The authors thank members of the Renee Reijo Pera laboratory for help during development of this protocol and during preparation of this manuscript. This work is supported by California Institute for Regenerative Medicine (CIRM) shared laboratory (CL-00518).

문화 미디어의 1 준비 <ol><li> 다음을 조합하여 마우스 배아 섬유 아세포 (MEF) 배지를 준비 : 445 ml의 DMEM, 50 ml의 소 태아 혈청 (FBS) 및 5 ㎖의 100X 페니실린 / 암피실린 원액. 이하 십사일 4 ° C에서 필터 살균 매체를 보관하십시오. </li><li> 준비 혈청 함유 다음 조합함으로써 hPSC 배지를 : 385 ml의 DMEM / F12, 100 ㎖ 녹아웃 혈청 여분 (KSR), 5 ㎖ 100X 비 필수 아미노산 원액, 5 ㎖ 100X 페니실린 / 암피실린 원액, 5 ㎖ 100X를 원액 메르 캅토 에탄올, 10 NG / ㎖의 bFGF. 더 이상 10 일 동안 4 ° C에서 필터 살균 매체를 보관하십시오. </li><li> 다음을 조합하여 mTeSR1의 무 혈청 배지를 제조 : 400 ml의 mTeSR1 기본 배지 100 ㎖의 5 배 보충, 및 5 ㎖ 100X 페니실린 / 암피실린 원액. 더 이상 10 일 동안 4 ° C에서 매체를 보관하십시오. </li><li> 10 배 콜라겐 IV 주식 솔루션을 준비 : 0.5 g 콜라게나 제 IV 전원을 달아50 ml의 DMEM / F12 및 필터 소독 그것을 녹인다. 분취 량을 확인하고 달 동안 C ° -20을 재고. </li><li> 0.1 % 젤라틴 용액을 준비 : 전원 (소 피부, B 형에서) 0.5 g 젤라틴을 달아 탈 물로 녹인다. 주 실온에서 오토 클레이브 멸균 솔루션을 유지합니다. </li><li> 다음을 조합하여 KSR 분화 배지를 제조 410 ㎖의 DMEM, 75 ml의 KSR을, 5 ㎖의 원액을 메르 캅토 에탄올 100X 비 필수 아미노산 원액, 5 ㎖ 100X 페니실린 / 암피실린 스톡 용액 5 ㎖를 100 배. 더 이상 10 일 동안 4 ° C에서 필터 살균 매체를 보관하십시오. 참고 : KSR은 분화 효율에 영향을 미칠 수있는 많은으로 많이 다릅니다. 그것은 차별화를위한 최선의 일을 찾기 위해 KSR의 여러 배치를 테스트하는 것이 좋습니다. </li><li> 다음을 결합하여 N2 차별화 매체를 준비합니다 : 98 ㎖의 DMEM, 1 ml의 100 배 N2 보충 및 1 ml의 100 배 페니실린 / 암피실린 원액을. 4에서 필터 살균 매체 유지없이 10 일 이상 C를 °. </li><li> 10 ㎖ 50X B27 보충제, 5 ㎖의 원액을 glutamax 100X, 100X 및 5 ㎖ 페니실린 / 암피실린 스톡 용액, 480 ml의 neurobasal 매체 : 다음을 조합하여 B27 분화 배지를 준비한다. 