作者感谢 Goureau 小组成员在这里所描述的方法的建立过程中的投入, 以及 g. Gagliardi 和 m. 拉·加里对他们的批判性阅读。这项工作得到了来自情报局 (GPiPS: ANR-2010-RFCS005 的赠款的支持;SightREPAIR: ANR-16-CE17-008-02), 视网膜法国协会和技术转让公司 SATT Lutech。它还在 Investissements d ' 艾文莉计划 (ANR-11-IDEX-0004-02) 的 LABEX LIFESENSES (ANR-10-LABX-65) 的框架内执行。

本文件所述的议定书遵循了 la 远景研究伦理学委员会的指导方针。根据目前的法国规定, 该研究所的视觉被允许操纵人体标本。标本处理遵循赫尔辛基的原则, 并在 "保护联合会 (CPP) 法国 V" 的伦理批准后的国家条例的情况下, 病人数据保护。
1. 培养培养基和菜肴的准备
<ol><li>
文化媒体
	<ol>
<li>使用 iPSC 培养基, 一种化学定义的培养基, 专门用于无馈线条件下的多潜能干细胞培养16。根据制造商的协议, 准备500毫升的培养基。</li>
		<li>使用基底 iPSC (Bi) 培养基, iPSC 化学定义培养基无成纤维细胞生长因子 2 (FGF2) 或转化生长因子β (TGFß)。</li>
		<li>准备500毫升的双介质, 辅以 1% N2 补充剂 (BiN2 培养基), 10 单位/毫升青霉素, 和10毫克/毫升链霉素。</li>
		<li>准备500毫升的 proneural N2-based 培养基 (ProN2 培养基) 由 DMEM 组成: 营养混合物 F-12 (DMEM/F12, 1:1, l-谷氨酰胺), 1% 个不必要的氨基酸, 1% N2 补充, 10 单位/毫升青霉素, 和10毫克/毫升链霉素。</li>
		<li>制备 proneural B27-based 培养基 (ProB27 培养基), 由 DMEM 组成: 营养混合物 F-12 (DMEM/F12, 1:1, l-谷氨酰胺), 1% 个不必要氨基酸, 2% B27 补充剂, 10 单位/毫升青霉素和10毫克/毫升链霉素。</li>
	</ol>
</li>
	<li>
培养容器的制备
	<ol>
<li>
人类 iPSC 文化
		<ol>
<li>在 1x PBS 中准备10毫升含有5微克/毫升及其与受精的及其与受精溶液。在10毫升的 1x PBS, 添加100µL 解冻及其与受精库存解决方案 (100x)。</li>
			<li>2毫升的及其与受精溶液每6厘米培养皿对应于0.5 µg/厘米2。在室温下孵化1小时 (RT)。用抽吸系统或5毫升吸管去除及其与受精溶液。</li>
			<li>添加4毫升的 iPSC 培养基每6厘米菜。</li>
			<li>在37摄氏度和 5% CO2的细胞培养孵化器中孵化菜肴, 使用前至少要30分钟。</li>
		</ol>
</li>
		<li>人类 iPSC 的视网膜色素 (hiRPE) 细胞培养</li>
		<li>根据制造商的协议, 使用多能干细胞合格的基质或基底来涂水井。添加足够的基质或基底覆盖整个生长表面积。例如, 在 T-25 cm2瓶中, 在24井板和3毫升矩阵中放入300µL 矩阵。</li>
		<li>至少在37摄氏度的1小时内孵化出涂层培养容器。使用真空吸入系统或5毫升吸管去除基体。</li>
		<li>在24井板上添加1毫升的 ProN2 培养基, 并将培养容器返回到孵化箱, 使培养基的温度和平衡最小30分钟。对于 T-25 厘米2烧瓶, 请参阅步骤5.2。</li>
	</ol>
</li>
</ol>2. 人类 iPSCs 的维护和扩大
<ol><li>
人类 iPSCs 的维护
	<ol>
<li>在15毫升管中准备3毫升的 iPSC 培养基, 在使用前将其保持在 RT 中至少15分钟。按照1.2.1 的描述, 准备一盘涂有6厘米的盘子。</li>
