Name: generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions
Uploaded: 2020-11-09T15:00:00
Description: Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

이 작업은 국립 보건원 (R00 AG054616에서 YAH, T32 GM136566에서 KC), 스탠포드 의과 대학 및 Siebel Fellowship (SC에 수여)의 보조금으로 지원되었습니다. YAH는 브라운 중개 과학 연구소의 중개 신경 과학 센터의 GFL 번역 교수입니다.

1. 인간 만능줄기세포로부터 인간 뉴런 유도
<ol><li>렌티바이러스 준비(~5일, 앞서 설명한 세부 프로토콜16)<ol><li>각 T75 플라스크에 ~100만 개의 HEK293T 세포를 플레이트하여 형질감염을 수행할 때 ~40% 합류하도록 합니다. 테트라사이클린 유도성 Ngn2 및 퓨로마이신 내성 유전자(PuroR, 동일한 TetO 프로모터 대조군 하에서), rtTA 및 3개의 헬퍼 플라스미드 pRSV-REV, pMDLg/pRRE 및 VSV-G(렌티바이?...

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H., Park, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reprogramming+methods+for+generation+of+induced-oligodendrocyte+precursor+cells.">Comparison of reprogramming methods for generation of induced-oligodendrocyte precursor cells.</a> Biomolecules &amp; Therapeutics (Seoul). 25 (4), 362-366 (2017).</li><li>Ehrlich, 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=Rapid+and+efficient+generation+of+oligodendrocytes+from+human+induced+pluripotent+stem+cells+using+transcription+factors.">Rapid and efficient generation of oligodendrocytes from human induced pluripotent stem cells using transcription factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (11), 2243-2252 (2017).</li><li>Rodrigues, G. M. 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=Defined+and+scalable+differentiation+of+human+oligodendrocyte+precursors+from+pluripotent+stem+cells+in+a+3D+culture+system.">Defined and scalable differentiation of human oligodendrocyte precursors from pluripotent stem cells in a 3D culture system.</a> Stem Cell Reports. 8 (6), 1770-1783 (2017).</li><li>Hu, B. Y., Du, Z. W., Li, X. J., Ayala, M., Zhang, S. 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시냅스 구조를 안정화시키고 수초화에 의한 염분 신호 전도를 촉진하는 물리적 및 대사적 지원 외에도 희소돌기아교세포 계통 세포는 뉴런 5,6,7과의 빠르고 역동적인 혼선을 통해 뉴런 활동 패턴을 형성할 수 있습니다. AD 병리학에서 희소돌기아교세포 반응은 처음에는 염증 및 산화 스트레스에 이차적인 것으로 간주되었지만...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.
Figure 3 was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig03.jpg" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded. <a href="https://www.jove.com/files/ftp_upload/61778/61778fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61778/61778fig3v2.png" /> 
Figure 3: Co-culture of iNs and iOPCs.&#160;(A) Representative bright field image of co-cultured iNs and iOPCs at Day 7, showing a proper density for further maturation. (B) Representative immunofluorescence image of iNs and iOPCs co-cultured for 28 days. Axonal marker neurofilament NF is shown in green and oligodendrocytic marker MBP in red. Right, a segment of iN axon ensheathed by iOL process (MBP+). (C) Synapse formation assayed in 4-week-old co-cultures. Cells were stained for Synapsin 1 (Syn1, green) and MAP2 (red), and synaptic puncta were quantified by confocal analysis of density along the dendritic segments as described17,18. (D) In our co-cultures of iNs and iOPCs (7 days of co-culturing), the expression of astrocyte markers, ALDHL1 and GFAP, is minimal (top), and the expression of microglia markers, TMEM119, TREM2, and CD33, is not detected (N.D.) by qPCR. The contamination from these two glial cell types is thus excluded.&#160;(E) Coculturing iOPC with iN leads to the formation of neuron-OPC synapses. The fluorescence-tagged post-synaptic marker PSD95-mCherry is expressed only in OPCs, and display a diffuse pattern in single cultures (left) but aggregate to form puncta in cocultures (right, indicated by arrows; Tuj1, neuronal marker). (F) The expression of well-characterized oligodendroglial genes that can sense and respond to neuronal activities in the pure cultures of iOPCs at Day 14.&#160;<a href="https://www.jove.com/files/ftp_upload/61778/61778fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The fourth&#160;paragraph was updated from:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).
to:
Co-culturing of iNs and iOPCs 
This protocol is optimized specifically for co-culturing iNs and iOPCs and allow our real-time monitoring of the inter-cellular communications between these two cell types along the course of neural development. The ideal plating densities for both cell types need to be decided with a series of cell number titration to achieve proper differentiation (Figure 3A). After 4 weeks in co-cultures, the iOPCs are expected to be adequately differentiated into OLs that are positive for specific markers such as MBP and extend processes to ensheath axons (Figure 3B). The co-culture system can robustly boost up the number of synapses, indicating that the iOPCs provide a neuronal support through physical contacts or release of trophic factors (Figure 3C). We can maintain the co-cultures in acceptable health condition for up to 6 weeks and observe that the synapse number and other neuronal attributes plateau around the fifth week. Of note, astrocytes and microglia are not present in our preparations and their absence can be documented by checking the expression of specific markers (Figure 3D).&#160;The iOPCs express a good number of well-characterized genes that can potentially respond to and mediate the activity-dependent signals from neighboring neurons, in a paracrine (e.g. neurotrophins and metabolites) and/or a synaptic manner (Figure&#160;3E and 3F).&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61778/generation-human-neurons-oligodendrocytes-from-pluripotent-stem-cells">Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions</a>.&#160;The Representative Results section has been updated.