더 이상 10 일 동안 4 ° C에서 필터 살균 매체를 보관하십시오. </li></ol> MEF 피더 세포에 인간 배아 줄기 및 hiPSCs의 2 문화 H9의 인간 배아 줄기는 WiCell 연구소에서 얻은하고 hiPSCs는 야마나카의 레트로 바이러스 매개 형질 도입을 통해 Reijo 페라 실험실에서 설립되었다 요인 OCT3 / 4, SOX2, KLF4 및 C-MYC 18. <ol><li> 적어도 일일 세포 통로 전에, 6 - 웰 플레이트의 1도에 1 ㎖의 0.1 % 젤라틴을 추가하여 일부 젤라틴 판을 준비하고 적어도 30 분 동안 37 ° C에서 번호판을 품어. </li><li> 배양 플레이트에서 흡 젤라틴 및 시드 조사 후에는 1.6 x 10 5의 세포 밀도로 플레이트 상 MEF 세포를 불 활성화/ 웰 MEF 배지에서 세포를 세는 혈구를 사용. 주 : 선택적으로 빠르면 3 일 정도 세포 통로 전에 MEF 피더를 준비합니다. </li><li> 통로 세포 MEF 피더 도금 후 일일 : hPSCs에서 흡인 매체 잘 / 1 ML에서 세포에 1 ㎎ / ㎖ 콜라게나 제 IV를 추가하고, 1 시간 동안 37 ° C에서 세포를 배양한다. </li><li> 이 부화하는 동안, PBS로 한번 MEF 피더 세척 한 다음도 1.5 ㎖ /에서 따뜻한 (37 ° C) 혈청 함유 hPSC 문화 매체를 추가 할 수 있습니다. </li><li> 미분화 식민지의 대부분이 판에서 분리 수 있도록 차별화 된 식민지가 연결된 상태를 유지하면서 1 시간 후, 배양 접시에 몇 번을 누릅니다. 조심스럽게 떠 식민지를 수집하고, 15 ML 튜브로 전송합니다. </li><li> 부드럽게 따뜻한 (37 ° C) DMEM / F12 한 번 접시를 씻어 같은 15 ML 튜브로 DMEM / F12을 전송합니다. </li><li> 3 분 동안 179 XG에 튜브를 원심 분리기. </li><li> 펠렛을 방해하지 않도록주의 튜브에서 대기음 매체. 전쟁 추가m hPSC 배지, 작은 클러스터로 휴식과 잘 섞는다까지 여러 번 아래로 세포를 피펫. </li><li> 일에 새로운 MEF 피더에 세포를 전송 : 3-1 : 6 비율. </li><li> 모든 5~6일 매일 통로 세포 매체를 변경합니다. </li></ol> 차별화를위한 세포의 3 준비 <ol><li> 세포의 준비를하기 전에 적어도 하루에 일부 마트 리겔 플레이트 (6 웰 플레이트의 일반적으로 세 우물)을 준비합니다. 1시 40분 찬 (4 ° C) DMEM / F12와 마트 리젤을 희석하고 6 웰 플레이트 잘 당 1 ㎖의 마트 리젤을 추가합니다. 4 ° C에서의 하룻밤 마트 리겔 판을 품어. 참고 : 하나는 파라 필름과 매트 리겔 판을 밀봉 한 것과 일주일 동안 4 ° C에서 그들을 재고 할 수 있습니다. </li><li> 사일 통과 한 후 세포를 (2 단계 참조)를 사용합니다. 판의 모든 차별화 된 식민지를 제거합니다. 배양 접시에서 대기음 매체는 5 분 동안 37 ° C에서 세포를 잘 한 ML accutase 당 추가하고, 부화. </li><li> 배양 동안 일부 젤을 준비6 - 웰 플레이트에 1 ㎖ 젤라틴 1 내지도 추가하여 플레이트 ATIN. 