		<li>在三十年代37摄氏度的水浴中, 从液氮罐或-150 °c 冷冻室解冻人体 iPSCs 的低温样品。</li>
		<li>用消毒液喷雾剂对低温小瓶进行仔细消毒。将解冻的人类 iPSCs 从 cryotube 转移到15毫升管, 其中3毫升的 iPSC 培养基在 RT 前预热。</li>
		<li>离心管在 110 x g 3 分钟, 用吸吸系统或5毫升吸管取出上清液。</li>
		<li>并用重悬细胞颗粒与1毫升的 iPSC 培养基从及其与受精涂层的6厘米培养皿使用2毫升吸管和转移细胞回到盘子。在改变培养基之前, 将盘子在37摄氏度和 5% CO2的情况下返回到孵化箱, 最小为24小时。 注: 一种岩石抑制剂, 如 Y-27632 在10µM, 可以添加到 iPSC 培养基中的6厘米培养皿, 以减少凋亡。</li>
		<li>每天改变媒体, 每周通过人类 iPSCs。</li>
	</ol>
</li>
	<li>
人类 iPSCs 的传代
	<ol>
<li>通过人类的 iPSC 在 70-80% 汇合, 经典后7天的文化。</li>
		<li>按照步骤1.2.1 中的描述, 为传代准备6厘米的菜肴。用汇合的 iPSCs 从盘子中取出 iPSC 培养基, 在 RT 中加入2毫升的离解溶液, 6 分钟。 注意: 孵化时间取决于 iPSC 克隆。尝试6分钟的孵化作为一个开始。</li>
		<li>使用真空吸入系统或5毫升吸管去除离解溶液, 并在 RT 中加入2毫升的 iPSC 培养基预热. 并用重悬 iPSC 殖民地吹打他们上下 5-10x 与1000µL 吸管。 注意: 避免单细胞从过量吹打分离。</li>
		<li>在新的6厘米培养皿中转移30到200µL 的悬浮细胞团簇。在改变培养基之前, 将盘子在37摄氏度和 5% CO2的细胞培养孵化器中返回到最小24小时。 注意: 传代所需的悬浮细胞团簇数量取决于 iPSC 克隆。</li>
		<li>每天改变媒体, 每周通过人类 iPSCs。</li>
	</ol>
</li>
</ol>3. 视网膜 Organoids 的产生
<ol><li>开始 iPSC 差异后的协议图解在图 1A , 当殖民地达到 60-70% 汇合 (图 1B)。</li>
	<li>每6厘米的盘子准备4毫升的双介质。将双介质加热至37摄氏度。将 iPSC 介质改为双介质。请注意此时间为0天 (D0)。</li>
	<li>在 D2, 将双介质中的培养基转换为 BiN2 培养基, 以前以摄氏37摄氏度的温度加热。每 2-3 天更换培养基。</li>
	<li>用 neuroepithelium 芽识别紧急自成视网膜 organoids, 如图 1C -1E所示。 注: 这些早期视网膜 organoids, 对应于他们的发展阶段, 视杯, 可以手动隔离的下游实验。</li>
</ol>4. 视网膜 Organoids 的成熟
<ol><li>在 D28, 准备6井板包含4毫升/井的 ProB27 介质最初补充 10 ng/毫升的 FGF2 (图 1A)。 注: 在将介质添加到印版之前, 立即添加 FGF2。 警告: 不要过滤 FGF2。</li>
	<li>手动从6厘米的盘子中恢复视网膜结构。要做到这一点, 隔离的结构, 使垂直条纹与针周围的神经上皮芽, 如图 1E所示, 并分离 organoids 通过轻轻地划伤他们与针。</li>
	<li>用1000µL 吸管吸入 10-15 organoids, 并将其转移到含有 ProB27 介质的6井板的单个井中。</li>
	<li>保持视网膜 organoids 在 ProB27 培养基中的漂浮培养条件在37°c 和 5% CO2的细胞培养孵化器。每 2-3 天改变一半的培养基。 注: 治疗 organoids 与 FGF2, 直到 D35。</li>
	<li>在 D35, 去除一半的培养基, 并添加新鲜的 prewarmed ProB27 培养基在37摄氏度, 没有 FGF2。</li>
	<li>根据视网膜细胞类型的出现, 每 2-3 天在需要的时间内改变一半的培养基, 如图 2所示, 以获得所需的视网膜细胞类型。</li>