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

희소돌기아교세포 전구세포(OPC), 수초 희소돌기아교세포 및 그 사이의 전이 유형을 포함한 희소돌기아교세포 계통 세포는 신경 발달 및 노화 전반에 걸쳐 중추신경계의 적절한 작동 및 유지를 위한 많은 중요한 기능에 적극적으로 참여하는 인간 뇌 세포1의 주요 그룹을 구성합니다.2,3,4 . 희소돌기아교세포는 백질에서 신경 활동 전달을 촉진하고 축삭 건강을 지원하기 위해 미엘린을 생성하는 것으로 잘 알려져 있지만, OPC는 수초화가 부족한 회백질에 풍부하고(~5%) 학습 행동 및 기억 형성을 제어하는 활동 의존적 신호 기능을 수행합니다. 5,6,7,8 . 알츠하이머병(AD) 및 기타 연령 관련 신경퇴행성 질환의 발병기전에서 희소돌기아세포 기능 및 기능 장애가 어떻게 연구되었는지는 연구되지 않았습니다9. 적절한 모델 시스템의 부적절 함과 실험 경로를 안내하는 일반 지식의 부족이 이러한 격차의 주요 원인입니다.
배아 줄기 (ES) 및 유도 만능 줄기 (iPS) 세포를 포함한 만능 줄기 세포로부터 인간 뇌 세포를 유도하는 최근의 돌파구에 비추어 볼 때, 현대 유전자 편집 도구와 결합 된 이러한 세포 모델은 뇌에서 세포 상호 작용의 복잡한 연결을 처리하는 강력한 도구로 등장했으며 인간 특이 적 질병 증상을 입증 할 수 있습니다10, 11. 개별 뇌 세포 유형이 동일한 AD 촉진 조건12,13에 직면하여 뚜렷하고 심지어 상충되는 효과를 나타낼 수 있다는 점을 고려할 때,이 줄기 세포 방법론은 뇌 세포 유형 모음에서 집계 판독 값만을 제공하는 확립 된 생체 내 또는 시험관 내 모델을 사용하여 이전에 놓친 세포 유형 별 정보를 고유하게 제공합니다. 지난 10년 동안 ES/iPS 세포의 트랜스분화 또는 다른 말기 분화 세포 유형(예: 섬유아세포)에서 직접 전환하여 인간 뉴런을 생성하기 위해 신뢰할 수 있는 프로토콜이 많이 개발되었습니다.14,15. 특히, 주요 신경인성 전사 인자(예를 들어, 뉴로게닌 2, Ngn2)16를 인간 만능 줄기 세포에 적용하면 신경교 세포와 공배양할 필요 없이 순수 배양을 위한 잘 특성화된 뉴런 세포 유형의 균질한 집단을 생성할 수 있다12,17,18. 유도 된 인간 희소 돌기 아교 세포의 경우, 시간과 자원에서 광범위한 효율성과 수요를 가진 1 차 대응 물과 매우 유사한 기능성 세포를 생성 할 수있는 몇 가지 발표 된 프로토콜이 있습니다 19,20,21,22,23,24,25,26,27,28 . 현재까지 이러한 프로토콜 중 어느 것도 희소돌기아교 세포가 AD 발병 기전에 어떻게 반응하고 영향을 미치는지 조사하는 데 적용되지 않았습니다.