이 판과 배양기에서 일일 전에 준비 마트 리겔 플레이트를 놓습니다. </li><li> 세포가 될 때 반 부동 5 분 배양 후 또는 피펫 세포 위아래로 여러 번 단일 세포로 만들 수 있습니다. </li><li> 3 분 동안 258 XG에 한 15 ml의 튜브에 세포와 원심 분리기를 수집합니다. </li><li> 재현 탁 된 hPSC 배지를 함유하는 혈청의 적절한 양의 세포, 및 2 μM의 최종 농도 Thiazovivin 추가. </li><li> 1에서 젤라틴 코팅 플레이트 상에 세포를 전송 : 30 분 동안 37 ° C에서 1 비율 및 배양 세포는 MEF 피더를 제거합니다. </li><li> 15 ML 원뿔 튜브에 세포를 전송, hPSC 매체를 포함하는 적절한 혈청을 추가하고 혈구와 세포를 계산합니다. </li><li> 3 분 동안 258 XG에서 세포를 원심 분리기. </li><li> 원심 분리기 중에 2 μM의 농도를 만들기 위해 mTeSR1 매체 처분시 Thiazovivin 추가. </li><li> 원심 분리기, 후매체를 spirate 및 / ㎖ 매체 1.8 × 10 5 세포가되도록 Thiazovivin와 적절한 mTeSR1 매체를 추가 할 수 있습니다. </li><li> , 인큐베이터에서 마트 리젤 판을 꺼내 마트 리젤을 대기음 6 웰 플레이트의 일에 잘 혼합 세포 2 ㎖를 추가합니다. NOTE : 세포 밀도가 3.6 × 104 세포 / 표면적이 cm이어야한다. </li><li> 인큐베이터에 번호판을 반환하고, 세포를 24 시간 동안 부착 할 수 있습니다. </li><li> 24 시간 세포를 도금 후, 오래된 매체를 대기음 잘 당 2 ㎖의에게 새로운 mTeSR1 매체를 추가하고 분화를 시작하기 전에 세포가 다른 24 시간 동안 성장 할 수 있습니다. </li></ol> 세포 분화 <ol><li> 마트 리겔 플레이트에 세포를 replating 후 분화 48 시간을 시작합니다. </li><li> 플레이트에서 기음 문화 매체는 PBS로 한 번 세포를 씻어 잘 당 다음과 같은 분화 배지 2 ㎖를 추가 : 10 μM SB431542 및 100 nm의 LDN-193 보충 KSR 차별화 매체189 마크 분화 D0로이 일. </li><li> D20에 D0에서 매일 매체를 변경합니다. 참고 : 한 번에 이일보다 더 사용하기 위해 미디어를 준비 할 수 있습니다. </li><li> 10 μM SB431542, 100 nm의 LDN-193189, 0.25 μM SAG, 2 μM의 purmorphamine, 50 NG / ㎖ FGF8b 보충 KSR 차별화 매체 : D1과 D2에 대한 다음과 같은 매체를 사용합니다. </li><li> 10 μM SB431542, 100 nm의 LDN-193189, 0.25 μM SAG, 2 μM의 purmorphamine, 50 NG / ㎖ FGF8b, 3 μM의 CHIR99021 보충 KSR 차별화 매체 : D3와 D4에 대한 다음과 같은 매체를 사용합니다. </li><li> 100 nm의 LDN-193189, 0.25 μM SAG, 2 μM의 purmorphamine, 50 NG / ㎖ FGF8b, 3 μM C​​HIR99021 보충 75 % KSR 차별화 매체 플러스 25 % N2 차별화 매체 : D5 및 D6에 대한 다음과 같은 매체를 사용합니다. </li><li> 100 nm의 LDN-193189와 3 μM C​​HIR99021 보충 50 % KSR 차별화 매체 플러스 50 % N2 차별화 매체 : D7과 D8에 대한 다음과 같은 매체를 사용합니다.