Name: directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells
Uploaded: 2017-11-01T12:30:00.000+00:00
Duration: 14 min 37 s
Description: Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

这项工作得到了美国华盛顿大学医学院血液科内科医学院的支持。T32HL007088 来自国家心脏、肺脏和血液研究所的支持。CMS 得到了美国血液学学者奖的支持。

1. 试剂 <ol> <li> 获取单元格线; 干细胞或 hiPSCs 1 、小鼠胚胎成纤维细胞 (MEFs) 29 、OP9-DL4 基质 30 、 31 . </li> <li> 试剂准备 <ol> <li> 在 PBS 中准备一个0.1% 瓦特/v 的明胶溶液。4、#176 灭菌, aliquoting 后进行消毒;
		<ol> <li> 准备明胶涂层 plasticware。外套 6-1.5 毫升的0.1% 明胶溶液的菜肴。室温孵育15分钟。使用前应立即抽出残留明胶溶液. </li>
		</ol> </li> <li> 通过使 IMDM 与15% 伏/v FBS 和1x 和 #160; 抗生素来准备 MEF 介质。补充2毫米 l-谷氨酰胺和400和 #181; M monothioglycerol (MTG) 之前使用和存储在4和 #176; C. </li>
		<li> 准备 hPSC 培养基作为 DMEM/F12 20% v/v 淘汰赛血清置换 (KOSR) 的解决方案, 1x、#160; 非必需氨基酸 (NEAA)、1x 和 #160; 抗生素、55、#181; M 和 #946;-基、2毫米-l-谷氨酰胺和 10 ng/毫升 bFGF。过滤器用2和 #181; m 过滤器和存储在黑暗中在4和 #176; C. </li>
		<li> 以5% 伏/v KOSR 和 4 mM HCl 为 DMEM/F12 的溶液制备无血清洗涤介质. 过滤器用2和 #181 进行灭菌; m 过滤器, 分, 和存储在黑暗中4和 #176; C. </li>
		<li> 1 毫克/毫升 dnasei i 溶液溶解 dnasei i 为 3-4 小时到 ice-cold, 无菌去离子水冰。过滤器用2和 #181; m 过滤器和存储等分在-20 和 #176; C. </li>
		<li> 使用洗涤介质准备停止解决方案: 添加 10% FBS 和10和 #181; g/毫升 dnasei i。停止解决方案应在使用前立即准备. </li>
		<li> 准备基底膜基体 ( 例如, , 基质, 简称为垫) 混合溶液. 
		注: 所有的步骤准备和使用的垫子混合物应在冰上或在4和 #176; c. 解冻结冰的垫在冰在4和 #176; c 隔夜。稀释垫1:1 通过加入10毫升 ice-cold IMDM 和冰寒意在4和 #176; C 过夜。稀释垫混合物与额外的20毫升 ice-cold IMDM 和寒冷的冰在4和 #176; C 过夜。分到 pre-chilled 管和存储在-20 和 #176; C 直到使用。当准备好使用, 解冻垫混合物隔夜在4和 #176;
		<ol> <li> 将垫子细胞培养皿涂上. 