여기에서는 인간 유도 뉴런(iN) 및 OPC/희소돌기아교세포(iOPC/iOL)의 단일 및 혼합 배양에 대한 개선된 프로토콜을 설명합니다. 여기에 설명된 iN 프로토콜은 널리 사용되는 Ngn2 접근법(16)에 기초하며, glia-free라는 추가적인 특징을 갖는다. 결과 iN은 균질하고 특징적인 피라미드 형태, 유전자 발현 패턴 및 전기 생리 학적 특징17,18을 갖는 피질 층 2/3 흥분성 뉴런과 매우 유사합니다 (그림 1). 만능 줄기 세포의 직접 분화의 근본적인 장벽 중 일부를 극복하기 위해 우리는 저용량 디메틸 설폭 사이드 (DMSO) 전처리29,30의 간단하고 효과적인 방법을 개발했으며 Douvaras 및 Fossati 32의 널리 채택 된 프로토콜을 기반으로 인간 ES / iPS 세포가 iOPC 및 iOL로 트랜스 분화하는 성향이 향상되었다고보고했습니다32 . 우리는 프로토콜을 더욱 단순화하고 강력한 분화 촉진 화합물인 클레마스틴 7,33,34를 통합하여 희소돌기아교 성숙 과정을 가속화했습니다. 그 결과(그림 2), iOPC는 2주 내에 생성될 수 있고(마커 O4에 대해 ~95% 양성) 4주 내에 iOL이 생성될 수 있습니다(성숙한 마커 MBP 및 PLP1 발현). 흥미롭게도, 우리는 iOPCs만이 놀라운 양의 아밀로이드 β (Aβ)를 분비한다는 것을 발견했으며, 이는 희소 돌기 아교 세포35,36에서 아밀로이드 전구체 단백질 (APP)의 풍부한 발현과 처리 프로테아제 β- 세크레타제 (BACE1)의 풍부한 발현을 보여주는 독립적 인 전사체 데이터와 일치합니다. 또한, 당사의 iN-iOPC 공동 배양 시스템은 MBP 양성 iOL 과정에 의한 축삭 외피를 촉진하고 시냅스 형성에 대한 상당한 지원을 제공합니다(그림 3). 따라서 아래에 자세히 설명된 프로토콜은 이전에 카탈로그화된 뉴런-희소돌기아교세포 공동 배양 방법에 비해 기술적 및 생물학적 이점을 가지고 있으며 AD의 신경퇴행을 더 잘 모델링할 수 있는 가능성을 가지고 있습니다.