</li><li> 100 nm의 LDN-193189와 3 μM의 CHIR99021 보충 25 % KSR 차별화 매체 플러스 75 % N2 차별화 매체 : 다음 D9에 대한 중간 및 D10을 사용합니다. </li><li> 3 μM C​​HIR99021, 10 NG / ml의 BDNF, 10 NG / ㎖ GDNF, 1 NG / ㎖ TGF3, 0.2 MM의 아스 코르 빈산 및 0.1 mM의 캠프로 보충 B27 차별화 매체 : D11과 D12에 대해 다음 매체를 사용합니다. </li><li> 10 NG / ml의 BDNF, 10 NG / ㎖ GDNF, 1 NG / ㎖ TGF3, 0.2 MM의 아스 코르 빈산 및 0.1 mM의 캠프로 보충 B27 차별화 매체 : 차별화의 나머지 부분에 대한 다음과 같은 매체를 사용합니다. </li><li> 분화에 대한 D16에서 일부 폴리-L-오르니 틴 / 라미닌 / 피브로넥틴 판을 준비합니다. 폴리-L-오르니 틴 감기 (4 ° C에서)와 원액을 15 ㎍ / ml의 PBS가, 하룻밤 37 ° C에서 접시를 6 웰 플레이트의 1도에 1 mL를 넣고 배양 희석. </li><li> , 폴리-L-오르니 틴 솔루션을 기음 판에게 멸균 수로 3 회 세척 한 후 공기가 판을 건조. </li><li> 라미닌 재고 졸을 희석1 ㎍ / ㎖의 감기 (4 ° C) PBS와의 ution는 6 - 웰 플레이트 잘 하나에 1 mL를 넣고 하룻밤 37 ° C에서 번호판을 품어. </li><li> , 라미닌 솔루션을 기음 판에게 멸균 수로 3 회 세척 한 후 공기가 판을 건조. </li><li> , 2 ㎍ / ㎖의 감기 (4 ° C) PBS와 피브로넥틴 원액을 희석 6 - 웰 플레이트의 1도에 1 mL를 넣고 하룻밤 37 ° C에서 번호판을 품어. </li><li> 파라 필름으로 밀봉 판과 최대 2 주부터 4 ° C에 배치합니다. </li><li> 분화 20 일 후, 폴리-L-오르니 틴 / 라미닌 / 피브로넥틴 플레이트에 세포를 replate. 기음 차별화 매체는, 6 웰 플레이트의 1도에 1 ml의 따뜻한 (37 ° C) accutase을 추가하고 5 분 동안 37 ° C에서 세포를 배양한다. </li><li> 배양 동안 인큐베이터에 폴리-L-오르니 틴 / 라미닌 / 피브로넥틴이 판을 배치합니다. </li><li> 5 분간 배양 한 후, 또는 세포가 될 때까지 아래로 수회 부드럽게 피펫 세포 세미 부동하나의 세포로합니다. </li><li> 15 ml의 원추형 튜브에 단일 세포를 수집하고, 팽창에 따라 달라질 셀 카운팅 neurobasal 매체의 적당량을 추가 (분화 11 일 후, 세포를 15 ~ 20 배로 확대있다). 혈구를 사용하여 세포를 카운트. </li><li> 3 분 동안 258 XG에서 세포를 원심 분리기. B27 분화 배지로 기음 상청액, 세포를 재현 탁하고 세포 농도 / ㎖ 배지 1 × 10 6 세포가되도록. </li><li> 접시에서 대기음 피브로넥틴은 PBS로 두 번 세척하고, 6 - 웰 플레이트의 한 웰에 2 ㎖의 세포를 추가합니다. NOTE : replating 대한 세포 밀도가 2 × 105 세포 / 표면적이 cm이어야한다. </li><li> 모든 부착되지 않은 죽은 세포를 제거하기 위해 24 시간 후 매체 변경합니다. </li><li> 주어진 실험에 대해 원하는 시점까지 매일 매체를 변경합니다. </li></ol>

