WO2011159050A2 - 인간 신경전구세포의 도파민 뉴우런으로의 분화방법 및 분화용 배지 - Google Patents
인간 신경전구세포의 도파민 뉴우런으로의 분화방법 및 분화용 배지 Download PDFInfo
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Definitions
- the present invention relates to a method for differentiating human neural progenitor cells into dopamine neurons, and more particularly, to dopamine of human neural progenitor cells comprising culturing human neural progenitor cells in a medium containing fusaric acid. A method of differentiation into neurons.
- the present invention also relates to a medium for differentiation of human neuroprogenitor cells into dopamine neurons.
- Parkinson's disease is characterized by selective degeneration of dopaminergic neurons in the substantia nigra region of the midbrain.
- Various therapies to replace dopamine neurons in Parkinson's disease patients through transplantation of fetal midbrain tissue have been studied (see Document 1).
- Transplanted fetal midbrain tissues survive long-term survival in the human brain, repairing dopamine neuronal distribution in the patient's striatum, and reducing motor symptoms following Parkinson's disease (see Document 2).
- Such a transplant may be effective for the treatment of Parkinson's disease, but its clinical application is limited to very few cases because it is difficult to obtain large amounts of abortion fetal tissues in humans.
- various cells have been studied as donor cell candidates for transplantation treatment of Parkinson's disease (see Document 3).
- hNPCs human neural progenitor cells derived from fetal midbrain tissue are cells for transplantation because they have long-term proliferative activity and have excellent self-proliferative ability and can differentiate into dopamine neurons. Applicability as a cell source is expected (refer documents 4 and 5).
- Parkinson's disease through dopamine neuron substitution (ie, transplantation)
- hNPCs As a method for differentiating hNPCs into dopamine neurons, a method of differentiating for 3 days by adding ascorbic acid and dibutyryl cyclic adenosine monophosphate (db-cAMP) to the medium (see Document 6); A method of differentiating for 3 weeks by adding Brain-derived neurotrophic factor (BDNF), dopamine, and Forskolin to the medium (see Document 7); And a method of differentiating with SHH (sonic hedgehog), fibroblast growth facter-8 (FGF-8), brain-derived neurotrophic factor (BDNF), and ascorbic acid for 3 weeks (see Document 8).
- BDNF Brain-derived neurotrophic factor
- FGF-8 fibroblast growth facter-8
- BDNF brain-derived neurotrophic factor
- the conventional differentiation method is not yet satisfactory in the differentiation efficiency, it requires a long time for differentiation, economically difficult due to the use of a medium containing a large amount of expensive additives such as SHH and FGF-8, and Parkinson's disease
- a medium containing a large amount of expensive additives such as SHH and FGF-8, and Parkinson's disease
- Parkinson's disease There are many limitations for clinical use due to the lack of technology to multiply the large number of cells needed to treat a patient.
- the present invention provides a method for differentiating human neural progenitor cells (hNPCs) into dopamine neurons with high differentiation efficiency and a medium for differentiation which can be usefully used for the differentiation.
- hNPCs human neural progenitor cells
- the present invention provides a method for effectively differentiating hNPCs into dopamine neurons using fusaric acid as a novel differentiation inducing agent, and a medium for differentiation which can be useful for the differentiation.
- an object of the present invention is to provide a method for differentiating human neuroprogenitor cells into dopamine neurons in a medium containing a new differentiation inducing agent.
- a method for differentiating human neural progenitor cells into dopamine neurons comprising culturing human neural progenitor cells in a medium containing fusaric acid.
- the medium is dibutyryl cyclic adenosine monophosphate (db-cAMP), forskolin, B27, SHH (sonic hedgehog) and FGF8 (fibroblast growth factor 8). It can be configured by adding fusarinic acid to the dopamine neuron differentiation medium containing a).