			注: 涂布垫板的所有步骤应在冰上进行。预冷6井和24井板在-20 和 #176; C 至少1小时。当准备好涂上盘子时, 把它们放在冰上。用 4 x-diluted 垫把每个井涂好, 去除并再利用任何多余的溶液。在冰上留 30-60 分钟, 并抽出多余的垫子。在37和 #176 中将涂布板擦干; C 5% CO 2 孵化器至少 3 h. 在涂层的 72 h 内使用. </li>
		</ol> </li> <li> 将10% 牛血清白蛋白 (bsa) 的溶液溶解在 ice-cold 中, 并将其分解为 10% w/v bsa, 蒸馏, 去离子水在4和 #176; c 为 3-4 h. 过滤器消毒用2和 #181; m 过滤入遮光瓶并且存放在4和 #176; c. </li>
		<li> 准备抗坏血酸溶液。缓慢溶解5毫克/毫升的 l-抗坏血酸的溶液在 ice-cold, 蒸馏, 去离子水在冰 3-4 h. 漩涡偶尔地直到溶化。过滤器用2和 #181; m 过滤器和存储等分在-20 和 #176; C. </li>
		<li> SFD 75:25 的 IMDM: 火腿和 #39 F-12, 然后加入 0.5% BSA, 1x 和 #160; B27, 0.5x 和 #160; N2 补充剂, 制备无血清分化培养基。存储在4和 #176; c. 添加1x 和 #160; L-谷氨酰胺, 50 和 #181; g/毫升抗坏血酸, 400 和 #181; M MTG, 150 和 #181; 在使用前立即的全息-转铁蛋白. </li>
		<li> 制备无血清干细胞培养基 ( (如 , StemPro, 称为 SP34) 根据制造商和 #39 的指示。存储在4和 #176; c. 添加1x 和 #160; L-谷氨酰胺, 50 和 #181; g/毫升抗坏血酸, 400 和 #181; M MTG, 150 和 #181; 在使用前立即的全息-转铁蛋白. </li>
		<li> 制备0.2% 胶原酶 ii 溶液通过溶解胶原酶 ii 到最后浓度0.2% 瓦特/v 在 1:4 FBS: PBS 溶液。溶解在37和 #176; c 水浴至少15分钟过滤器, 以2和 #181; m 过滤和存储等分在-20 和 #176; c. </li>
		<li> 在使用前立即准备荧光活化细胞分类 (FBS) 缓冲液作为 5% IMDM 的解决方案. </li>
		<li> 准备 OP9 介质作为 #945 的解决方案;-记忆, 20% FBS, 2 毫米 l-谷氨酰胺, 和1x 和 #160; 抗生素。过滤器以 2 #181; m 过滤和存储在4和 #176; C. </li>
		<li> 获取 methylcellulose-based 媒体 ( 如 , MethoCult, 在此称为 pre-aliquoted) 为3毫升的数量或作为一个100毫升瓶。如果使用100毫升, 在到达时, 分为2.5 毫升等分在14毫升锥形管. 
		注: 所有等分应存储在-20 和 #176; C. 等分应在使用前立即解冻. </li>
	</ol> </li> </ol> 2。胚层分化的 hPSCs <ol> <li> 将 MEFs 上的 hPSC 单元格线增长为 70-80% 70-100, 在37和 #176 中; C 孵化器, 5% CO 2 。
	注意: hPSCs 应该有尖锐的, 未分化的边界. </li>
	<li> 在传代 hPSCs 前准备 plasticware 24 小时. 
	注: 涂覆的6个盘子的总数量在不同的 hPSC 线。通常情况下, 三 6-好菜是足够的每 6-好菜的汇合 hPSCs 在 MEFs。
	<ol> <li> 执行以不同比例的汇合 hPSCs: 垫井 (1:4、1:3、1:2、 等 ) 在第一次分化之前的试验馈线损耗, 以确定在随后的区分中应使用多少席涂覆的 6-好的菜肴. 
		注:24 小时后在垫子上电镀后, hPSCs 应该是 80-90% 汇合, 与细胞团簇约 10-20 细胞大小. </li>
	</ol> </li> <li> 将吸入 hPSC 介质, 并添加 1 ml 37 和 #176; C 0.25% 胰蛋白酶-EDTA 每井1分钟, 并添加1毫升停止介质到每个井。用刮板机刮掉盘子上的细胞。每井加1毫升洗涤缓冲器. 
	<ol> <li> 使用2毫升血清学吸管, 研制 3-5 次, 以分解大块的细胞, 并将细胞转移到50毫升锥形管。离心 hPSCs 在 330 x g 为 5 min. </li>
	</ol> </li> <li> 重 hPSC 介质的总容量中的单元格, 相当于每张涂布 plasticware 的2毫升。添加2毫升/井 hPSCs 到 plasticware 和孵化过夜在一个37和 #176; C 孵化器与 5% CO 2 . </li>
	<li> 24 小时后, 抽吸介质并替换为37和 #176; C 0.05% 胰蛋白酶-edta 为1分钟, 抽出胰蛋白酶-edta 并添加1毫升停止介质。用刮板机用力刮细胞。每井加1毫升洗涤介质。2毫升血清吸管, 研制细胞 1-2 倍, 并转移到50毫升锥形管。离心机细胞在 220 x g 为 5 min. 
