells, B is total viable cell number, C is% LDH release to total cytoplasmic LDH, and D is total DAPI + cells % Cells with heavy degraded and aggregated apoptosis nuclei, E for% TUNEL + cells, and F for% degraded caspase 3+ cells, respectively.

Figure 8 shows in vivo of Nurr1-induced DA cells ( In In vivo ) graph of survival, differentiation and function, A is a graph of quantification of TH + cells in transplanted tissues at 4 and 8 weeks after transplantation, and B is N, NHB, NM transplanted tissues at 8 weeks of transplantation. It is a graph comparing the DA level, C is a graph of the behavior recovery results based on amphetamine-induced rotation, and D is a graph of the footfoot test results.

Figure 9 is a graph showing the differentiation of Mash1 and neurogenin 1 and 2 on Nurr1-induced TH expression, A is a graph showing the results of immunoblot analysis of TH and TuJI culture on day 2 of differentiation, B And C are graphs showing morphological analysis of TH + cells derived from Nurr1 + LacZ and Nurr1 + Mash1 cultures on day 10 of differentiation.

The present invention relates to a method for differentiating neural progenitor cells into mature dopaminergic neurons.

Since neural stem cells are present in every detailed tissue of the embryonic central nervous system during embryonic development, the neural stem cells can be isolated and cultured by cutting the desired embryonic cranial nerve tissue, and in adults, the subventricular zone (SVZ) or hippocampus ( Adult neural stem cells can be isolated and cultured from hippocampus, and studies using these neural stem cell cultures can provide a great deal of information for understanding the processes of normal brain nervous system development from neural stem cells.

Such neural stem cells show a large difference in their characteristics depending on the site of separation and the number of fetus days of the animal used to isolate the neural stem cells.For example, when culturing neural stem cells isolated at 14 days of rat cultivation, differentiation is induced. Differentiation into neurons occurs, whereas neural stem cells isolated from rat fetuses after 18 days of age show differentiation characteristics, mainly toward astrocytes. In addition, there are many differences in cell growth patterns and responses to various cytokines.

On the other hand, neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease (senile dementia), demyelinating disease, amyotrophic lateral sclerosis (ALS), and quadriplegia due to spinal injury are associated with specific neurons in the nerve tissue. It is caused by permanent loss and dysfunction, and since these nerve tissues are quite limited in their reconstruction when damaged, there is no fundamental treatment for these diseases.

In the case of Parkinson's disease, a representative neurodegenerative disease, secretion of dopamine neurotransmitters from the transplanted dopaminergic neurons into the striated body shows a clear recovery of function, so that embryonic cells can be transplanted into such patients In anticipation of in vivo induction of dopaminergic neurons from CNS stem cells in In vitro ) manufacturing is of great interest (Olanow, et al., Trends) Neurosci ., 1996, 19: 102-109; Piccini, et al., Nat . Neurosci ., 1999, 2: 1137-1140; Freeman, et al., N. Engl . J. Med ., 1999, 341: 988-992).

Wagner et al. Have successfully differentiated neural stem cell lines (neural dopaminergic cell lines) into dopaminergic neurons using Nurr1, but this differentiation requires cofactors derived from type I mesenchymal glial cells as well as neurons in the above. The use of stem cell lines makes them unsuitable for cell transplantation for Parkinson's disease (Wagner, et al., Nature Biotechnology , 1999, 17: 653-659.

Accordingly, the present inventors have disclosed the results of the differentiation rate into dopaminergic neurons when Nurr1 is treated to neural progenitor cells from neural stem cells through overexpression of Nurr1 as Korean Patent Application No. 2003-14662. . However, this also has drawbacks that are not sufficient to induce mature and functionally mature dopamine neurons. The present invention solves these problems.

Surprisingly, it was found that a combination of Nurr1 and Bcl-XL, Sony Hedgehog (SHH), or BHLH family of neurogenic transcription factors was used in neural progenitor cells to show morphological and functional synergistic effects upon differentiation.

That is, an object of the present invention is to provide a method for differentiating morphologically and functionally mature dopaminergic neurons.

In a first aspect, the present invention provides a method of differentiating neural progenitor cells into mature dopaminergic neurons. This method is accomplished by treating neural progenitor cells with Nurr1, Bcl-XL and Sony hedgehog (SHH). The result is a form of synergistic action and a functional effect.

In another aspect, the present invention provides a method of differentiating neural progenitor cells into mature dopaminergic neurons. This method is achieved by treating neuronal progenitor cells with Nurr1 and bHLH family neurogenic transcription factors. The result is a form of synergistic action and a functional effect.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated concretely.

Synergistic Compositions Useful for the Treatment of Parkinson's Disease

In one aspect, the present invention provides novel compositions useful for treating Parkinson's disease. The composition comprises Nurr1 and a second therapeutic agent selected from Bcl-XL, Sony Hedgehog (SHH), and a bHLH family of neurogenic transcription factors. It has been surprisingly found that the combination of Nurr1 with the second therapeutic agent exhibits a synergistic form and a functional effect.

A. Nurr1

As is known, Nurr1 is a transcription factor belonging to the steroid / thyroid hormone receptor expressed in midbrain dopaminergic neurons (Law, et al., Mol. Endocrinol ., 1992, 6: 2129), dopamine Expressed in sexual neurons (Zetterstrom, et al., Mol . Brain Res ., 1996, 41: 111), are believed to play a role in the development of midbrain dopaminergic neurons.

 B. Bcl-XL and Sony Hedgehog (SHH)

In another aspect the present invention provides a composition comprising Nurr1 with Bcl-XL and Sony Hedgehog (SHH). Surprisingly, it has been found that the combination of Nurr1 with Bcl-XL and Sony Hedgehog (SHH) enhances morphological and functional effects.

Bcl-XL is a type of anti-apoptotic protein and has a SHH mediated effect on neuronal precursors including midbrain DA neurons and neurons after cell division.

SHH stands for Sony hedgehog, and the term "sonic hedgehog (SHH)" used in the present invention is one of many genes required for the development of midbrain dopaminergic neurons (Ye, et al. , Cell, 1998, 93: 755-766.

C. Neuronal Transcription Factors of the bHLH Family

In another aspect, the present invention provides a composition comprising a neuronal transcription factor of Nurr1 and bHLH family. Surprisingly, it was found that the combination of the Nurr1 and bHLH family of neurogenic transcription factors showed synergistic morphology and functional effects.