<ol>
	<li>Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Will+embryonic+stem+cells+be+a+useful+source+of+dopamine+neurons+for+transplant+into+patients+with+Parkinson's+disease.">Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson's disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 1755-1757 (2002).</li><li>Perrier, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+midbrain+dopamine+neurons+from+human+embryonic+stem+cells.">Derivation of midbrain dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 12543-12548 (2004).</li><li>Park, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+analyses+of+human+embryonic+stem+cell-derived+dopamine+neurons.">In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.</a> Journal of Neurochemistry. 92, 1265-1276 (2005).</li><li>Buytaert-Hoefen, K. A., Alvarez, E., Freed, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tyrosine+hydroxylase+positive+neurons+from+human+embryonic+stem+cells+after+coculture+with+cellular+substrates+and+exposure+to+GDNF.">Generation of tyrosine hydroxylase positive neurons from human embryonic stem cells after coculture with cellular substrates and exposure to GDNF.</a> Stem Cells. 22, 669-674 (2004).</li><li>Zeng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+differentiation+of+human+embryonic+stem+cells.">Dopaminergic differentiation of human embryonic stem cells.</a> Stem Cells. 22, 925-940 (2004).</li><li>Yan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+dopaminergic+neuronal+subtypes+from+human+embryonic+stem+cells.">Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells.</a> Stem Cells. 23, 781-790 (2005).</li><li>Schulz, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+to+dopaminergic+neurons+in+serum-free+suspension+culture.">Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.</a> Stem Cells. 22, 1218-1238 (2004).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+dopaminergic+neurons+in+vitro+from+human+embryonic+stem+cells+treated+with+neurotrophic+factors.">Generation of dopaminergic neurons in vitro from human embryonic stem cells treated with neurotrophic factors.</a> Neuroscience Letters. 359, 99-103 (2004).</li><li>Cho, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 3392-3397 (2008).</li><li>Cooper, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+ES+and+Parkinson's+disease+iPS+cells+into+ventral+midbrain+dopaminergic+neurons+requires+a+high+activity+form+of+SHH,+FGF8a+and+specific+regionalization+by+retinoic+acid.">Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.</a> Molecular and Cellular Neurosciences. 45, 258-266 (2010).</li><li>Chambers, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+neural+conversion+of+human+ES+and+iPS+cells+by+dual+inhibition+of+SMAD+signaling.">Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.</a> Nature Biotechnology. 27, 275-280 (2009).</li><li>Sanchez-Danes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+A9+midbrain+dopaminergic+neurons+by+lentiviral+delivery+of+LMX1A+in+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells.">Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem cells and induced pluripotent stem cells.</a> Human Gene Therapy. 23, 56-69 (2012).</li><li>Fasano, C. A., Chambers, S. M., Lee, G., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+derivation+of+functional+floor+plate+tissue+from+human+embryonic+stem+cells.">Efficient derivation of functional floor plate tissue from human embryonic stem cells.</a> Cell Stem Cell. 6, 336-347 (2010).</li><li>Kriks, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+neurons+derived+from+human+ES+cells+efficiently+engraft+in+animal+models+of+Parkinson's+disease.">Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.</a> Nature. 480, 547-551 (2011).</li><li>Xi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+midbrain+dopamine+neurons+from+primate+pluripotent+stem+cells.">Specification of midbrain dopamine neurons from primate pluripotent stem cells.</a> Stem Cells. 30, 1655-1663 (2012).</li><li>Kirkeby, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+regionally+specified+neural+progenitors+and+functional+neurons+from+human+embryonic+stem+cells+under+defined+conditions.">Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.</a> Cell Reports. 1, 703-714 (2012).</li><li>Mendez, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+analysis+of+functional+fetal+dopamine+cell+suspension+transplants+in+the+striatum+and+substantia+nigra+of+patients+with+Parkinson's+disease.">Cell type analysis of functional fetal dopamine cell suspension transplants in the striatum and substantia nigra of patients with Parkinson's disease.</a> Brain: a Journal of Neurology. 128, 1498-1510 (2005).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Nishimura, K., Takahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+application+of+stem+cell+technology+toward+the+treatment+of+Parkinson's+disease.">Therapeutic application of stem cell technology toward the treatment of Parkinson's disease.</a> Biological &amp; Pharmaceutical Bulletin. 36, 171-175 (2013).</li><li>Smidt, M. P., Burbach, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+make+a+mesodiencephalic+dopaminergic+neuron.">How to make a mesodiencephalic dopaminergic neuron.</a> Nature Reviews. Neuroscience. 8, 21-32 (2007).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin+and+bFGF+cooperate+to+maintain+the+pluripotency+of+human+embryonic+stem+cells+in+the+absence+of+feeder+layers.">Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers.</a> Biochemical and Biophysical Research Communications. 330, 934-942 (2005).</li><li>Chen, J. K., Taipale, J., Young, K. E., Maiti, T., Beachy, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+modulation+of+Smoothened+activity.">Small molecule modulation of Smoothened activity.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 14071-14076 (2002).</li></ol>