</ol>5. 人 iPSC 视网膜色素 (hiRPE) 细胞的生成和扩增
<ol><li>
人类 hiRPE 细胞的生成
	<ol>
<li>按照图 3A中的协议图解, 当菌落达到 60-70% 汇合时, iPSCs 进行分化。</li>
		<li>准备每6厘米的4毫升的双层培养基和温暖的双介质在37摄氏度。将 iPSC 介质改为双介质。请注意此时间为0天 (D0)。</li>
		<li>在 D2, 将双介质中的区域性转换为以前在摄氏37摄氏度处加热的 BiN2 介质。每 2-3 天更换培养基。</li>
		<li>在 D28, 改变培养基到一个新鲜的 ProN2 培养基以前温暖的37摄氏度, 如图 3A所示。 注意: 此步骤可在步骤4中描述的 organoid 领料后完成。</li>
		<li>每 2-3 天更改 ProN2 介质。</li>
		<li>在 D42, 通过观察色素斑块来识别紧急 hiRPE 细胞, 如图 3B所示。 注意: hiRPE 细胞的色素沉着会出现在 D35 与 D56 之间, 具体取决于 iPSC 克隆。</li>
	</ol>
</li>
	<li>
hiRPE 细胞的扩增
	<ol>
<li>在 D42, 准备一个24井板以前涂上的矩阵, 如步骤1.2.2 中所述, 并包含1毫升的 ProN2 介质每井。在37°c 和 5% CO2在使用之前安置板材为15分钟在孵化器。</li>
		<li>手动从6厘米的盘子中恢复 hiRPE 补丁。要隔离斑块, 用针在色素上皮周围做一个垂直的条纹, 然后用针轻轻地搔它, 将床单分离。使用1000µL 吸管吸入10色素斑块, 并将其转移到24井板的单个井中。</li>
		<li>在改变培养基之前, 在37摄氏度和 5% CO2的细胞培养孵化器中保持 hiRPE 补丁在 ProN2 培养基中。注意这个 hiRPE 细胞通道为 0 (hiRPEp0)。每 2-3 天用 ProN2 培养基改变培养基。</li>
		<li>当细胞汇合时, 传递 hiRPEp0 细胞。<ol>
<li>使用5毫升吸管, 用真空吸尘系统去除介质, 并用1毫升的 PBS 清洗每个井1x。</li>
			<li>使用真空吸入系统丢弃 PBS, 添加200µL 每井0.25% 胰蛋白酶, 并孵化它至少15分钟的孵化器在37摄氏度。添加800µL ProB27 培养基, 使胰蛋白酶失效。 注: 不推荐使用 ProN2 培养基停止胰蛋白酶活性。</li>
			<li>将 hiRPE 细胞的表吹打上下分离。将细胞悬浮在15毫升管中, 在 110 x g 处离心5分钟。 注: 可以用 ProN2 培养基进行额外洗涤, 以去除 ProB27 培养基的过量。</li>
			<li>取出上清, 并轻轻并用重悬细胞颗粒在2毫升的 ProN2 培养基前预热37摄氏度。用单元格计数器计数单元格。</li>
			<li>采取125万细胞, 并将其放置在基质涂层 T-25 cm2瓶 (见步骤 2.2.2) 包含5毫升的 ProN2 培养基预热37摄氏度。注意这个 hiRPE 细胞通道为 1 (hiRPEp1)。</li>
		</ol>
</li>
		<li>将 hiRPE 细胞保存在细胞培养孵化器中的 ProN2 培养基中, 在37摄氏度和 5% CO2处, 在改变培养基前保持48小时。</li>
		<li>每 2-3 天更改媒体。当 hiRPEp1 细胞到达汇合处时, 执行下一段或超低温保存。</li>
	</ol>
</li>
</ol>6. 视网膜 Organoids 和 hiRPE 细胞的超低温保存
<ol><li>
全视网膜 organoids
	<ol>
<li>选择5到20视网膜 organoids 使用转移吸管和放置在一个低温小瓶。用1000µL 吸管去除任何多余的介质, 而不触及管子底部的 organoids, 并添加250µL 的冷冻存介质 (见材料表)。</li>