- db-cAMP dibutyryl cyclic adenosine monophosphate
- forskolin forskolin
- B27 sonic hedgehog
- FGF8 fibroblast growth factor 8
- the medium may be NB medium containing fusarinic acid, db-cAMP, forskolin, and B27, preferably 50uM to 4mM of fusarinic acid, 50uM to 4mM N-B medium containing db-cAMP, 5-20 uM of forskolin, and 0.5-5% by weight of B27.
- the medium may be an NB medium comprising 100 uM of fusaric acid, 100 uM of db-cAMP, 10 uM of forskolin, and 2% by weight of B27.
- the culturing may be preferably performed under low oxygen partial pressure conditions of 2-10% oxygen partial pressure.
- a medium for differentiation of human neuroprogenitor cells into dopamine neurons there is provided a medium for differentiation, wherein the medium is NB medium containing fusarinic acid, db-cAMP, forskolin, and B27. .
- the differentiation medium may be an NB medium containing 50 uM to 4 mM fusarinic acid, 50 uM to 4 mM db-cAMP, 5 to 20 uM forskolin, and 0.5 to 5 wt% of B27, preferably 100 uM It can be NB medium containing fusarinic acid of, 100uM db-cAMP, 10uM of forskolin, 2% by weight of B27.
- the differentiation method of the present invention can be usefully applied to the production of dopamine neurons for the treatment of neurological disorders including Parkinson's disease.
- Figure 1 shows the results of induction of differentiation into dopamine neurons in various media compositions, and then measured TH and Tuji through DAPI staining.
- Fig. 2 shows the results of measuring the expression levels of TH and Tuji after proliferating hNPCs, inducing differentiation into neurons with or without adding 100 uM of fusaric acid in the medium.
- 3 shows the results of proliferating hNPCs and inducing differentiation into neurons with or without adding 100 uM of fusaric acid in the medium, followed by immunostaining and RT-PCR analysis.
- Figure 4 shows the result of measuring the TH expression of the differentiated cells after inducing differentiation into dopamine neurons in a medium of different types of differentiation inducing agents.
- Figure 6 shows the results of measuring the effect of fusarinic acid when inducing differentiation in the presence of MPP.
- Fig. 7 shows the expression of TH and Tuji after the proliferation and passage of hNPCs to induce differentiation from individual hNPCs obtained in the early (seventh), middle (11th) and late (17th). The result of the measurement is shown.
- Figure 8 shows the proliferation and passage of hNPCs to induce differentiation from each hNPCs obtained in the early (stage 7), middle (11th passage), late (stage 17), and then immunostaining for each cell.
- TH indicates the result of measuring expression.
- FIG. 9 shows the neural stem cell markers nestin, which are differentiated from the hNPCs obtained in the early (seventh), middle (11th), and late (17th) by proliferating and subcultured hNPCs.
- Ki67 a proliferative cell marker
- Tuj1 a mature neuron marker
- FIG. 10 shows the results of FACS analysis before and after differentiation from induction of differentiation from early (stage 7) hNPCs.
- Figure 11 shows the proliferation and passage of hNPCs to induce differentiation from each hNPCs obtained in the early (stage 7), middle (11th passage), late (stage 17), RT-PCR for the obtained cells And Western blot analysis is shown.
- Figure 12 shows the results of evaluation through immunostaining the characteristics of the cells obtained by inducing differentiation according to the differentiation method of the present invention.
- Figure 13 shows the results of evaluation of the characteristics of the cells obtained by inducing differentiation according to the differentiation method of the present invention through RT-PCR, Western blot analysis, and immunostaining.
- the present invention provides a method for differentiating human neural progenitor cells into dopamine neurons, comprising culturing human neural progenitor cells in a medium containing fusaric acid.
- human neural progenitor cells have been previously reported in methods such as Storch et al. 2001; Milosevic et al. Can be obtained according to the method reported in 2006, 2007, etc.
- human neural progenitor cells refers to human neural progenitor cells which are separated from the human body, that is, separated into the body and proliferated by conventional cell culture.
- the human neural progenitor cells may have stem cell characteristics and may be differentiated into neurons, astrocytes, and oligodendrocytes.