In the present invention, as the bHLH family of transcription factors, a neurogenic transcription factor (neurogenic transcription factor) may be used. As a neurogenic transcription factor gene, for example, neurogenin 1 gene (GenBack Accession No .: U63842, U67776) , Neurogenin 2 gene (Genbank Accession No .: U76207, AF303001), Neuro D gene (Genbank Accession No .: U24679, AB018693), Mash1 gene (Genbank Accession No .: M95603, L08424), MATH3 gene (Genbank Accession No. CDNA obtained from sequencing information such as D85845) and E47 gene (Genbank Accession No .: M65214, AF352579) can be cloned or synthesized, and if the same or similar activity can be exhibited, some of these sequences can be modified or deleted. And substituted may also be used. Most preferably, the Mash1 gene is used.

In the present invention, as a method of increasing the levels of Nurr1 and bHLH transcription factors present in neural progenitor cells, bHLH transcription factors may be introduced into the cells directly or by using an appropriate carrier. More preferably, the Nurr1 and bHLH transcription factors may be introduced. A method of introducing a gene expressing the active fragment into the neural progenitor cells may be used.

Such Nurr1, SHH, Bcl-XL and Mash1 genes are generally transformed using the same amount. As a result, when used in neural progenitor cells, they can differentiate into morphologically and functionally mature dopaminergic neurons. In addition, the mature dopaminergic neurons thus obtained have the advantage that they can be effectively applied as a therapeutic agent for Parkinson's disease.

How to differentiate neural progenitor cells into mature dopaminergic neurons

The present invention provides a method of differentiating neural progenitor cells into mature dopaminergic neurons by treating the neural progenitor cells.

In the method of the present invention, the neural progenitor cells may be applied to any born days or brain regions, but may be ventral midbrain, cortex or lateral ganglionic eminence (stripe primitive). It is preferable to use neural progenitor cells isolated from striatum anlage), and more preferably to use those isolated from fetuses. In addition, usable animals can be any mammal including humans. In a preferred embodiment of the present invention, the neural progenitor cells are born 12 days (fetus day 12, hereinafter abbreviated as 'E12'), born 14 days (hereinafter abbreviated as 'E14') or born 16 days (hereinafter 'E16') Neural progenitor cells isolated from the midbrain, cerebral cortex or lateral ganglion ridges of rat fetuses were used.

The treatment of Nurr1 and other cofactors (hereinafter referred to as 'cofactor transduction') in neural progenitor cells isolated and cultured from the cerebral cortex of E14 rats is more effective than tyrosine hydrolase (Tyrosine) than without cofactor transduction. The fiber length of cells expressing hydroxylase (hereinafter referred to as 'TH') (hereinafter abbreviated as 'TH +') increased by 3-7 times or more (see FIGS. 3C and 4C). Since dopaminergic neurons differentiated from neural progenitor cells express TH, the degree of differentiation of neural progenitor cells into dopaminergic neurons can be determined by measuring the number of TH + cells. Neurons expressing TH include adrenergic and noradrenergic neurons in addition to dopaminergic neurons. Dopaminergic neurons do not express a protein called DBA (dopaminergic beta-hydroxylase), whereas adrenergic or noradrenergic neurons are distinguished in that they express DBH protein in addition to TH. TH + induced by the method of the present invention The cells are not adrenergic or noradrenergic neurons but all differentiate into dopaminergic neurons.

In the present invention, in order to process Nurr1 and other cofactors in neural progenitor cells, the gene is transfected into a plasmid containing the Nurr1 gene having the nucleotide sequence described in the sequence list or expressed by infecting the neural progenitor cells with a retrovirus expression vector. Let's do it.

In the present invention, when B-27 serum-free supplement (B-27 serum-free supplement, Life Technologies, USA) or ascorbic acid is added to Nurr1, the B-27 serum-free adjuvant and ascorbic acid are treated. Differentiation efficiency into dopaminergic neurons is increased by 3-5 times compared to no treatment. The final concentration of ascorbic acid added to the neural progenitor cells is usually preferably added 50 to 500 μM, more preferably 100 to 400 μM. In addition, the final concentration of the B-27 serum-free adjuvant added to the neural progenitor cells is usually preferably added 0.4 to 10%, more preferably 1 to 5%. In addition, it is preferable to simultaneously add the B-27 serum-free adjuvant and ascorbic acid to the neural progenitor cells.

As described above, morphologically and functionally differentiation into mature dopaminergic neurons is possible. In the present invention, "maturation" refers to the morphological maturation of the dendritic protrusions and length of the neurons and to form synapses with surrounding neurons and to secrete the neurotransmitters produced by them through synapses. It is defined as mature.

Specifically, young neurons that are not mature are tubulin III (tublin III) or MAP2 (a + b), which are neuronal marker proteins, and GABA, glutamate, dopamine, serotonin, and neurotransmitters. It does not occur, such as to form a synapse with other neurons. In addition, morphologically, young neurons extend only on both sides and have short branches.

However, mature neurons undergo morphological maturation such as dendritic dendritic branches and longer lengths. In addition, it also forms synapses with neighboring nerve cells and secretes neurotransmitters produced directly through synapses, which can be confirmed through electrophysiological analysis.

In the present invention, when ascorbic acid is added to primary neurons, the number of dendritic protrusions of dopaminergic neurons increases and their length increases, and in the electrophysiological analysis of mEPSC (miniature excitatory post-synaptic current) It can be seen that Nurr1 and other cofactors cause morphological and functional maturation of neurons, including dopaminergic neurons, by showing a significant increase in size and frequency compared to untreated cells (FIGS. 3, 4, 6 to 6). 9).

In order to determine whether dopaminergic neurons differentiated by Nurr1 and other cofactors of the present invention can be used for cell therapy of Parkinson's disease, the present inventors have applied the treatment of Nurr1 and other cofactors to rat fetal cortical neuronal progenitor cells. Differentiation-induced dopaminergic neurons were transplanted into the striatum of striatum in Parkinson's disease model rats and their improvement was investigated using a rotation test. As a result, the number of rotations after cell transplantation treated with Nurr1 and other cofactors was significantly reduced, and the settlement and differentiation of cells in the transplanted tissues were well induced. From the above results, it can be seen that dopaminergic neurons induced by differentiation by Nurr1 and other cofactors can be used for cell therapy of Parkinson's disease.