		<li>在-80 摄氏度的基异丙醇冷冻容器中冷冻小瓶, 至少4小时. 将冷冻瓶转移到-150 °c 冷藏柜或液氮罐中进行长期贮存。</li>
	</ol>
</li>
	<li>
对于 hiRPE 细胞
	<ol>
<li>在1通道, 当细胞汇合时, hiRPE 细胞离解。 注意: 不推荐 hiRPEp0 细胞的超低温保存。</li>
		<li>使用真空吸入系统从 T-25 cm2瓶中吸取培养基, 并用5毫升吸管将其1x 与3毫升的 PBS 清洗。</li>
		<li>使用真空吸入系统去除 PBS, 加入1毫升0.25% 胰蛋白酶, 并在15分钟内将其孵化为37摄氏度。加入5毫升的 ProB27 培养基以停止胰蛋白酶活性。 注意: 不推荐使用 ProN2 培养基来灭活胰蛋白酶。</li>
		<li>用10毫升的吸管将 hiRPE 细胞吹打。用单元格计数器计数单元格。将细胞悬浮在15毫升管中, 在 110 x g 处离心5分钟。 注: 可使用 ProN2 培养基进行额外洗涤, 以去除 ProB27 培养基的过量。</li>
		<li>用真空吸入和轻轻并用重悬细胞在250µL 的冷冻培养基中获得200万细胞。</li>
		<li>每低温小瓶转移250µL 细胞悬浮液。以异丙醇为基础的冷冻容器中的小瓶冷冻, 最低为4小时,-80 摄氏度。</li>
		<li>将冷冻小瓶转移到-150 摄氏度冷藏柜或液氮贮罐中, 用于长期贮存。</li>
	</ol>
</li>
</ol>7. 视网膜 Organoids 和 hiRPE 细胞的解冻
<ol><li>
全视网膜 organoids 的解冻
	<ol>
<li>温暖的 ProB27 培养基在37°c (每一个低温小瓶需要2毫升)。</li>
		<li>从液氮罐或-150 °c 冷藏柜中, 将含有视网膜 organoids 的低温小瓶在37摄氏度的水浴中解冻, 三十年代. 用消毒剂喷雾剂小心消毒低温小瓶。</li>
		<li>打开药瓶, 加入1毫升预热的 ProB27 培养基。用转移吸管将 organoids 转移到1.5 毫升管。</li>
		<li>通过吹打, 轻轻地移开培养基, 而不接触管底部的视网膜结构。再用1毫升的预热 ProB27 培养基清洗 organoids 1 次。</li>
		<li>吸入 organoids 与转移吸管并且安置他们在1井一个6井板包含 ProB27 中等预热和平衡在孵化器在37°c 和 5% CO2。 注: 放置 10-15 视网膜 organoids 每井6井板。</li>
		<li>根据图 2所述, 在需要的时间内, 每 2-3 天改变一半的培养基以获得所需的视网膜细胞类型。</li>
	</ol>
</li>
	<li>
hiRPE 细胞的解冻
	<ol>
<li>温暖的 ProN2 培养基在37°c (每一个低温小瓶需要8毫升)。</li>
		<li>从液氮罐或-150 °c 冷冻机, 解冻一个低温样品的 hiRPEp1 细胞由孵化成水浴在37°c 在三十年代. 用消毒剂喷雾剂小心消毒低温小瓶。</li>
		<li>打开管并加入1毫升预热的 ProN2 培养基, 将细胞悬浮液转移至15毫升, 含2毫升预热的 ProN2 培养基。离心机5分钟, 110 x 克。</li>
		<li>取出上清, 并轻轻并用重悬细胞颗粒在2毫升的 ProN2 培养基前预热37摄氏度。</li>
		<li>用单元格计数器计数单元格。</li>
		<li>采取125万细胞和放置在基质涂层 T-25 cm2瓶 (见步骤 2.2.2) 包含5毫升的 ProN2 培养基预热37摄氏度。在这个阶段, 注意段落作为 2 (hiRPEp2)。</li>
		<li>在改变培养基前, 将细胞培养孵化器中的 hiRPEp2 细胞保持在37摄氏度和 5% CO2的 ProN2 培养基中。</li>
		<li>每 2-3 天更改媒体。当 hiRPE 细胞到达汇合处时, 执行下一段或超低温保存。 注意: 在培养2周后, 可以收集 8-1000万 hiRPE 细胞进行进一步的实验。</li>
	</ol>
</li>
</ol>