- Differentiation method according to the present invention is carried out using a medium containing fusaric acid as the differentiation inducing agent.
- the fusarinic acid has 5-butylpicolinic acid (5-butylpicolinic acid) has the structure of formula (1).
- Fusaric acid is a known substance and can be prepared by a known method or purchased commercially (for example, Sigma-Aldrich, etc.).
- the medium may be configured by adding fusarinic acid to the conventional dopamine neuron differentiation medium.
- dopamine including the conventional dibutyryl cyclic adenosine monophosphate (db-cAMP), forskolin, B27, sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8)
- db-cAMP dibutyryl cyclic adenosine monophosphate
- SHH sonic hedgehog
- FGF8 fibroblast growth factor 8
- db-cAMP, forskolin, B27, SHH, FGF8 and the like are all commonly used materials, can be purchased commercially, for example db-cAMP and forskolin can be purchased from Sigma, B27 is GIBCO (B-27 minus-AO supplement) and SHH and FGF8 are available from R & D system and PeproTech, respectively.
- the medium consisting of the addition of fusarinic acid to the conventional dopamine neuron differentiation medium is 50uM ⁇ 4mM db-cAMP, 5uM ⁇ 20uM phospholine, 0.5% ⁇ 5% B27, 25ng / ml ⁇ 500ng / It can be NB medium (Neurobasal media) containing ml SHH, FGF8 of 10ng / ml ⁇ 200ng / ml, and 50uM ⁇ 4mM fusarinic acid, NB medium is the basic medium can be purchased commercially from Invtorgen, etc. have.
- the medium used in the differentiation method of the present invention may be a medium excluding SHH and FGF8, which are limited in use at a high cost.
- a medium to which only fusarinic acid was added without SHH and FGF8 exhibited higher dopamine neuron differentiation efficiency compared to a medium containing all of SHH, FGF8, and fusarinic acid.
- the medium may be NB medium containing fusarinic acid, db-cAMP, forskolin, and B27, preferably 50uM to 4mM of fusarinic acid, 50uM to 4mM NB medium comprising db-cAMP, 5-20 uM of forskolin, and 0.5-5% by weight of B27. More preferably, the medium may be NB medium containing 50 uM to 1 mM fusarinic acid, 50 uM to 1 mM db-cAMP, 5 to 15 uM forskolin, and 0.5 to 3 wt% B27.
- the medium may be NB medium comprising about 100uM of fusaric acid, about 100uM db-cAMP, about 10uM of forskolin, about 2% by weight of B27.
- the medium may further include an antibiotic such as gentamicin or an amino acid such as L-glutamine.
- an antibiotic such as gentamicin or an amino acid such as L-glutamine.
- the antibiotics and amino acids can be appropriately replaced with other substances.
- the culturing may be performed using the culture conditions used in the conventional differentiation method.
- the culturing may be performed at about 37 ° C. for 7-14 days, preferably about 7 days. It has been found by the present invention that higher culture efficiency can be achieved when the culture is incubated under hypoxia (ie, hypoxia conditions). Therefore, in the differentiation method of the present invention, the culturing may be preferably carried out under low oxygen partial pressure conditions of 2 to 10% oxygen partial pressure.
- Dopamine neurons obtained by the differentiation method according to the present invention can be harvested by a conventional method, for example, after separating the cells using an enzyme such as Accutase (PAA), the obtained cells for about 5 minutes at 1000rpm By removing the supernatant by centrifugation, differentiated neural progenitor cells can be harvested.
- PAA Accutase
- the present invention also provides a medium for differentiation of human neuroprogenitor cells into dopamine neurons, wherein the medium is NB medium containing fusarinic acid, db-cAMP, forskolin, and B27.
- the differentiation medium may be configured by adding fusarinic acid to the conventional dopamine neuron differentiation medium as described above.