The authors declare that they have no competing financial interests.

(인간 배아 줄기 및 hiPSCs 모두 포함) 인간 만능 줄기 세포는 대부분을 생성하기 위해 체외에서 분화 할 수없는 모든 경우, 이전의 연구 (19)에서 설명 된 DA 신경 세포는 우리 몸의 세포 유형. 여기서, 다른 실험실 14,15부터 배아 도파민 뉴런의 발달과 기술 (20)로부터 발행 프로토콜에 기초하여, 우리는 인간 배아 줄기 및 hiPSCs에서 DA 뉴런의 생성을위한 배양 조건을 최?...

도파민 (DA) 신경 세포는 중뇌, 시상 하부, 망막, 후각 전구 등 여러 가지 뇌 영역에서 찾을 수 있습니다. 전뇌로의 선조체 추체 외로 돌출하고 모터 시스템을 형성함으로써 흑색질의 유리체 compacta (SNpc) 제어 동작 및 운동 뉴런 A9 DA. A9 DA 뉴런의 변성은 두 번째 가장 일반적인 인간의 신경 퇴행성 질환과 현재 불치 파킨슨 병 (PD)에 연결됩니다. 세포 대체 요법은 PD (1)의 치료를위한 가장 유망한 전략 중 하나이고, 따라서 인간 배아 줄기 세포 (인간 배아 줄기) 및 모두 포함한 인간 다 능성 줄기 세포 (hPSCs)로부터 A9 DA 신경 세포를 유도에 많은 관심이 있었다 최근 인간의 유도 만능 줄기 세포 (hiPSCs). 많은 연구는 다양한 방법을 사용하여 hPSCs에서 A9 DA 뉴런을 유도하기 위해 노력하고 있습니다. 신경을 통해 최초의 보고서 생성 된 모든 DA 뉴런장미 전구 단계입니다. 으로 또는 2-4주, L. STUDER 동료 성공적 신경 로제트 인간 배아 줄기를 생산하는 유도 다른 세 그룹의 외부 성장 인자없이 2 MS5 PA6 또는 3-5 기질 세포와 공동 배양. 그런 다음 기계적 박리 또는 추가 차별화를위한 소화 효소에 의해이 로제트를 강화. 다른 보고서에서 연구자들은 문화의 차별화 6-9 부동 배아 체를 통해 신경 로제트 (EB)를 생성합니다. 이후 연구진은 그들이 세포 외 매트릭스에 인간 배아 줄기 및 hiPSCs 도금 단일 층을 기반으로 차별화 방법 (10, 11)을 설립 생체 배아 DA 신경 세포의 개발을 모방 DA 뉴런에 hPSCs의 분화를 유도하는 문화에서 서로 다른 성장 인자를 추가 . 이러한 모든 연구는 DA 신경 세포의 일부 특성 티로신 수산화 효소 (TH) 발현 세포를 수득되지만, 전 분화 과정은, 시간과 노동력 소모일반적으로 비효율적 인, 그리고 더 중요한 것은, 이러한 신경 세포의 A9 ID는 LMX1a 자궁외 식 (12)를 제외한 대부분의 연구에서 입증되지 않았다. 최근, 새로운 바닥 판 (FP) 기반 프로토콜은 다음 DA 신경 세포의 잠재력 FP 전구체가 먼저 분화의 초기 단계에서 소닉 고슴도치와 정식 Wnt 신호 경로의 활성화에 의해 생성 된에 13-16, 개발되었다 이러한 FP 세포는 더 DA 뉴런에 지정되었습니다. 이 프로토콜은 더 효율적이지만, 여전히 몇 가지 문제가있다; 예를 들면, 전체의 분화 과정은 장시간 (적어도 35 일) 소요 종속 피더 셀 (15)은, 또는은 또는 EB A9 아이덴티티 14 입증되지 않았다 (16) 좌우된다. 여기서, 생체 배아 DA 신경 발달과 다른 연구자들의 발표 된 결과에서 지식에 기초하여, 우리는 EFF위한 배양 조건을 최적화 한인간 배아 줄기 및 hiPSCs 모두에서 DA 뉴런의 icient 생성. 우리는 첫번째 작은 분자 CHIR99021 작은 분자 SAG와 purmorphamine와 신호 소닉 고슴도치 정식 Wnt 신호의 활성화에 의해 FP 전구 세포를 생성합니다. 이러한 FP 세포는 FOXA2, LMX1a, 코린,의 Otx2 및 네 스틴을 표현한다. 우리는 다음 등 BDNF, GDNF를 포함 성장 인자와 DA 뉴런 이러한 FP 세포를 지정했습니다. 그들은 Calbindin 17 동안 네거티브 GIRK2 대해 긍정적으로 생성 DA 뉴런 A9 세포 유형이다. 이 프로토콜은 매우 효율적이고 재현성 피더 셀 또는 EB 독립적이다. 이 프로토콜을 사용하여, 하나 또는 체외 PD의 모델링 또는 PD에 대한 잠재적 치료제를 테스트 PD 환자의 hiPSCs에서 인간 배아 줄기 또는 세포 이식 연구에 대한 정상인의 hiPSCs 미만 사주에서 DA 신경 세포를 유도 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="778">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10569-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">26140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">penicillin/ampicillin</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DMEM/F12</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10565-018</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">KSR</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10828-028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Non-essential amino acid</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">b-mercaptoethanol</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">bFGF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">233-FB-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">mTesR1</td>
			<td style="width:184px;">STEMCELL Technologies</td>
			<td style="width:119px;">5850</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Collagenase IV</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Gelatin</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">N2 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">B27 supplement</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Neurobasal</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">21103-049</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Glutamax</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">35050-061</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">PBS</td>
			<td style="width:184px;">Life Technologies</td>