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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=Concise+review:+Patient-specific+stem+cells+to+interrogate+inherited+eye+disease.">Concise review: Patient-specific stem cells to interrogate inherited eye disease.</a> STEM CELLS Translational Medicine. 5, 132-140 (2016).</li><li>Nakano, T., Ando, 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=Self-formation+of+optic+cups+and+storable+stratified+neural+retina+from+human+ESCs.">Self-formation of optic cups and storable stratified neural retina from human ESCs.</a> Cell Stem Cell. 10, 771-785 (2012).</li><li>Reichman, S., Terray, 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=From+confluent+human+iPS+cells+to+self-forming+neural+retina+and+retinal+pigmented+epithelium.">From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 8518-8523 (2014).</li><li>Meyer, J. S., Howden, S. E., 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=Optic+vesicle-like+structures+derived+from+human+pluripotent+stem+cells+facilitate+a+customized+approach+to+retinal+disease+treatment.">Optic vesicle-like structures derived from human pluripotent stem cells facilitate a customized approach to retinal disease treatment.</a> Stem Cells. 29, 1206-1218 (2011).</li><li>Zhong, X., Gutierrez, 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=Generation+of+three-dimensional+retinal+tissue+with+functional+photoreceptors+from+human+iPSCs.">Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.</a> Nature Communications. 5, 4047 (2014).</li><li>Mellough, C. B., Collin, 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=IGF-1+Signalling+plays+an+important+role+in+the+formation+of+three+dimensional+laminated+neural+retina+and+other+ocular+structures+from+human+embryonic+stem+cells.">IGF-1 Signalling plays an important role in the formation of three dimensional laminated neural retina and other ocular structures from human embryonic stem cells.</a> Stem Cells. 33, 2416-2430 (2015).</li><li>Gonzalez-Cordero, A., Kruczek, 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=Recapitulation+of+human+retinal+development+from+human+pluripotent+stem+cells+generates+transplantable+populations+of+cone+photoreceptors.">Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors.</a> Stem Cell Reports. 9, 820-837 (2017).</li><li>Eiraku, M., Takata, N., 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=Self-organizing+optic-cup+morphogenesis+in+three-dimensional+culture.">Self-organizing optic-cup morphogenesis in three-dimensional culture.</a> Nature. 472, 51-56 (2011).</li><li>Meyer, J. S., Shearer, R. 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=Modeling+early+retinal+development+with+human+embryonic+and+induced+pluripotent+stem+cells.">Modeling early retinal development with human embryonic and induced pluripotent stem cells.</a> Proceedings of the National Academy of Sciences of the United States of America. , 16698-16703 (2009).</li><li>Reichman, S., Slembrouck, 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+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+ips+cells+in+xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human ips cells in xeno-free and feeder-free conditions.</a> Stem Cells. 35, 1176-1188 (2017).</li><li>Chen, G., Gulbranson, D. R., 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=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nature Methods. 8, 424-429 (2011).</li><li>Croze, R. H., Buchholz, D. E., 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=ROCK+inhibition+extends+passage+of+pluripotent+stem+cell-derived+retinal+pigmented+epithelium.">ROCK inhibition extends passage of pluripotent stem cell-derived retinal pigmented epithelium.</a> Stem Cells Translational Medicine. 3, 1066-1078 (2014).</li><li>Eberle, D., Schubert, S., Postel, K., Corbeil, D., Ader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+integration+of+transplanted+CD73-positive+photoreceptor+precursors+into+adult+mouse+retina.">Increased integration of transplanted CD73-positive photoreceptor precursors into adult mouse retina.</a> Investigative Ophthalmology &amp; Visual Science. 52, 6462-6471 (2011).</li></ol>