As described above, dopaminergic neurons differentiated by treating Nurr1 and other cofactors in neural progenitor cells may be usefully used for cell therapy of neurodegenerative diseases including Parkinson's disease.

As such, the neural progenitor cells into which the Nurr1 and the cofactor of the present invention are introduced or the dopaminergic neurons differentiated from the neural progenitor cells are Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotriophic lateral sclerosis. It can be usefully used for cell therapy and gene therapy for brain neurological diseases including stroke and ischemia.

In this case, as a method of obtaining an excess of neurons included in the cell therapy composition, the Nurr1 transcription factor gene and other cofactors are first introduced into the isolated neural progenitor cells and selected, and then proliferated under appropriate conditions in vitro, Dopaminergic neurons obtained by differentiation can be used, or the neural progenitor cells can be sufficiently propagated, and then Nurr1 transcription factor genes and other cofactors can be introduced and differentiated into dopamine neurons.

In addition, as an example of a gene therapy method using dopaminergic neurons differentiated from neural progenitor cells of the present invention, genes related to specific brain diseases (eg, dopamine synthesis) in the neural progenitor cells or dopaminergic neurons differentiated therefrom. To introduce the desired gene into the animal nervous system by additionally introducing a gene to allow expression of the tyrosine hydroxynase gene) and transplanting the neural progenitor cells or the dopaminergic neurons differentiated therefrom into brain tissue of the animal. It can be used in the method. Thus, neurons differentiated by the methods of the present invention can be useful tools for introducing genes into the nervous system of animals.

The therapeutic composition comprising the dopaminergic neurons produced by the method of the present invention as an active ingredient can be injected into a patient's body according to a known method, for example Freed CR ( N Engl J Med . 2001,344: The clinical methods published by 710 can be used.

The single dose of dopaminergic neurons can be increased or decreased in consideration of various related factors such as the disease to be treated, the severity of the disease, the route of administration, and the weight, age and sex of the patient.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Primary Culture of Rat Fetal Cortical Progenitor Cells

The inventors cultured neural progenitor cells from rats to differentiate into dopaminergic neurons. All animal experiments were performed according to the NIH guidelines.

Specifically, cortex was cut from E 14 Sprague-Dawley rats (KOATECK, purchased in Korea), and the isolated tissue was cut into CMF-HBSS (Ca ²⁺ / Mg ²⁺ −). cells were physically detached by grinding in free HBSS) (Invitrogen, Carlsland, Calif.).

Cells can be covered in slipslip (for non- passaged (P0) culture, 12 mm diameter, Carolina Biological Supply Company, Burlington, NC) or 10 cm tissue culture dish (for passaged (P1) culture, Corning, NY, polyornithine / fibronectin Pre-coated with) to 20,000 cells / cm ² . The neural progenitor cells were grown under serum-free N2 medium containing 20 ng / ml of basic fibroblast growth factors (bFGF) (R & D Systems, Minneapolis, MN).

Precursors covering 60-80% of the bottom (typically 3-4 days in vitro bFGF-proliferation) were incubated under CMF-HBSS for 1 hour, then physically detached and the N2 + bFGF (P1 single cell) Incubated on a pre-coated cover slip.

For some experiments, precursors incubated for 1 hour in CMF-HBSS were detached using a cell lifter (Corning) and incubated directly on polyornithine / fibronectin coated cover slips without further grinding (P1 cluster).

Retroviral transduction was performed on P1 or P0 cells when covering 50-60% of the bottom (typically 1-2 days after cell placement). Cell differentiation was induced by suspension of bFGF in serum-free N2 medium containing 200 mM ascorbic acid (AA, Sigma, St Louis, Mo.). The culture was maintained at 37 ° C. in a 5% CO ₂ incubator, the medium was changed every other day, and bFGF was added daily before differentiation. For some experiments, Sony hedgehog (SHH, R & D systems) or cyclopamine (Toronto Research Chemicals, North York, Canada) was added to the culture.

Retro Viral Products and Infections

The expression vector used in this study was made based on the retroviral vector pIRES-LacZ or pIRES-GFP based on Korean Patent 344090.

The retroviral vectors pNurr1-IRES-LacZ and pNurr1-IRES-GFP (N) expressing Nurr1 were made by inserting Nurr1 cDNA digests amplified by PCR above the IRES fractions of pIRES-LacZ and pIRES-GFP, respectively. .

Nurr1 sequence in pNurr1-IRES-LacZ vector was replaced with N-SHH (Seo Haeyoung, Ajou University, Korea), Smo-M2 (Arnon Resenthal, Genentech, Inc.), Bcl-XL, Mash1, Ngn1 or Ngn2 Was prepared. Bi-cistronic (pNurr1-IRES-SHH (NH), pNurr1-IRES-Bcl-XL (NB), and pNurr1-IRES-Mash1 (NM)) or tri-cistronic vector (pNurr1-IRES-SHH-IRES-Bcl-) XL (NHB)) was prepared by replacing LacZ of pNurr1-IRES-LacZ with each DNA lysate.

The retroviral vector was transfected into 293gpg packaging cells (Lipofectamine, Invitrogen) and obtained 72 hours after inoculation of the supernatant containing the viral fraction (VSV-G pseudotyped recombinant retrovirus). Viral titrator was adjusted to 5 × 10 ⁶ particles / ml. Neuroprecursors were incubated with viral supernatant for 2 hours in the presence of polybrene (1 mg / ml), incubated overnight in N2 + bFGF and then differentiated the next day by discontinuation of bFGF.

Co-expression studies were performed by infecting cells with bi- / tri-cistronic vectors or mixtures of each viral preparation (1: 1, v: v).

Cell Viability Assay

The total number of viable cells was observed during the whole experiment under phase contrast microscope. Cells with degraded and aggregated apoptotic nuclei were visually observed by DAPI staining. Activated caspase 3 assay, lactate dehydrogenase (LDH) assay (Promega, Madison, WI), terminal deoxynucleotidyl transferase-mediated dUTP nick terminal labeling for cell viability (TUNEL) Assay (Roche, Mannheim, Germany) and immunotoxicity chemistry.

LDH activity in the medium was measured by Cytotox 96 kit (Promega) according to the manufacturer's recommendations. The results are expressed in% of maximum LDH release obtained by cell dissolution in 1% Tripton X-100.