			<td style="width:119px;">10010-023</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Growth factor reduced matrigel</td>
			<td style="width:184px;">BD Biosciences</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:308px;">Accutase</td>
			<td style="width:184px;">MP Biomedicals</td>
			<td style="width:119px;">1000449</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Thiazovivin</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-361380</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SB431542</td>
			<td style="width:184px;">Tocris Bioscience</td>
			<td style="width:119px;">1614</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">LDN-193189</td>
			<td style="width:184px;">Stemgent</td>
			<td style="width:119px;">04-0074</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">SAG</td>
			<td style="width:184px;">EMD Millipore</td>
			<td style="width:119px;">566660-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Purmorphamine</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-202785</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">FGF8b</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">423-F8-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">CHIR99021</td>
			<td style="width:184px;">Cellagen Technology</td>
			<td style="width:119px;">C2447-2s</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">BDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">248-BD-025</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">GDNF</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">212-GD-010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">TGF-beta3</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">243-B3-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Ascorbic acid</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">cAMP</td>
			<td style="width:184px;">Sigma-Aldrich</td>
			<td style="width:119px;">D0627</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:308px;">Mouse anti human NESTIN antibody</td>
			<td style="width:184px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-23927</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human OTX2 antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9566</td>
			<td style="width:167px;">1/2000 dilutiion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Goat anti human FOXA2 antibody</td>
			<td style="width:184px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2400</td>
			<td style="width:167px;">1/200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">rabbit anti human LMX1a antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB10533</td>
			<td style="width:167px;">1/1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human TH antibody</td>
			<td style="width:184px;">Pel Freez</td>
			<td style="width:119px;">P40101</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Chicken anti human TH antibody</td>
			<td style="width:184px;">Millipore</td>
			<td style="width:119px;">AB9702</td>
			<td style="width:167px;">1/500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Mouse anti human TUJ1 antibody</td>
			<td style="width:184px;">Covance</td>
			<td style="width:119px;">MMS-435P</td>
			<td style="width:167px;">1/2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human GIRK2 antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab30738</td>
			<td style="width:167px;">1/300 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Rabbit anti human Calbindin antibody</td>
			<td style="width:184px;">Abcam</td>
			<td style="width:119px;">ab25085</td>
			<td style="width:167px;">1/400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Centrifuge</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;">5804</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