沙赖希曼, 奥利弗 Goureau, 和何塞-阿兰-萨赫勒是发明人在等待的专利相关的视网膜细胞从人类多潜能干细胞的产生。

该协议描述了如何在无异和无饲养条件下, 从人类多能干细胞中产生视网膜 RGCs 和感光细胞的视网膜色素 organoids。与良好的生产实践 (GMP) 过程兼容, 这里提出的方法允许大量生产 iPSC 视网膜细胞作为 RPE 细胞, RGCs, 和光传感器, 以发展干细胞为基础的治疗和药物未来治疗视网膜退行性疾病的发现方法。整个视网膜 organoids 或 hiRPE 细胞的超低温保存也为建立中间细胞库提供了一个重要的优势, 这是今?...

视网膜是中枢神经系统的一个组成部分, 在外伤或疾病后自发再生的能力有限。因此, 退行性病变导致明确的视网膜细胞丢失, 如年龄相关黄斑变性 (AMD), 视网膜色素变性 (RP), 青光眼和糖尿病视网膜病变, 通常导致不可逆转的失明。抢救视网膜退化是一个主要的挑战, 以干细胞为基础的治疗, 旨在取代受损或丢失的细胞是最有希望的方法1,2,3。多潜能干细胞作为人类胚胎干细胞 (ESCs) 细胞或人类诱导的多潜能干细胞 (iPSCs) 有能力在培养中无限期扩展, 它们有可能产生任何细胞类型。我们对视网膜发育的认识和改善人 iPSC 分化的体外协议的进展, 导致了视网膜 organoids7,8,9的产生, 10,11,12。所有的主要视网膜细胞, 包括视网膜神经节细胞 (RGCs), 感光细胞, 视网膜色素上皮 (RPE), 已经成功地分化为人类 ESCs 和 iPSCs4,5, 6. 以 Eiraku等为基础, 建立了 SFEB (胚状体样骨料) 的无血清培养方法。13、在定义的胞外基质成分7、10、14中, 可从 ESC 或 iPSC 衍生的胚体样骨料中获得视网膜 organoids 的自形成。但是这些协议是错综复杂的, 需要大量的步骤并不总是与大规模生产细胞的治疗方法或药物筛选。因此, 人类视网膜细胞生成方法的选择是至关重要的, 该方法需要健壮、可伸缩性和高效性。
在这里, 根据我们以前的出版物15, 我们描述了一个简单和高效的视网膜细胞的每一个步骤, 通过视网膜 organoid 自形成从黏附人 iPSCs 培养在无饲养和异的条件。从人类 iPSCs 的常规文化出发, 该协议只需要一个简单的连续培养基改变, 以允许在4周内生成 iPS 源性 RPE (hiRPE) 细胞和 neuroretinal 结构。人工隔离后, hiRPE 可以扩展, 视网膜结构可以培养为漂浮 organoids, 在那里视网膜祖细胞能够按照与体内人一致的顺序依次分化为所有视网膜细胞类型。retinogenesis。最后, 对于研究进展或临床翻译, 我们描述了一种冷冻方法, 允许长期储存的整个视网膜 organoids 和 hiRPE 细胞不影响其表型特征和功能。

<table><tbody><tr><td>Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td><td>ThermoFisher Scientific</td><td>A14700</td><td>Coating</td></tr><tr><td>CTS Vitronectin (VTN-N) Recombinant Human Protein, Truncated</td><td>ThermoFisher Scientific</td><td>A27940</td><td>Coating</td></tr><tr><td>Essential 8 Medium</td><td>ThermoFisher Scientific</td><td>A1517001</td><td>medium</td></tr><tr><td>Essential 6 Medium</td><td>ThermoFisher Scientific</td><td>A1516401</td><td>medium</td></tr><tr><td>CTS (Cell Therapy Systems) N-2 Supplement</td><td>ThermoFisher Scientific</td><td>A1370701</td><td>supplement CTS</td></tr><tr><td>N-2 Supplement (100X)</td><td>ThermoFisher Scientific</td><td>17502048</td><td>supplement</td></tr><tr><td>B-27 Supplement (50X), serum free</td><td>ThermoFisher Scientific</td><td>17504044</td><td>supplement</td></tr><tr><td>CTS B-27 Supplement, XenoFree</td><td>ThermoFisher Scientific</td><td>A1486701</td><td>supplement CTS</td></tr><tr><td>DMEM/F-12</td><td>ThermoFisher Scientific</td><td>11320074</td><td>medium</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100X)</td><td>ThermoFisher Scientific</td><td>11140035</td><td>supplement</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>ThermoFisher Scientific</td><td>15140122</td><td>antibiotic</td></tr><tr><td>CellStart CTS</td><td>ThermoFisher Scientific</td><td>A1014201</td><td>Matrix CTS</td></tr><tr><td>Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix</td><td>ThermoFisher Scientific</td><td>A1569601</td><td>Matrix</td></tr><tr><td>Gentle Cell Dissociation Reagent</td><td>Stemcell Technologies</td><td>7174</td><td>dissociation solution</td></tr><tr><td>Cryostem Freezing Media</td><td>clinisciences</td><td>05-710-1D</td><td>Cryopreservation medium</td></tr><tr><td>Fibroblast growth factor 2 (FGF2)</td><td>Preprotech</td><td>100-18B</td><td>FGF2</td></tr><tr><td>Fibroblast growth factor 2 (FGF2) animal free</td><td>Preprotech</td><td>AF-100-18B</td><td>FGF2 Xeno free</td></tr><tr><td>AGANI needle 23G</td><td>Terumo</td><td>AN*2332R1</td><td>Needle</td></tr><tr><td>Flask 25 cm&amp;sup2; Tissue Culture Treated</td><td>Falcon</td><td>353109</td><td>T-25 cm&amp;sup2;</td></tr><tr><td>24 well plate Tissue Culture Treated</td><td>Costar</td><td>3526</td><td>24-well plate</td></tr><tr><td>6 well plate Tissue Culture Treated</td><td>Costar</td><td>3516</td><td>6-well plate</td></tr></tbody></table>