<table>
	<tbody>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>ThermoFisher</td>
			<td>PHG 0266</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>MilliporeSigma</td>
			<td>A9501</td>
			<td></td>
		</tr>
		<tr>
			<td>Clemastine</td>
			<td>MilliporeSigma</td>
			<td>SML0445</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 medium</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>ThermoFisher</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>MilliporeSigma</td>
			<td>D3072</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ScienCell</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>H1 human ES cells</td>
			<td>WiCell</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR plus</td>
			<td>STEMCELL Technologies</td>
			<td>5825</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal A medium</td>
			<td>ThermoFisher</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>ThermoFisher</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-AA</td>
			<td>R&#38;D Systems</td>
			<td>221-AA-010</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>VWR</td>
			<td>71002-812</td>
			<td></td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>MilliporeSigma</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>pRSV-REV</td>
			<td>Addgene</td>
			<td>12253</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>ThermoFisher</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
		<tr>
			<td>SAG</td>
			<td>Tocris</td>
			<td>4366</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff Neural Progenitor Freezing Media</td>
			<td>STEMCELL Technologies</td>
			<td>5838</td>
			<td></td>
		</tr>
		<tr>
			<td>STEMdiff SMADi Neural Induction Kit</td>
			<td>STEMCELL Technologies</td>
			<td>8581</td>
			<td></td>
		</tr>
		<tr>
			<td>T3 triiodothyronine</td>
			<td>MilliporeSigma</td>
			<td>T6397</td>
			<td></td>
		</tr>
		<tr>
			<td>Tempo-iOlogo: Human iPSC-derived OPCs</td>
			<td>Tempo BioScience</td>
			<td>SKU102</td>
			<td></td>
		</tr>
		<tr>
			<td>TetO-Ng2-Puro</td>
			<td>Addgene</td>
			<td>52047</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human neurons and oligodendrocytes from pluripotent stem cells for modeling neuron-oligodendrocyte interactions

알츠하이머 병 (AD) 및 기타 신경 퇴행성 질환에서 희소 돌기 부전은 일반적인 초기 병리학 적 특징이지만 특히 뇌의 회백질에서 질병 발달 및 진행에 어떻게 기여하는지는 거의 알려지지 않았습니다. 희소돌기아교세포 계통 세포의 기능 장애는 희소돌기아교세포 전구체 세포(OPC)의 수초화 결핍 및 자가 재생 장애로 특징지어집니다. 이 두 가지 결함은 병리학의 축적을 따라 뉴런과 희소돌기아교세포 사이의 상호 작용이 중단되어 적어도 부분적으로 발생합니다. OPC는 CNS 발달 동안 수초 희소돌기아교세포를 생성합니다. 성숙한 뇌 피질에서 OPC는 주요 증식 세포 (전체 뇌 세포의 ~ 5 %를 구성)이며 신경 활동 의존적 방식으로 새로운 미엘린 형성을 제어합니다. 이러한 뉴런-희소돌기아교세포 통신은 적절한 도구의 부족으로 인해 특히 AD와 같은 신경퇴행성 상태의 맥락에서 상당히 과소 연구되고 있습니다. 최근 몇 년 동안 우리 그룹과 다른 사람들은 인간 만능 줄기 세포에서 개별적으로 기능성 뉴런과 희소돌기아교세포를 생성하기 위해 현재 사용 가능한 프로토콜을 개선하기 위해 상당한 진전을 이루었습니다. 이 원고에서는 뉴런-희소돌기아교세포 연결을 모델링하기 위한 공동 배양 시스템의 구축을 포함하여 최적화된 절차에 대해 설명합니다. 우리의 예시적인 결과는 뇌 아밀로이드증 및 시냅스 무결성에 대한 OPC/희소돌기아교세포의 예상치 못한 기여를 시사하고 AD 연구를 위한 이 방법론의 유용성을 강조합니다. 이 환원 주의적 접근법은 뇌 내부의 고유 한 복잡성에서 특정 이종 세포 상호 작용을 해부하는 강력한 도구입니다. 여기에서 설명하는 프로토콜은 신경 퇴행의 발병 기전에서 희소돌기전 결함에 대한 향후 연구를 용이하게 할 것으로 예상됩니다.