RT-PCT and immunoblot analysis

Total RNA preparation, cDNA synthesis and RT-PCT reactions were performed according to commonly known methods. Information on primer sequences (front and back), heat treatment temperature, PCR cycles and product size (base pair) is as follows: Otx1 (5'-GCTGTTCGCAAAGACTCGCTAC-3 ', 5'-ATGGCTCTGGCACTGATACGGATG-3', 62 ° C, 30cycles , 425 bp); Emx2 (5′-TTCGAACCGCCTTCTCGCCG-3 ′, 5′-TGAGCCTTCTTCCTCTAG-3 ′, 59 ° C., 35cycles, 188 bp); Pax6 (5′-CCAAAGTGGTGGACAAGATTGCC-3 ′, 5′-TAACTCCGCCCATTCACTGACG-3 ′, 58 ° C., 35cycles, 419 bp); SHH (5′-GGAAGATCACAAACTCCGAAC-3 ′, 5′-GGATGCGAGCTTTGGATTCATAG-3 ′, 58 ° C., 32 cycles, 354 bp); Smo (5′-TGCTGTGTGCTGTCTACATGCC-3 ′, 5′-TCTTGGGGTTGTCTGTCCTCAC-3 ′, 58 ° C., 32 cycles, 240 bp); Ptc (5′-GGCAAGTTTTTGGTTGTGGGTC-3 ′, 5′-CCATGTAACCTGTCTCCGTGATAAG-3 ′, 58 ° C., 35cycles, 355 bp); Bcl-XL (5′-CAAGCTTTCCCAGAAAGGAT-3 ′, 5′-TGAAGAGTGAGCCCAGCAGA-3 ′, 58 ° C., 33cycles, 702 bp); Synapsin (5′-CCACCCCCACAAGGCCAGCAACA-3 ′, 5′-GGTCCCCCGGCAGCAGCAATGATG-3 ′, 58 ° C., 29 cycles, 512 bp); Synaptophysin (5′-TGGTATCCTACCGCATTC-3 ′, 5′-ACTCACCTCATAGCTCC-3 ′, 58 ° C., 26 cycles, 379 bp); GAP43 (5′-AGAAAGCAGCCAAGCTGAGGAGG-3 ′, 5′-CAGGAGAGACAGGGTTCAGGTGG-3 ′, 58 ° C., 26cycles, 167 bp); Rho8 (5′-ACGGGAAGCAGGTAGAGTTG-3 ′, 5′-GATGGGCACATTTGGACAG-3 ′, 58 ° C., 21 cycles, 191 bp); Scg10 (5′-CTACCCGGAGCCTCGCAAC-3 ′, 5′-ACCTGGGCCTCCTGAGACTTC-3 ′, 58 ° C., 21 cycles, 231 bp); GAPDH (5′-GGCATTGCTCTCATTGACAA-3 ′, 5′-AGGGCCTCTCTCTTGCTCTC-3 ′, 25 ° C., 60 cycles, 165 bp).

Proteins were extracted from cultured cell lysates, electrophoresed and protein transferred into nitrocellulose films as commonly known. The blot was anti-rat neuronal-specific class III β-tubulin (TuJ1, Covance, Richmond, CA, 1: 1,000), TH (Sigma, 1: 5,000), β-actin (Abcam, Cambridge, UK, 1 : 5,000) probes with an antibody followed by anti-rat or rabbit IgG conjugated with peroxidase (Cell Signaling Technology, Beverly, MA, 1: 2,000). The resulting bands were visually observed with an ECL detection kit (Amersham Pharmacia, Buckinghamshire, UK).

DA Level Observation and DA Absorption Assay

HPLC analysis for the determination of DA levels was carried out with a slight modification of commonly known methods. To observe in vitro DA release from cultured cells, differentiated progenitor cells in 24-well cultures with 500 μl N2 + AA medium or 15 minutes (stimulated release) for 24 hours or 15 minutes (basic release). ) Were cultured in the same medium combined with 56 mM KCl. The medium was then collected and stabilized in 0.1 N perchloroic acid containing 0.1 mM EDTA and extracted by aluminum absorption.

To determine the DA content in the transplanted tissue of the transplanted animal, the tissue obtained at the center of the transplant (approximately 2 mm diameter size) was homogenized in 0.2 N perchloro acid containing 0.1 mM EDTA and then centrifuged for 10 minutes. The supernatant was filtered through a centrifugal filtration device (microcon YM-10, Millipore Co., Mass., USA) at 12,000 g for 10 minutes and used for DA level measurement.

DA was separated by reversed phase m-Bondapak C18 column (150 × 3.0 mm, Eicom, Japan) at a flow rate of 0.5 ml / min. Electroactive compounds were analyzed at +750 mV using analytical cells and amperometric detectors (ECD-300, Eicom).

DA levels were calculated using a catecholamine standard mixture comprising an internal standard (50 nM methyl-DOPA) and 1-50 nM external standard (DA) injected immediately before and immediately after each experiment. DA carrier (DAT) -mediated specific DA uptake was performed as recently known in connection with human ES cell-derived DA neuronal characterization (Park, et al., J. Neurochem ., 2005, 92: 1265).

Electrophysiology

Biochemical analysis was performed by recording whole cells using standard whole cell patch clamp technique. Nurr1-expressing cells were identified by GFP expression (Nurr1-IRES-GFP). Co-expression studies of Mash1 or SHH and Bcl-XL in Nurr1-expressing cells were performed according to the method described above.

After 10 days of in vitro differentiation, each GFP and Nurr1-expressing cells were selected for biochemical analysis. The patch electrode has a resistance of 2-4 MΩ. The cell membrane condenser and series of resistors were electronically canceled (typically> 80%) using a patch-clamp amplifier (Axopatch-200A; Axon Instruments, Foster City, Calif.).

Current protocol generation and data acquisition was performed using pClamp 8.2 software on an IBM computer equipped with an analog-to-digital converter (Digidata 1322A; Axon Instruments). All experiments were performed at room temperature (21-24 ° C.). To record the membrane potential in current clamp mode, the patch pipette solution was (in mM) KCl 134, MgCl ₂ 1.2, MgATP 1, Na ₂ GTP 0.1, EGTA 10, Glucose 14 and HEPES 10.5 (to pH 7.2 with KOH). Adjusted). Bath solutions included (in mM) NaCl 134, KCl 5, CaCl ₂ 2.5, MgCl ₂ 1.2, Glucose 14 and HEPES 10.5 (adjusted to pH 7.4 with NaOH).