- the differentiation medium is NB medium containing 50 uM to 4 mM fusarinic acid, 50 uM to 4 mM db-cAMP, 5 to 20 uM forskolin, and 0.5 to 5 wt% B27, as described above. And, more preferably, NB medium containing 50 uM to 1 mM fusarinic acid, 50 uM to 1 mM db-cAMP, 5 to 15 uM forskolin, and 0.5 to 3 wt% B27.
- the medium may be NB medium comprising about 100 uM of fusaric acid, about 100 uM of db-cAMP, about 10 uM of forskolin, and about 2% by weight of B27.
- the medium may further include an antibiotic such as gentamicin or an amino acid such as L-glutamine.
- an antibiotic such as gentamicin or an amino acid such as L-glutamine.
- the antibiotics and amino acids can be appropriately replaced with other substances.
- Human neural progenitor cells were isolated from a 14-week-old fetus obtained from preterm birth due to maternal uterine myotonia. Prior to obtaining the fetal sample, informed consent was obtained from the parent of the fetus in advance and also approved by the Cha Hospital Institutional Research Ethics Committee for their collection and their use for research purposes.
- Human neuroprogenitor cells obtained above were inoculated in a single layer at a density of 30,000 cells / cm 2 in a culture dish coated with 15 ug / ml poly-L-ornithine and 4 ug / ml fibronectin.
- NB medium containing differentiation inducers such as 2% B-27 minus-AO supplement (GIBCO), 10 uM of forskolin, 1 mM db-cAMP and 1 mM fusarinic acid, 7 Incubated for days.
- Differentiated cells, or dopamine neurons were harvested as follows: Remove the culture medium from the culture vessel, wash with buffer, treat cells for 30 minutes with Accutase (PAA), separate cells from the culture dish, and then return to buffer Washed. The obtained cells were centrifuged at 1000 rpm for about 5 minutes to remove supernatant and harvested dopamine neurons induced differentiation.
- PAA Accutase
- Total cell RNA was extracted from hNPCs using triazole and chloroform.
- cDNA was synthesized using Superscript II RTase, Oligo-d (T) primers, DTT and dNTPs from 500 ng total RNA according to the manufacturer's instructions.
- PCR reactions were performed using a SYBR-Green SYBR-Green mixture in a final volume of 20 ⁇ l containing 1 ⁇ l of cDNA and 1 ⁇ l of 10 pM primer. Expression of TH, DAT, GFAP, Nurr1, Tuj1, and Lmx1a were analyzed using RPL22 as internal control.
- Quantitative real-time PCR was performed using the LightCycler System and the amplification was monitored and analyzed by measuring the binding of the fluorescent dye SYBR Green with double stranded DNA.
- Target DNA was amplified by performing a total of 40 cycles under conditions of 10 seconds at 95 ° C., 10 seconds at 60 ° C., and 20 seconds at 72 ° C., and a final extension was performed at 72 ° C. for 10 minutes. The results were shown relative to gene RPL22 by comparative Ct method. Primers used in the polymerase chain reaction are shown in Table 1 below.
- hNPCs were washed three times with PBS and fixed for 10 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it was blocked by reacting with PBS containing 3% Normal goat serum, 0.2% Triton X-100 and 1% BSA for 1 hour at room temperature.
- Anti-TH (rabbit-anti-TH, Pelfreez), Tuj1 (mouse anti-Tuj1 Millipore, CA.USA), nestin (rabbit anti-nestin, COVANCE, CA, USA), GFAP (mouse-anti-GFAP Millipore, CA) USA), Ki67 (mouse anti-Ki67, Leica), O4 (mouse-anti-O4, Millipore), Sox2 (rabbit- anti-Sox2, Abcam), VMAT2 (rabbit anti-VMAT2, Abcam), Pitx3 (rabbit anti -Pitx3, Millipore), DAT (rabbit anti-DAT, Santa cruze), Nurr1 (rabbit anti-Nurr1, Santa cruze), NeuN (mouse-anti-NeuN, Millipore), GIRK2 (rabbit-anti-GIRK2, Alomone lab) , Cal28K (mouse-anti-Cal28K, Sigma), Glutamate (rabbit-anti-Glutamate, Sigma), GABA (
- mice (Alexa Fluor TM 594), anti-rabbit (Alexa Fluor TM 488), anti-rabbit (Alexa Fluor TM 594) and incubated, and stained with DAPI (4'-6-Diamidino- 2-phenylindole) with (co understaining).