인간 만능 줄기 세포에서 인간 유도 뉴런의 직접 생성 시작하는 인간 다 능성 줄기 세포가 iN 또는 iOPC / iOL의 성공적인 생성을 위해 높은 수준의 다 능성을 나타내는 것이 매우 중요합니다. 따라서, 세포는 본 원고에 기재된 유도 프로토콜 중 하나를 시작하기 전에 Oct4 및 SOX2와 같은 특정 마커에 대해 염색되어야 한다(도 1A). 인간 H1 세포는 Zhang et al.에 의해 이?...

Watch this Scientific Journal Video about Generation of Human Neurons and Oligodendrocytes from Pluripotent Stem Cells for Modeling Neuron-Oligodendrocyte Interactions at JoVE.com

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

신경 퇴행의 뉴런-아교 세포 상호 작용은 부적절한 도구와 방법으로 인해 잘 이해되지 않습니다. 여기에서는 인간 만능 줄기 세포에서 유도 뉴런, 희소돌기아교세포 전구세포 및 희소돌기아교세포를 얻기 위한 최적화된 프로토콜을 설명하고 알츠하이머병에서 세포 유형 특이적 기여를 이해하는 데 있어 이러한 방법의 가치에 대한 예를 제공합니다.

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 ...

The protocols described here, are expected to facilitate future studies on oligodendroglial defects, in the pathogenesis of neuro degeneration. This is an improved method that can be used to generate functional human oligodendrocyte lineage cells in only three to four weeks, and establish a co-culture system to model the neuron oligodendrocyte connections. The protocol can be used to study not only Alzheimer's disease, but any other disease associated aspect of neuron oligodendrocyte interactions.To begin, plate approximately one million HEK293T cells in each T75 flask to achieve 40%confluency, then transfect them with plasmids expressing tetracycline inducible NGN2 and puromycin resistant tetracycline transactivator gene, along with the three helper plasmids. Use PEI for transfection. Change the media after 16 hours.Harvest released viral particles by collecting culture media every day and replacing it with fresh media for three days. Pool the collected media for purification, then filter the virus through a 0.22 micrometer filter and centrifuge at 49, 000 times G for 90 minutes. After centrifugation, re-suspend the pellet in the appropriate volume of PBS glucose.Use commercially available H1 human ES cells to perform neuron induction. Culture the cells on extracellular matrix solution coated 6-well plates with ESL maintenance medium and incubate the plates at 37 degrees Celsius with 5%carbon dioxide. Two days before induction of differentiation, detach ES cells by adding one milliliter of cell detachment solution and incubating at room temperature for 10 minutes.Transfer the cells to a tube, then wash the well with two milliliters of media and add it to the same tube. Centrifuge the cells at 300 times G for five minutes. Then re-suspend the pellet in media and plate the cells onto matrix coated 6-well plates at the seating density of one times 10 to the fifth cells per well.On the next day, add lentiviruses, expressing NGN2 plus puro. R, and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add two micrograms per milliliter doxycycline in DMEM/F-12 medium supplemented with N2 and without morphogens.On day one, add puromycin in fresh DMEM/F-12, plus N2 and doxycycline to the final concentration of one microgram per milliliter. Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and re-plate them on 24-well plates coated with matrix solution.Maintain them in MBA B27 medium without doxycycline. Culture pure induced neurons on the plates, coated with extracellular matrix-based solutions. To perform neural progenitor cell generation, culture H1 human ES cells and transdifferentiate them into neural progenitor cells, as described in the text manuscript.On day negative one, seed 0.5 to one million cells in ES cell maintenance medium into each well of a 6-well plate coated with growth factor reduced matrix solution. On day zero, treat cells for 24 hours with ES cell maintenance medium supplemented with 2%DMSO. On days one through six, change the full media with warm neural induction medium containing some add inhibitors from a commercial kit.On day seven, passage NPCs using cell detachment solution and plate them at a seating density of one to two times 10 to the fifth cells per well of a 24-well plate. Assay the differentiation efficiency by immunohistochemical staining for absence of pluripotentcy marker and presence of NPC markers. To generate the oligodendrocyte precursor cells, plate the NPCs in warm neural induction medium with some add inhibitors from a commercial kit.On day eight, prepare a solution of 1%DMSO in the OPC differentiation medium, and treat the plated NPCs for 24 hours. On day nine, replace the