Immunostaining on Cultured Cells and Cerebral Cleavage

Cultured cells or cold cut cerebral sections were fixed in 4% paraformaldehyde in PBS for 20 minutes and reacted with primary antibody overnight at 4 ° C. Primary antibodies include anti-rabbit TH (Pel-Freez, Rogers, AR, 1: 250), TuJ1 (Covance, Richmond, CA, 1: 2,000), anti-rat TH (Sigma, 1: 10,000), anti-GFP (Roche, Mannheim, Germany, 1: 400) and microtubule associated protein 2 (a + b) (MAP2, Sigma, 1: 500), anti-rabbit active caspase 3 (Cell signaling, Beverly, MA, 1: 200 ) Was used.

Secondary antibodies with appropriate fluorescent-tack (Jackson Immunoresearch Laboratories, West Grove, Pa.) Were used for visual observation. By mounting the cell and tissue section in VECTASHIELD ^ⓡ containing (Vector Laboratories, Burlingame, CA) DAPI was used a fluorescence microscope (Nikon, Tokyo, Japan) or Carl Zeiss LMS 510 confocal microscope (Carl Zeiss, Jena, Germany) Analyzed.

Morphological analysis of neurite growth

Neurites length of TH + cells were measured as commonly known. Specifically, 20-50 TH + cells were tested independently three times with random selection from at least three cultures. The cells were photographed on an Axiovert phase contrast microscope equipped with an Axiocam digital camera system, and the neurite length from the somatic cell to the tip of the branch was measured using an Axiovision image analyzer (Carl Zeiss). Total neurite length was defined as the combined length of all neurite per cell.

Surgical Process and Behavior Experiments

Animals were treated according to the National Institutes of Health guidelines. Female, Sprague-Dawley rats (200-250 g) were unilaterally implanted injured by 6-OHDA (Sigma) into the black matter and MFB (median forebrain bundle).

Four weeks after injury, animals were tested for drug-induced rotational imbalance (amphetamine 3 mg / kg i.p., Sigma). Rotation ratings were monitored for 60 minutes in an automated rotometer system (Med Associate Inc., St. Albans, Vermont). Animals with 5 / minor side for injury were selected for transplantation.

Forelimb akinesia was measured three times prior to cell transplantation by a 'footfoot test' and repeated every week for 8 weeks after cell transplantation. Footsteps of the non-resected cell while the animal was moving backwards (90 cm for 10 seconds). Counted The percent foot foot on the injured and intact side was quantified (3 trials / time average).

On day 2 after retroviral infection, the precursors propagated with bFGF were physically degraded and 3 μl of cell suspension (1.5 × 10 ⁵ cells / μl in PBS) was purified using a KDS310 nanopump (KD Scientific Inc., Holliston, Mass.). A 22 G needle was injected into the ipsilateral striatum for 5 minutes. Cells were adjusted to 3-digit (Bregma related AP, ML and V and dura (1) -0.03, -0.30, -0.58 (2) -0.03, -0.40, -0.58 (3) 0.03, -0.35, at 3.5 mm -0.58 incisor bar set).

The needle was left for 5 minutes after each injection. Cyclosporin A (10 mg / kg, i.p.) was injected daily into the rats from one day to three weeks after transplantation.

Histological Procedure

Animals were anesthetized (50 mg / kg phenobarbitol) and sprayed intracardially in 4% paraformaldehyde in PBS. The cerebrum was emerged with 20% sucrose in PBS and then cut to 35 μm on frozen microtome. Glass-rich sections were performed according to commonly known TH-immunohistochemistry procedures. Measurement of TH + cell number is also commonly known.

Cell counting and statistical analysis

Cell counts were performed by uniform random selection of 5-20 microscopic fields / well, 3-6 wells / experimental conditions, and the data obtained were expressed as mean ± SEM. Statistical comparisons were performed using ANOVA with Tukey post hoc analysis (SPSS 12.0; SPSS Inc., Chicago, IL) when two or more groups were included.

Example 1 Neuronal Differentiation of Nurr1-Induced DA Cells from Unpassed (P0) and Passed (P1) Rat Fetal Neuronal Progenitor Cells>

The expression of DA neurotransmitters in a number of neural progenitors by the expression of Nurr1 was confirmed, but the level of neural differentiation varied widely between cells. Long-lived adult neural precursors expressing Nurr1 alone did not exhibit neuronal properties. However, it has been reported that neurons were identified in a subpopulation of Nurr1-induced fetal neural precursors that proliferated for a short time. These findings suggest that neuronal differentiation in Nurr1-induced DA cells may depend on the duration of in vitro proliferation or the developmental period of progenitor cells.

Our previous study on Nurr1-induced neural precursors was performed on physical and cultured passaged (P1) cells after proliferation for several days in vitro. These conditions were chosen to ensure no neural precursors (P1) (> 95% nestin + cells, <1% of TuJ1 + neurons) compared to neural precursors (P0) that already comprise at least 15% differentiated neurons.

Hereinafter, the neuronal differentiation level of Nurr1-induced DA cells at P0 and P1 was tested and the results are summarized in FIG. 2. Precursors were transduced with Nurr1-retrovirus on the last day of progenitor cell proliferation (PO: day 3 in vitro, P1: day 6 in vitro).

Cells were differentiated for an additional 3-10 days prior to analysis. First, it was tested whether Nurr1 affects cellular apoptosis and morphology. For Nurr1-type introduced P0 and P1 cultures, cell apoptosis was increased compared to LacZ-type introduced control cultures. After 3 days of differentiation, the percentage of cells with apoptotic nuclei (digested or aggregated) increased significantly in Nurr1-form introduction compared to lacZ-form introduced control cultures: 16.5 ± 1.0%: 10.9 ± 0.8% in P0 culture. ; 15.7 ± 0.8% in P1 culture: 10.6 ± 0.5% (n = 35-60 microscope field, 3-8 cover glass, p> 0.001).

As shown in FIG. 2A, neurite length was significantly reduced in cells transduced with Nurr1 (Nurr1-IRES-GFP) compared to control culture (LacZ-IRES-GFP). This result is thought to be caused by decreased cell viability due to apoptosis, but it is not yet clear.