- DAPI 4,6-Diamidino- 2-phenylindole
- Proteins were extracted with RIPA buffer [10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2, 0.1% NP-40] containing Proteinase inhibitor (PI) (Roche Molecular Biochemicals). Protein concentration was measured using the BCA method (Pierce), and proteins were separated on SDS-10% polyacrylamide gels and then transferred to PVDF membranes.
- RIPA buffer 10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2, 0.1% NP-40
- PI Proteinase inhibitor
- the membrane was blocked for 2 hours at room temperature with 5% skim milk in TBS-T buffer, followed by anti-TH (rabbit-anti-TH, Pelfreez, 1: 1000), Tuj1 (rabbit anti-Tuj1 COVANCE, 1: 5000), Nestin (rabbit anti-Nestin, Abcam, 1: 1000), Sox2 (rabbit- anti-Sox2, Abcam, 1: 1000), Bcl2 (mouse anti-Bcl2, Santa cruze, 1: 200), PCNA (mouse anti-PCNA, Santa cruze, 1: 1000), VMAT2 (rabbit anti-VMAT2, Abcam, 1: 1000), Pitx3 (rabbit anti-Pitx3, Millipore, 1: 2000), DAT (rabbit anti-DAT, Santa cruze, 1: 200), Nurr1 (rabbit anti-Nurr1, Santa cruze, 1: 250), Actin (rabbit anti-Actin, Santa cruze, 1: 5000) incubated overnight at 4 with primary antibody. The membrane was incubated
- Interleukin 1 beta, db-cAMP, and fusaric acid (Fus) were added to Neurobasal medium [Differentiation medium (DM) control] at various concentrations and combinations to induce differentiation into dopamine neurons by adding hNPCs to the medium.
- TH and the neuronal marker Tuji known as a marker of dopamine neurons, were measured by DAPI staining. From the results of FIG. 1, it can be seen that the differentiation rate is highest when the media containing db-cAMP and fusarinic acid are added together with the highest expression levels of TH and Tuji, respectively, when db-cAMP and fusarinic acid are 100 uM, respectively. It can be seen that a high differentiation rate can be achieved.
- HNPCs were propagated by adding hNPCs in DMEM / F12 (1: 1) Glutamax medium, and again 2% B-27 minus-AO supplement (GIBCO), 10 uM forskolin, 1 mM db- in Neurobasal (NB) medium.
- Glutamax medium Glutamax medium
- NB Neurobasal
- cAMP B-27 minus-AO supplement
- differentiation was induced by neurons with or without adding 100 uM of fusaric acid.
- the expression levels of TH and Tuj1 were measured as shown in FIG. 2, and the results of immunostaining chemical analysis and RT-PCR are shown in FIG. 3. From the results of FIGS. 2 and 3, it can be seen that when fusarinic acid (FA) was added, TH, a marker of dopamine neurons, was much expressed, and Tuj1, a neuronal marker, was also increased.
- FA fusarinic acid
- hNPCs were propagated by adding hNPCs in DMEM / F12 (1: 1) Glutamax medium, and 2% B27, 200ng / ml SHH TH expression was measured by inducing differentiation in Neurobasal (NB) medium (Invitrogen) using, 25 ng / ml FGF8, 10 uM of forskolin, 100 uM of fusaric acid, and 100 uM of db-cAMP. Same as FIG. 4. From the results of FIG. 4, it can be seen that the treatment of fusarinic acid instead of SHH and FGF8 was most effectively differentiated into dopamine neurons. In addition, the addition of fusaric acid in addition to SHH and FGF8 further increased the differentiation into dopamine neurons.