defined xeno-free and feeder-free culture conditions for the generation of human ipsc-derived retinal cell models

多潜能干细胞的专业细胞的生产为再生医学的发展提供了有力的工具。使用人类诱导的多能干细胞 (iPSCs) 对神经退行性疾病研究特别有吸引力, 其中包括视网膜白斑, iPSC 视网膜细胞模型标志着一个重要的步骤, 以了解和消除失明。在本文中, 我们描述了一个简单和可扩展的协议, 以生成, 成熟, cryopreserve 视网膜 organoids。在媒介变化的基础上, 该方法的主要优点是避免了在 iPSCs 的引导分化过程中通常需要的多次和耗时的步骤。模拟视网膜发育的早期阶段由被定义的媒介的连续的变动在黏附人的 iPSC 文化, 这个协议允许同时产生自已形成的 neuroretinal 结构和视网膜色素上皮 (RPE) 细胞在4周内重现性和高效的方式。这些结构包含视网膜祖细胞 (rpc) 可以很容易地分离, 以进一步成熟的漂浮培养条件, 使 rpc 分化为七视网膜细胞类型存在于成人视网膜。此外, 我们还描述了快速保存视网膜 organoids 和视网膜色素上皮细胞长期贮存的方法。结合起来, 这里描述的方法将是有用的生产和银行人类 iPSC 的视网膜细胞或组织的基础和临床研究。

在无馈线条件下培养的人类 iPSC 分化的第一步是关闭使用双介质的自我更新机械, 以鼓励自发分化 (图 1A)。然后, 在 D2, 双介质辅以 N2 补充, 以指导分化 iPSCs 细胞对神经和视网膜血统。这个过程导致 neuroretinal 芽的出现在 D28 附近 (图 1C - 1E)。自形成的 neuroretinal 结构可以用针来隔离, 如<strong class="xfi...

Watch this Scientific Journal Video about Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models at JoVE.com

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

多潜能干细胞的专业视网膜细胞的产生是干细胞治疗视网膜疾病的一个转折点。本文介绍了一种有效生成视网膜 organoids 和视网膜色素上皮的简单方法, 用于基础、平移和临床研究。

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

defined-xeno-free-feeder-free-culture-conditions-for-generation-human

Research

JoVE Journal

Developmental Biology

9.2K 次观看.  Sorbonne Université, INSERM, CNRS, F-75012 Paris, France. 多潜能干细胞的专业视网膜细胞的产生是干细胞治疗视网膜疾病的一个转折点。本文介绍了一种有效生成视网膜 organoids 和视网膜色素上皮的简单方法, 用于基础、平移和临床研究。

视频: Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

视网膜色素上皮细胞的干细胞高效的推导

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

科研

发育生物学

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

生物学

Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority of RP patients have progressive photoreceptor degeneration leading to peripheral vision loss, night blindness, and finally, total blindness. To date, thousands of mutations in more than 90 genes have been reported to be associated with RP. Currently, there are few animal models available for all the affected genes and different types of mutations, which largely hampers the deciphering of the mechanisms underlying the gene/mutation pathology and limits treatment and drug development. Patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids (ROs) have provided a better system to model the human early-onset disease than cells and animals. In order to study RP, those patient-derived 3D retinal organoids were utilized to recapitulate the clinical phenotypes of RP. In the RP patient-derived ROs, Rhodopsin mislocalization was clearly displayed. Compared with other animal models, patient iPSC-derived retinal organoid models more closely recapitulated RP features and represent an ideal approach for investigating the disease pathogenesis and for drug development.