media with fresh OPC differentiation medium without DMSO, then feed the cells every other day until day 15. If the the cells reach confluence before day 15, passage them to the seating density of one to two times 10 to the fifth cells per well.On day 14, plate OPCs in OPC differentiation medium at a density of one to two times 10 to the fifth cells per well, in a 24-well plate. Test cells for the presence of OPC specific markers by IHC staining or qPCR, and for the absence of NPC markers. On day 15, replace media with OL maturation medium.Change the medium every other day or every day. When cells reach 90%confluence, split them at a one to three ratio for up to two passages or until cell division slows down substantially. If OPCs divide too fast and reach confluencey in less than three days, add Ara-C at a concentration of two to five micromolar for one to three days.On day 14, plate the IOPCs at a density of one times 10 to the fifth cells per well in a 24-well plate in OPC differentiation medium. On the next day, detach the induced human neurons with cell detachment solution. Add neurons onto the cultured OPCs, plating at the seating density of two times 10 to the fifth cells per well.Use co-culture medium containing neurobasal A medium, 2%B27 supplement, and 100 nanograms per milliliter T3 triiodothyronine. Change the medium on the next day and then every other day. If OPCs proliferate too fast and reach confluency in less than three days, add two to five micromolar Ara-C.It is important that the starting human pluripotent stem cells exhibit a high degree of pluripotentcy for the successful generation of induced neurons, or OPCs and oligodendrocytes. Therefore, cells should be stained for specific markers, such as OCT-4 and SOX2, before starting either of the induction protocols. Human H1 cells were used to obtain induced excitatory forebrain neurons.Pure neurons in culture started expressing synapse in one at day 14 to 16. IOPCs were generated in two weeks and mature oligodendrocytes in four to five weeks. DMSO treatment enriched the number of cells in the early G1 phase for better signaling Integration.Specific OPC markers were detected as early as two weeks after plating of ES H1 cells. The IOPC population at two weeks was fairly homogenous, with over 95%of cells positive for 04 staining and a high level of immunoreactivity for other markers. After initiation of oligodendrocyte maturation, specific oligodendrocyte markers were detected starting at day 28.The ideal plating densities for induced neurons, and IOPCs, need to be determined with a series of cell number titrations to achieve proper differentiation. After four weeks, in co-cultures, the IOPCs should be adequately differentiated into oligodendrocytes that are positive for specific markers such as MBP. This co-culture system can robustly boost the number of synapses, indicating that the IOPCs provide a neuronal support through physical contact or release of trophic factors.This described procedure can be further sophisticated by adding different brain cell types. For example, stem cells derived human astrocytes to model novel aspects of neuron glial interactions in health and disease.

Research

JoVE Journal

Neuroscience

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

폐기 인간 태아 두피 조직에서 신경 줄기 세포의 생성

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

연구

신경과학

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

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 ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

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

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

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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

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 ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

성인 인간 피부 섬유 아 세포에서 유도 된 신경의 고수익 문화의 간단한 세대

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

PiggyBac 벡터를 사용 하 여 기능적 척추 및 두개골 모터 뉴런으로의 인간 유도 만능 줄기 세포 (iPSCs)의 전환

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

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

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 ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

지역별 인간 만능줄기세포 유래 성상세포 및 뉴런을 이용한 ALS 모델링을 위한 전기생리학적 플랫폼 구축

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...