- HNPCs were propagated by adding hNPCs in DMEM / F12 (1: 1) Glutamax medium, Neurobasal (NB) medium containing 2% of B27, 10 uM of forskolin, 100 uM of fusarinic acid, and 100 uM of db-cAMP (Invitrogen) G)
- hNPCs induce differentiation under oxygen partial pressure condition (Hypoxia condition) and 21% oxygen partial pressure condition (Normoxia condition) of 3%
- the TH expression is measured as shown in FIG. 5, it can be seen that differentiation into dopamine neurons is more efficient when differentiation is induced under hypoxia (Hypoxia condition) than when differentiation is induced under normal oxygen condition (Normoxia condition).
- MPP 1-methyl-4-phenylpyridium
- HNPCs were added to DMEM / F12 (1: 1) Glutamax medium to propagate hNPCs, and the culture medium was changed every 2 days and continuously passaged at 3% oxygen partial pressure.
- HNPCs were added to NB medium (Invitrogen), followed by incubation for 7 days at 3% oxygen partial pressure to induce differentiation.
- Tuj1 positive cells before differentiation were increased from about 10% to 40-45% after differentiation, and there was no change in late passage (see FIG. 7B).
- the results were the same in the immunostaining results for TH-positive cells, and differentiation efficiency increased further with later passages (see FIG. 8).
- the expression of nestin and ki67 decreased, whereas Tuj1 expression increased (see FIG. 9). This indicates that stem cell characteristics decrease as the differentiation progresses, and at the same time, the differentiated cells gradually differentiate and increase mature neurons.
- hNPCs were added to NB medium (Invitrogen) containing 2% B27, 10 uM forskolin, 100 uM fusarinic acid, and 100 uM db-cAMP, followed by incubation for 14 days at 3% oxygen partial pressure. Differentiation was induced.
- the expression of TH from the obtained cells and the expression of the mature dopamine neuron markers NeuN, VMAT2, Nurr1, and Pitx3 are shown in FIG. 12A, and the TH expressed is A9 or A10.
- the type of Girk2 and cal28K expression was measured by immunostaining results are shown in FIG.
- TH When TH is simultaneously expressed in cells expressing Girk2, it may be regarded as A10 type when TH is simultaneously expressed in cells expressing cal28K. 12, it can be seen that the dopamine neurons obtained according to the differentiation method of the present invention are A9 type neurons as mature dopamine neurons.
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Abstract
Description
유전자명 | 프라이머 | 서열 | 서열번호 | product size |