As shown in Figure 2b, in P0 and P1 cultures, Nurr1 transduction induced induced expression of TH, expression of specific markers in DA neurons. Morphology of Nurr1-induced DA cells showed a significant increase in the total length of TH + fibers in P0 culture compared to P1 culture (P0: 84.0 ± 4.9 mm; P1: 14.2 ± 5.7 mm, n = 40-50, p <0.001 ). Consistent with data on morphological differentiation, the majority of TH + cells expressed the neuronal markers TuJ1 and MAP2 in P0 cultures compared to P1 cultures.

As shown in FIG. 2C, on day 3 of in vitro differentiation, TuJ1 simultaneously expressed TH + cells at 37.3 ± 2.4% (P0) and 8.6 ± 1.2% (P1) (n = 25, p <0.001). MAP2 expression results in vitro differentiation on day 10 were within 65.0 ± 4.7% of all TH + cells at P0 and 11.7 ± 1.8% of TH + cells at P1 (n = 30, p <0.001). This demonstrates that Nurr1-TH + cells in P0 culture exhibit higher neuronal differentiation compared to P1 culture.

Example 2: SHH-Mediated Morphological Differentiation of Nurr1-TH + Cells

The main differences between P0 and P1 cultures are that longer periods of proliferation of P1 in vitro progenitor cells (in 6-day P1: in 3-day P0) and physical passage of P1 (P0 cells are not passaged). have.

First, it was tested whether the physical passage procedure affected the reduced level of neuronal differentiation in P1 to P0 cultures. Regular passage procedures were based on physical grinding of cell clusters into single cell suspensions after CMF-HBSS inoculation.

In order to examine the effect of cell interactions with cells, the cell clusters were rearranged identically without grinding. As shown in Figure 3a, under these conditions, the morphological differentiation of Nurr1-TH cells was not significantly altered in the P1 cluster compared to the P0 culture, whereas the P1 culture with unicellular suspension (P1 single cell) was much worse than P0. (P0 culture (P0): 67.5 ± 4.2 mm, P1 cluster (P1C): 43.0 ± 11.7 mm, P1 single cell (P1D): 9.3 ± 0.7 mm, n = 25-30). These results demonstrate that disruption of cell-to-cell contact during passage has a negative effect on neuronal maturation of Nurr1-induced DA cells.

The reduced length of TH + fibers in the P1 culture is not due to the loss of cellular processes during physical grinding. Similar levels of physical grinding for the ground cortex were applied at the time of preparation compared to passaged cells for P1 culture. As a result, no significant difference in cell morphology was observed for 6-24 hours after cell transplantation compared to P0 and P1 cultures (data not shown).

Molecular understanding of the differences observed between crushed and uncrushed P1 Nurr1-induced DA cells is provided. Gene expression gradients were made from neuroprecursors in P0, P1 single cells and P1 cluster cultures. The candidate marker tested, SHH expression, was maintained in P1 cluster culture, while completely lost in P1 single cells.

As shown in FIG. 3C, expression of P1 isolated culture with SHH (500 ng / ml) was found to increase the TH fiber length in Nurr1-induced DA cells (SHH treatment: 46.0 ± 3.6 mm, untreated control: 11.8 ± 1.3 mm, n = 45, p <0.001). Smo-M2 gene expression with persistently low SHH receptors was comparable with that of the 22 kD SHH N-terminal domain (Nurr1-IRES-SHH vector, NH, 43.3 ± 4.8 mm, n = 20, data Not shown).

Furthermore, as shown in FIG. 3B, neurite products of Nurr1-DA cells in P0 cultures were strongly inhibited upon treatment with the well characterized SHH inhibitor cyclopamine compared to the control (cyclopamine: 6.5 ± 3.0 mm; control: 76.0 ± 3.6 mm , n = 30, p <0.001). Treating the P0 culture with SHH or treating the P1 culture with cyclopamine did not induce a significant change in TH + fiber length compared to the untreated control culture (data not shown). This suggests that loss of endogenous SHH during passage adversely affects the differentiation of Nurr1-induced DA cells.

Example 3 Effect of Bcl-XL on Improved Differentiation of Nurr1-DA Cells in Neural Precursors Induced from Late Developmental Stages

The in vitro proliferation period was then tested to influence the morphological differences observed between P0 and P1 Nurr1-TH cells. An important factor affecting the differentiation of neural precursors is higher cell density promoted differentiation through soluble factors selected from the precursors.

The cells were then cultured at low density (4,000 cells / 10 cm culture, ~ 80 cells / cm ² ) and then transduced with Nurr1 retroviruses on day 2 and 5 of the proliferation (passage is not included in any culture). The paracrine effect was minimized. Analyzes were performed upon further 5 days of differentiation on days 2 and 5 of the transduced culture. At least 20 cell colonies (average size,> 1 mm) were observed per 10 cm dish.

Although the diameter of the colonies grew from 2-5 days of proliferation (doubled with diameter), the total cell density in the culture was very low due to paracrine factor interference. As shown in FIG. 4 a, in contrast to the P0 and P1 culture data, neurite products showed a longer proliferation period (5 days transduced cells: 6.6 ± 1.0 mm, 2 days transduced cells: 0.6 ± 0.1 mm, n = 20, p <0.001) increased in culture.

It was found that early fetal neural precursors broaden the in vitro early selection properties of precursors derived from late development. Thus, results on improved morphological maturation in the expanded precursors over in vitro prolongation may affect changes in progenitor phase. Therefore, it is intended to confirm that Nurr1 transduction of rat cortical (cortical) precursors isolated from various developmental periods (E12-E18) produces similar differences.

As shown in FIG. 4B, it was found that the neural precursors derived from the late developmental phase had higher cell differentiation rate compared to the early developmental phase (E18 P0 culture: 101.7 ± 5.2 mm E16 P0: 98.2 ± 3.0 mm E14 P0: 74.7 ± 8.4 mm E12 P0: 63.5 ± 5.1 mm, n = 35-40, p <0.001). These data are believed to be because neural progenitors influence the degree of neural differentiation in Nurr1-induced DA cells.

Anti-apoptotic proteins Bcl-2 and Bcl-XL are believed to be factors that enhance DA neuronal differentiation. Interestingly, increased Bcl-XL expression was increased in precursors isolated from late fetuses at stages associated with improved morphological maturation. We tested whether Bcl-XL expression enhances neuronal differentiation in Nurr1-DA cells (Nurr1-IRES-Bcl-XL vector, NB).

As shown in Figure 4c, it was confirmed that the transduced cells NB significantly increased neurite length compared to Nurr1-IRES-LacZ transduced cells (N) (NB: 62.0 ± 5.2 mm versus N: 10.3 ± 0.6 mm, n = 20-25, p <0.001).