	注: 胰蛋白酶-EDTA 处理时间长度ld 由每 hPSC 线的调查员确定。胰蛋白酶的处理应尽可能长时间地应用, 而不会引起单细胞分离。这一步应该将所有的 hPSCs 从垫子涂层的盘子中分离出来, 但不会导致单细胞离解。结果的 EBs 平均应为 6-10 单元. </li>
	<li> 吸出介质。使用5毫升血清吸管, 轻轻重20毫升洗涤介质中的细胞。离心机在 220 x g 为 5 min. </li>
	<li> 在0天的媒体中轻轻重 EBs ( 表 1 )。使用10毫升血清吸管, 在6井 low-adherent 细胞培养皿的每一个井中分配2毫升的分化介质, 包含 EBs。放置到37和 #176; C 5% CO 2 5% O 2 (气体) 孵化器. 
	注: 0 天介质: SFD 介质辅以 10 ng/mL BMP4 ( 表 1 )。每两个盘子的 hPSCs, 使用一个6井 low-adherent 细胞培养皿. </li>
	<li> 24 小时后, 添加 2 mL 天1媒体 ( 表 1 )。在气体孵化室过夜. 
	注: 1 天媒体: SFD 培养基辅以 10 ng/毫升 BMP4 和 10 ng/毫升 bFGF ( 表 1 ). </li>
	<li> 18 小时后, 用5毫升血清吸管轻轻地收割 EBs, 并在 100 x g 处旋转5分钟, 并将整个养殖与10毫升 IMDM。在 100 x g 旋转5分钟, 吸气介质. 
	注意: 碎片将存在于文化中。这种离心步骤在低速 (100 x g) 将清除碎片. </li>
	<li> 轻轻地重 SFD 介质中的 EBs, 并辅以天2介质 (表 1), 然后将2毫升每井分配到6井 low-adherent 细胞培养板中。在37和 #176 孵化; C 气体孵化30小时 
	注: 2 天培养基:10 ng/毫升 BMP4, 5 ng/毫升 bFGF, 以及适当的 WNT 激动剂或拮抗药 ( 表 1 )。
	<ol> <li> 添加3和 #181; M CHIR99021 以指定最终的造血祖细胞。另外, 添加3和 #181; M IWP2 和 1 ng/毫升激活 A 来指定原始造血祖细胞. </li>
	</ol> </li> <li> 继续进行造血祖规范的3节. </li> </ol>
 3. CD34 的规范 + 造血祖细胞 <ol> <li> 在30小时后, 将 EBs 与2毫升血清吸管分离, 在3天的分化。转到50毫升锥形管和离心机在 330 x g 5 分钟轻轻重和洗涤与10毫升的 IMDM。再次离心的细胞在 330 x g 为 5 min. </li>
	<li> 在3天的媒体 ( 表 2 ) 中重了 EBs, 并将2毫升分配到 low-adherent 6 井板的每个井中。孵育细胞在37和 #176; C 气体孵化器为 72 h. 
	注: 3 天培养基: SP34, 辅以 15 ng/毫升 VEGF 和 5 ng/毫升 bFGF ( 表 2 )。如果3天的介质 pH 值发生显著变化 ( 即 , 黄色-橙色介质), 则每井添加更多介质。或者, 在0天的每个井使用较少的 EBs. </li>
	<li> 在72小时的孵育后, 将2毫升的日6介质 ( 表 2 ) 添加到每个井中。孵育在37和 #176; C 气体孵化器为额外的 48 h. 
	注: 6 天媒体: SP34 培养基辅以 15 ng/毫升 VEGF, 5 ng/毫升 bFGF, 200 ng/毫升, 4 的 EPO, 20 ng/毫升 IL-6, 10 ng/ml IL-11, 和 50 ng/ml IGF-1 ( 表 2 )。如果在步骤3.1 中添加了更多的介质, 则在步骤3.2 中添加相同数量的介质, 以便最终的细胞因子浓度保持不变. </li>
	<li> 在差异化的8天隔离 CHIR99021-treated EBs。继续执行4节. </li>
	<li> IWP2-treated EBs 是在8天的分化成50毫升锥形管收获。离心机在 330 x g, 5 分钟轻轻重在8天的媒体 ( 表 2 )。在37和 #176 中孵育24小时; C 5% CO 2 在低粘着的盘子里孵化。继续执行4节. 