TH | 센스 프라이머 | 5'-agccctaccaagaccagacg-3' | 1 | 132bp |
안티센스 프라이머 | 5'-gcgtgtacgggtcgaactt-3' | 2 | ||
Tuj1 | 센스 프라이머 | 5'-gggcctttggacatctcttc-3' | 3 | 90bp |
안티센스 프라이머 | 5'-cctccgtgtagtgacccttg -3' | 4 | ||
Nestin | 센스 프라이머 | 5'-cagctggcgcacctcaagatg-3' | 5 | 208bp |
안티센스 프라이머 | 5'-agggaagttgggctcaggactgg-3' | 6 | ||
Sox2 | 센스 프라이머 | 5'-gccgagtggaaacttttgtc-3' | 7 | 264bp |
안티센스 프라이머 | 5'-gttcatgtgcgcgtaactgt-3' | 8 | ||
Musashi1 | 센스 프라이머 | 5'-acagcccaagatggtgactc-3' | 9 | 191bp |
안티센스 프라이머 | 5'-ccacgatgtcctcactctca-3' | 10 | ||
Lmx1a | 센스 프라이머 | 5'-tgcttagcccaggactttca-3' | 11 | 136bp |
안티센스 프라이머 | 5'-tgaagatggagggagagctg-3' | 12 | ||
PAX6 | 센스 프라이머 | 5'-ccaaagtggtggacaagattgcc-3' | 13 | 419bp |
안티센스 프라이머 | 5'-taactccgcccattcactgacg-3' | 14 | ||
VMAT2 | 센스 프라이머 | 5'-atccagaccaccagaccagag-3' | 15 | 616bp |
안티센스 프라이머 | 5'-ccccatccaagagcaccaagg-3' | 16 | ||
Pitx3 | 센스 프라이머 | 5'-tgtcattctcagatgcaggcac-3' | 17 | 400bp |
안티센스 프라이머 | 5'-tgaccgagttaaggcgaac-3' | 18 | ||
DAT | 센스 프라이머 | 5'-tgcgtgccacatcaataaca-3' | 19 | 170bp |
안티센스 프라이머 | 5'-aacatccttcactcagtattgctaa-3' | 20 | ||
Nurr1 | 센스 프라이머 | 5'-cgaccaagacctgctttttg-3' | 21 | 125bp |
안티센스 프라이머 | 5'-attgcaacctgtgcaagacc-3' | 22 | ||
GIRK2 | 센스 프라이머 | 5'-gggcaaacccttctcttctc-3' | 23 | 212bp |
안티센스 프라이머 | 5'-ggcactttgcactttcatca-3' | 24 | ||
Lmx1a | 센스 프라이머 | 5'-tgcttagcccaggactttca-3' | 25 | 136bp |
안티센스 프라이머 | 5'-tgaagatggagggagagctg-3' | 26 | ||
RPL22 | 센스 프라이머 | 5'-cacgaaggaggagtgactgg-3' | 27 | 116bp |
안티센스 프라이머 | 5'-tgtggcacaccactgacatt-3' | 28 |
Claims (9)
- 인간 신경전구세포를 푸사린산(fusaric acid)을 포함하는 배지 중에서 배양하는 것을 포함하는, 인간 신경전구세포의 도파민 뉴우런으로의 분화방법.
- 제1항에 있어서, 상기 배지가 디부티릴 시클릭 아데노신 모노포스페이트(dibutyryl cyclic adenosine monophosphate, db-cAMP), 포스콜린(forskolin), B27, SHH(sonic hedgehog) 및 FGF8(fibroblast growth factor 8)을 포함하는 도파민 뉴우런 분화용 배지에 푸사린산을 가하여 이루어진 것임을 특징으로 하는 분화방법.
- 제1항에 있어서, 상기 배지가 푸사린산, db-cAMP, 포스콜린, 및 B27을 포함하는 NB 배지인 것을 특징으로 하는 분화방법.
- 제3항에 있어서, 상기 배지가 50uM ∼ 4mM의 푸사린산, 50uM ∼ 4mM의 db-cAMP, 5∼20 uM의 포스콜린, 및 0.5∼5 중량%의 B27을 포함하는 NB 배지인 것을 특징으로 하는 분화방법.
- 제3항에 있어서, 상기 배지가 100uM의 푸사린산, 100uM의 db-cAMP, 10uM의 포스콜린, 2 중량%의 B27을 포함하는 NB 배지인 것을 특징으로 하는 분화방법.
- 제1항 내지 제5항 중 어느 한 항에 있어서, 상기 배양이 산소분압 2∼10 %의 저산소 분압 조건에서 수행되는 것을 특징으로 하는 분화방법.
- 인간 신경전구세포의 도파민 뉴우런으로의 분화용 배지로서, 상기 배지가 푸사린산, db-cAMP, 포스콜린, 및 B27을 포함하는 NB 배지인 분화용 배지.
- 제7항에 있어서, 상기 배지가 50uM ∼ 4mM의 푸사린산, 50uM ∼ 4mM의 db-cAMP, 5∼20 uM의 포스콜린, 및 0.5∼5 중량%의 B27을 포함하는 NB 배지인 것을 특징으로 하는 분화용 배지.
- 제8항에 있어서, 상기 배지가 100uM의 푸사린산, 100uM의 db-cAMP, 10uM의 포스콜린, 2 중량%의 B27을 포함하는 NB 배지인 것을 특징으로 하는 분화용 배지.
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