Example 4 Various Actions of bHLH Factor in Nurr1-Induced DA Neuronal Differentiation

Neuronal bHLH transcription factors are believed to play an important role in morphological and functional maturation of cellular neurons after progenitor neuronal specificity and cell division. The present inventors confirmed that the known neurogenic effects of bHLH protein can overcome the limited neuronal contribution and maturation in Nurr1-induced DA cells.

As shown in Figure 9a, all bHLH protein showed a similar effect on the total neuronal differentiation evaluated by TuJ1, while there was a significant difference in the differentiation of Nurr1-induced DA cells. As shown in Figures 9B and 9C, expression of Nurr1-IRES-Mash1 retrovirus (NM) results in a significant increase in morphological differentiation of Nurr1-DA cells without affecting TH + cell yield (NM: 147.4 ± 50.9 mm versus N: 9.0 ± 1.8 μm).

As shown in FIG. 9A, co-expression of Ngn1 or Ngn2 belonging to the Nurr1 and atonal family causes almost complete loss of TH + cells. Among the total cells, Nurr1 + Ngn1 was 1.1 ± 0.6% and Nurr1 + Ngn2 was 2.8 ± 0.9%, whereas Nurr1 + LacZ (N) was 41.1 ± 2.0%.

These results indicate that bHLH protein has a remarkable effect on neuronal specificity and maturation, while Ngn1 and Ngn2 have inhibitory effects on DA neuronal specificity of Nurr1-induced neuronal precursors.

Example 5: Bcl-XL / SHH and Mash1 Regulate Differentiation of Nurr1-DA Cells, respectively>

SHH and Bcl-XL are Nurr1-DA cells (Nurr1-IRES-SHH-IRES-Bcl-XL, NHB) 82.5 ± 1.5 mm, NH: 38.0 ± 5.8 mm, NB: 62.2 ± 6.1 mm, n = 25-40) Has an additional effect on my neurite outgrowth. However, no effect was found when Mash1 was simultaneously expressed with Bcl-XL or SHH, or when Bcl-XL + SHH and Mash1 were simultaneously expressed in Nurr1 induced DA cells (data not shown).

Thus, it is inferred that the expression of Bcl-XL and SHH or the expression of Mash1 acts to regulate neuronal and dopaminergic differentiation in Nurr1 induced DA cells. To demonstrate this, we tested other factors, such as glial cell line-derived neurotrophic factor (GDNF), Wnt-5a, Pax2, DAT, and tissue inhibitor of metalloproteinase (TIMP), but did not find any effect. (Data not shown).

Example 6 Neuron and Dopaminergic Properties of Nurr1-Induced DA Cells

It was then observed whether morphological maturation was involved in the expression of the mature neuronal marker MAP2. % Quantification of Nurr1-induced DA cell expression MAP2 was performed by confirming morphological data on cell differentiation and the results are summarized in FIG. 5.

As shown in FIG. 5, TH + / MAP2 + cells among TH + cells were 67.5 ± 4.1% (NHB), 71.0 ± 2.4% (NM) and 11.8 ± 2.1% (N). The effects of NHB and NM on neurite outgrowth and maturation were largely related to the growth cone development (GAP43), neurite by-products (Rho8 and Scg10), and the transcripts associated with synapsin and synaptophysin.

DA neuronal function in front of NHB, NM or N transduced cell synapses was assessed by measuring in vitro DA release. HPLC analysis showed no detectable DA level at baseline level (15 min exposure to medium) but increased DA release upon cell sensitization (15 min exposure in medium containing 56 mM KCl). As shown in FIG. 6A, while all conditions showed KCl induced DA release, there was a significant quantitative difference between groups. DA level is NHB 2558.2 ± 152.4 pg / ml; NM: 3049.9 ± 377.6 pg / ml; And N: 561.2 ± 50.2 pg / ml. Differences in function in DA metabolism were further assessed by measuring DA's reuptake of DAT-mediated high affinity.

As shown in FIG. 6B, the resorption in Nurr1 culture was up to detection limit (N: 0.23 ± 0.01 fmol / min / well), while NHB and NM transplanted cells showed positive DA uptake: NHB: 4.59 ± 0.50 fmol / min / well; NM: 2.90 ± 0.61 fmol / min / well).

As shown in FIG. 6C, electrophysiological analysis of Nurr1-induced DA cells (NHB group) showed that well-developed sodium and potassium channels in cells with differentiated neuromorphisms (data not shown), action potentials with extended polarization current Potential action by injection, demonstrated the function of differentiated nerves.

In addition, as shown in FIG. 6D, as the intensity of polarization current increased, there was a time proportional decrease in membrane deflection, indicating metabolic and modified properties for nigrostriatum DA neurons. These cells also show current inward polarization, as shown in FIG. 6E, which can reproduce deformation in the current-clamp mode. As shown in Figures 6F-H, similar biochemical patterns can also be observed in cells infected with NM.

Example 7: In vitro of Nurr1-induced DA cell viability by Bcl-XL / SHH, and Mash1 in vitro Line Effect>

Survival of transplanted DA neurons is in preclinical studies in animal models of PD. The effect of this transduction on the viability of the precursor was observed.

% Cells with apoptotic nuclei were Bcl- compared to LacZ-transduced control cultures (11.3 ± 0.6%, N = 40 microscopy field, LacZ-transformed control in remarkably different 4 cover glass from p <0.001). It was reduced in cultures transduced with XL (4.2 ± 0.2%) or SHH (7.5 ± 0.6%).

We then tested the effects of Bcl-XL, SHH, and Mash1 on the survival of Nurr1-form introduced precursor cells. NHB transduced cells showed significant cell viability compared to Nurr1 alone (N) culture. Data are TH + cell total (A), cell total (B), LDH release (C),% cells with apoptotic nuclei (D),% TUNEL + cells (E) and cells positive for activated caspase 3 (F) As provided in FIG. 7.

As shown in Fig. 7, cell survival in culture transduced with Nurr1 + SHH + Bcl-XL (NHB) was more pronounced by SHH and Bcl-XL than NH or NB alone in cell survival. Presented. Coexpression of Mash1 in Nurr1-transformed cells (NM) also improved.

<Example 8: Fresh vegetable ( In vivo)  Transplant Research>

The final step in the experiment is to identify the ability, integration and function of Nurr1-induced DA cells for the in vivo survival of mice with Parkinson's disease.