In this protocol, retinitis pigmentosa patient induced pluripotent stem cell (iPSC)-derived 3D retinal organoids were generated. Those organoids successfully recapitulated some clinical phenotypes of the retinitis pigmentosa disease.

Retinitis pigmentosa (RP) is a rare and inherited retinal degenerative disease with a prevalence of approximately 1/4,000 people worldwide. The majority ...

Generation of Retinal Organoids from Healthy and Retinal Disease-Specific Human-Induced Pluripotent Stem Cells

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative therapies. This protocol describes a simpler, robust, and stepwise method of generating retinal organoids in a hybrid culture system consisting of adherent monolayer cultures during the first 4 weeks of retinal differentiation till the emergence of distinct, self-organized eye field primordial clusters (EFPs). Further, the doughnut-shaped, circular, and translucent neuro-retinal islands within each EFP are manually picked and cultured under suspension using non-adherent culture dishes in a retinal differentiation medium for 1-2 weeks to generate multilayered 3D optic cups (OC-1M). These immature retinal organoids contain PAX6+ and ChX10+ proliferating, multipotent retinal precursors. The precursor cells are linearly self-assembled within the organoids and appear as distinct radial striations. At 4 weeks after suspension culture, the retinal progenitors undergo post-mitotic arrest and lineage differentiation to form mature retinal organoids (OC-2M). The photoreceptor lineage committed precursors develop within the outermost layers of retinal organoids. These CRX+ and RCVRN+ photoreceptor cells morphologically mature to display inner segment-like extensions. This method can be adopted for generating retinal organoids using human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). All steps and procedures are clearly explained and demonstrated to ensure replicability and for wider applications in basic science and translational research.

This protocol describes an efficient method of differentiating hiPSCs into eye field clusters and generating neuro-retinal organoids using simplified culture conditions involving both adherent and suspension culture systems. Other ocular cell types, such as the RPE and corneal epithelium, can also be isolated from mature eye fields in retinal cultures.

从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官

Pluripotent stem cells can generate complex tissue organoids that are useful for in vitro disease modeling studies and for developing regenerative ...

增强人胚胎干细胞的 3D 神经视网膜分化

Generating Neural Retina from Human Pluripotent Stem Cells

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of retinopathy. Unfortunately, ethical barriers hinder the collection of evidence from humans. Recently, numerous studies have shown that human pluripotent stem cells (PSCs) can be differentiated into retinal organoids (ROs) using different induction protocols, which have enormous potential in retinopathy for disease modeling, drug screening, and stem cell-based therapies. This study describes an optimized induction protocol to generate neural retina (NR) that significantly reduces the probability of vesiculation and fusion, increasing the success rate of production until day 60. Based on the ability of PSCs to self-reorganize after dissociation, combined with certain complementary factors, this new method can specifically drive NR differentiation. Furthermore, the approach is uncomplicated, cost-effective, exhibits notable repeatability and efficiency, presents encouraging prospects for personalized models of retinal diseases, and supplies a plentiful cell reservoir for applications such as cell therapy, drug screening, and gene therapy testing.

作者聚焦：用于疾病建模的简单高效的神经视网膜类器官生产

The present protocol describes an optimized 3D neural retina induction system that reduces the adhesion and fusion of retinal organoids with high repeatability and efficiency.

从人类多能干细胞生成神经视网膜

Retinopathy is one of the main causes of blindness worldwide. Investigating its pathogenesis is essential for the early diagnosis and timely treatment of ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

定义的无异和无饲养培养条件的人 iPSC 视网膜细胞模型的生成

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Generation of Human Patient iPSC-derived Retinal Organoids to Model Retinitis Pigmentosa

从健康和视网膜疾病特异性人诱导的多能干细胞中生成视网膜类器官

从人类多能干细胞生成神经视网膜

从人类多能干细胞生成神经视网膜

从人类多能干细胞生成神经视网膜

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备