directed dopaminergic neuron differentiation from human pluripotent stem cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

분화 프로토콜의 개요는도 1에 도시된다. 여기에 제시된 분화 프로토콜의 효율은 출발 세포의 상태에 의존한다. 따라서, 첫 번째는 분화 단일 세포로 hPSCs 해리 전에 분화 콜로니를 제거하며, 둘째, 하나는 모든 경우에, MEF 피더 세포 젤라틴 - 코팅 된 플레이트상에서 세포를 배양하여, 대부분 고갈 것을 확인하는 것이 중요 30 분, 그리고 세 번째, 마트 리겔 플레이트 상에 적절한 밀도?...

Watch this Scientific Journal Video about Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells at JoVE.com

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

인간의 다 능성 줄기 세포에서 도파민 신경 세포의 분화를 감독

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

directed-dopaminergic-neuron-differentiation-from-human-pluripotent

Research

JoVE Journal

Neuroscience

16.3K 조회수.  Stanford University School of Medicine. We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson’s disease.

비디오: Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

작은 분자와 유도 Pluripotent 인간 배아 줄기 세포에서 인간의 연결을 Progenitors과 뉴런의 효율 유도

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

연구

신경과학

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

인간 만능의 줄기 세포에서 Telencephalic Glutamatergic 뉴런의 사양

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

인간의 신경의 집계의 cGMP-해당 확장 방법은 줄기 및 전구 세포는 다 능성 줄기 세포 또는 태아 뇌 조직에서 파생 된

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

인간 다 능성에서 신경 능선 세포의 전구 세포의 공급이없는 유도는 줄기 세포

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

신형성 및 시냅스 성숙의 고함량 스크리닝을 위한 인간 다능성 줄기 세포 유래 뉴런의 분화 후 리포팅

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

시험관 내 마우스 배아 줄기 세포로부터의 신경 분화

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

인간 유도 만능 줄기 세포에서 뇌 미세 혈관 내피 세포의 파생, 확장, 냉동 보존 및 특성화

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it contributes to disease development and progression, particularly in the gray matter of the brain, remains largely unknown. The dysfunction of oligodendrocyte lineage cells is hallmarked by deficiencies in myelination and impaired self-renewal of oligodendrocyte precursor cells (OPCs). These two defects are caused at least in part by the disruption of interactions between neuron and oligodendrocytes along the buildup of pathology. OPCs give rise to myelinating oligodendrocytes during CNS development. In the mature brain cortex, OPCs are the major proliferative cells (comprising ~5% of total brain cells) and control new myelin formation in a neural activity-dependent manner. Such neuron-to-oligodendrocyte communications are significantly understudied, especially in the context of neurodegenerative conditions such as AD, due to the lack of appropriate tools. In recent years, our group and others have made significant progress to improve currently available protocols to generate functional neurons and oligodendrocytes individually from human pluripotent stem cells. In this manuscript, we describe our optimized procedures, including the establishment of a co-culture system to model the neuron-oligodendrocyte connections. Our illustrative results suggest an unexpected contribution from OPCs/oligodendrocytes to the brain amyloidosis and synapse integrity and highlight the utility of this methodology for AD research. This reductionist approach is a powerful tool to dissect the specific hetero-cellular interactions out of the inherent complexity inside the brain. The protocols we describe here are expected to facilitate future studies on oligodendroglial defects in the pathogenesis of neurodegeneration.

The neuron-glial interactions in neurodegeneration are not well understood due to inadequate tools and methods. Here, we describe optimized protocols to obtain induced neurons, oligodendrocyte precursor cells, and oligodendrocytes from human pluripotent stem cells and provide examples of the values of these methods in understanding cell-type-specific contributions in Alzheimer&#8217;s disease.

뉴런-희소돌기아교세포 상호작용 모델링을 위한 만능줄기세포로부터 인간 뉴런 및 희소돌기아교세포 생성

In Alzheimer&#8217;s disease (AD) and other neurodegenerative disorders, oligodendroglial failure is a common early pathological feature, but how it ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

인간 미세아교세포 유사 세포: 유도만능줄기세포로부터의 분화 및 인간 시냅토좀을 이용한 체외 생세포 식균작용 분석

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

인간 줄기 세포 이식 및 검증을 위한 생체 외 모델로서의 성인 인간 피질의 기관형 배양

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...