Precursors transfected with N, NM and NHB were transplanted into the injured striatum of rats with unilateral 6-OHDA injuries. Histological analysis was performed at 4 and 8 weeks of transplantation. As shown in FIG. 8A, the NHB and NM transduced precursors showed a marked increase in the number of TH + cells surviving in the striatum relative to Nurr1 alone cells (N).

At 8 weeks of implantation, the average number of TH + cell / animal was NHB: 7339 ± 6 NM: 5758 ± 0 TH + cell / animal, N: 1461 ± 8 TH + cell / animal (N = 5 animals / group; p <0.05 for NHB and NM versus N). In addition to the cell survival effect, there was a significant difference in the morphological maturity of the transplanted TH + cells. NM- or NHB-induced TH + cells in grafts show mature neuronal morphology with multiple long processes that extend into host striatum, whereas N-transduced TH + cells are morphologically immature without significant neurites. It was. The graft was negative for proliferation marker PCNA without signs of tumor formation.

In vivo (In In vivo ) graft function was further assessed by DA level. HPLC analysis showed a significant increase in DA levels in NHB or NM grafts compared to N grafts: DA levels per tissue were NHB: 316.3 ± 8.6 mg / mg protein in graft, NM: 286.8 ± 2.5 mg / mg; N: 49.6 ± 4.7 mg / mg (N = 3 p <0.05 for both NHB and NM versus N).

Behavior analysis was performed specifying the amphetamine-induced rotational behavior (FIG. 8C) and the valdimdim test (FIG. 8D). Overall, animals transplanted with NM or NHB-transformed cells showed a marked increase in these parameters (see FIG. 8C, FIG. 8D, Tables 1 and 2 below). Nurr1 alone (N) could not confirm significant behavioral recovery (see FIGS. 8C and 8D).

Amphetamines: Number of animals recovering> 60% at rotation week PBS, n = 8 N, n = 10 NHB, n = 25 NM, n = 15 2 0 (0) 1 (10) 0 4 (20) 4 1 (12) 1 (10) 6 (24) 4 (20) 6 1 (12) 1 (10) 9 (36) 5 (33) 8 1 (12) 1 (10) 9 (36) 5 (33)

% Of animals recovering> 60% in footsteps week PBS, n = 8 N, n = 10 NHB, n = 25 NM, n = 15 One 0 (0) 0 (0) 0 (0) 0 (0) 2 0 (0) 0 (0) 1 (4) 2 (13) 3 0 (0) 0 (0) 8 (32) 7 (47) 4 0 (0) 1 (10) 21 (84) 12 (80) 5 1 (12) 1 (10) 24 (96) 14 (93) 6 1 (12) 1 (10) 23 (92) 14 (93) 7 1 (12) 1 (10) 23 (92) 15 (100) 8 1 (12) 1 (10) 24 (96) 14 (93)

These data suggest that SHH and BclXL expression or Mash1 expression enhances in vitro and in vivo functions of Nurr1-induced DA cells, and that genetic engineering may be an important strategy in the development of donor cell sources suitable for intracellular PD therapy. Can be.

As described above, dopaminergic neurons differentiated and proliferated from neural progenitor cells according to the method of the present invention are cell replacement therapy and gene therapy for treating cerebral nervous system diseases such as Parkinson's disease, Alzheimer's disease, stroke and ischemia. It can be widely used as a material for therapy or for drug research or various research in new drug development.

<110> Industry-Universoty Cooperation Foundation Hanyang University <120> A method for differentiation of neural precusor cells to matured          dopaminergic neuron <130> dp20060283 <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Otx1 <400> 1 gctgttcgca aagactcgct ac 22 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Otx1 <400> 2 atggctctgg cactgatacg gatg 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Emx2 <400> 3 ttcgaaccgc cttctcgccg 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Emx2 <400> 4 tgagccttct tcctctag 18 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Pax6 <400> 5 ccaaagtggt ggacaagatt gcc 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Pax 6 <400> 6 taactccgcc cattcactga cg 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for SHH <400> 7 ggaagatcac aaactccgaa c 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> backward primer for SHH <400> 8 ggatgcgagc tttggattca tag 23 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Smo <400> 9 tgctgtgtgc tgtctacatg cc 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Smo <400> 10 tcttggggtt gtctgtcctc ac 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Ptc <400> 11 ggcaagtttt tggttgtggg tc 22 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Ptc <400> 12 ccatgtaacc tgtctccgtg ataag 25 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Bcl-XL <400> 13 caagctttcc cagaaaggat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Bcl-XL <400> 14 tgaagagtga gcccagcaga 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Synapsin <400> 15 ccacccccac aaggccagca aca 23 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Synapsin <400> 16 ggtcccccgg cagcagcaat gatg 24 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Synaptophysin <400> 17 tggtatccta ccgcattc 18 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Synaptophysin <400> 18 actcacctca tagctcc 17 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAP43 <400> 19 agaaagcagc caagctgagg agg 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> backward primer for GAP43 <400> 20 caggagagac agggttcagg tgg 23 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Rho8 <400> 21 acgggaagca ggtagagttg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Rho8 <400> 22 gatgggcaca tttggacag 19 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Scg 10 <400> 23 ctacccggag cctcgcaac 19 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> backward primer for Scg 10 <400> 24 acctgggcct cctgagactt c 21 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 25 ggcattgctc tcattgacaa 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> backward primer for GAPDH <400> 26 agggcctctc tcttgctctc 20  

Claims (17)

Neuroprogenitor cells from the ventral midbrain, cortex or lateral ganglionic eminence (striated anlage) of rodent embryos, By simultaneously treating Nuclear Receptor related 1 (Nurr1), sonic hedgehog (SHH) and Bcl2-associated X protein long form (Bcl-XL), Obtaining dopamine neurons in vitro from the neural progenitor cells, The dopamine neuron has a 3 to 7 times increase in the total length of the fibers of the dopamine neuron compared to the early immature neurons in the number and length of dendritic dendritic morphology, and functionally A method for obtaining dopamine neurons, characterized in that it forms a synapse. delete delete delete The method of claim 1, wherein the neural progenitor cells are isolated from the cerebral cortex of rat embryos 12 days to 16 days old. The method of claim 1, wherein said treatment is performed by gene transfer or transduction. delete delete delete delete delete delete delete delete delete delete delete

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