	注: 8 天媒体: SP34 媒体辅以 100 ng/毫升, 2 的 EPO, 10 ng/毫升 IL-6, 5 ng/毫升 IL-11, 25 ng/毫升 IGF-1, 30 ng/毫升 TPO, 10 ng/毫升 Flt3-L, 30 ng/ml IL-3 ( 表 2 ). </li>
</ol> 4。ebs 与造血祖分离酶解离的研究 <ol> <li> 将 ebs 与血清学吸管分离为50毫升锥形管。离心机在 330 x g, 5 分钟从上清液中抽出. </li>
	<li> 在 EBs 中加入2毫升的0.25% 胰蛋白酶-EDTA。5 s 的漩涡孵化37和 #176; C 水浴8分钟添加5毫升的停止解决方案。离心机在 330 x g, 5 分钟从上清液中抽出. </li>
	<li> 重5毫升胶原酶 II. 的 EBs漩涡为 5 s 和孵育在一个37和 #176; C 水浴。60分钟后, 涡流为5秒, 并添加5毫升的停止解决方案。在 330 x g 处旋转5分钟, 从上清液中抽出. </li>
	<li> 在1毫升的外地资产管制的缓冲区, 重的细胞。通过 40 #181; m 细胞过滤器。这将消除任何不细胞团簇. </li>
	<li> 对单元格进行计数。分 1 x 10 6 细胞变成14毫升锥形管。旋转细胞在 330 x g 为5分钟重的细胞在浓度为 5 x 10 6 细胞/毫升在外地资产管制缓冲区。分和池10% 的每个条件下的单元格流细胞颜色控制。在冰上, 在黑暗中孵育抗体的细胞30分钟. 
	注意: 请参见 材料表 中建议的荧光组合。
	<ol> <li> 对于 IWP2-treated 的单元格, 使用 anti-CD34 和 anti-CD43 抗体. </li>
		<li> 对于 CHIR99021-treated 单元格, 请使用 anti-CD34、anti-CD43、anti-CD73 和 anti-CD184 抗体. </li>
	</ol> </li> <li> 使用 "资产管制" 缓冲区清洗两次单元格。在 330 x g 上旋转5分钟, 重最后浓度为 5 x 10 6 细胞/毫升. </li>
	<li> 通过流式细胞仪分析或分离细胞。对于原始造血祖细胞, 分析 CD34 和 CD43 表达式 ( 图 2A )。对于最终造血祖细胞, 隔离他 (CD34 + CD43 和 #8722; CD73 和 #8722; CD184 和 #8722; ) ( 图 2B )。收集含有冷的控制的缓冲区的管中的细胞. </li>
	<li> 为了评估从孤立他的最终造血潜能, 继续到5节的 endothelial-to-hematopoietic 过渡, 或7节 T 淋巴化验。对于原始的造血 CFC 化验, 继续6节. </li>
</ol> 5。Endothelial-to-hematopoietic 转换 <ol> <li> 旋转隔离的 CD34 + CD43 和 #8722; CD73 和 #8722; CD184 和 #8722; 在 330 x g 中的 5 min 重细胞在20万细胞/mL 和 #160;( 中的单元格表 2 )。在50和 #181 中分布单元格; 等分在 96 low-adherent 细胞培养板中。在37和 #176 的夜间孵育细胞; C 5% CO 2 5% O 2 气体孵化器. 
	注: 媒体: SP34 培养基辅以 5 ng/毫升 VEGF, 5 ng/毫升 bFGF, 100 ng/毫升, 2 的 EPO, 10 ng/毫升 IL-6, 5 ng/毫升 IL-11, 25 ng/毫升 IGF-1, 30 ng/毫升 TPO, 10 ng/毫升 FLT3-L, 30 ng/ml IL-3, 10 ng/毫升 BMP4, 20 ng/毫升嘘, 10 和 #181; g/毫升血管紧张素 II, 和100和 #181; M 洛沙坦钾在 2 x 10 5 单元格/ml ( 表 2 )。
	根据需要, <li> 准备24井垫涂层板 (参见步骤 1.2.7.1). </li>
	<li> 这些细胞将在一夜之间聚集成 6-10 细胞团簇。对于每50和 #181; reaggregated 细胞的体积, 在第二天早上轻轻地将细胞转移到一个24层的垫子涂层的中心。轻轻地把盘子放在37和 #176; C 气体孵化器为 4-6 小时, 直到细胞被连接. </li>
	<li> 添加1毫升的新鲜他的媒体, 每个井后, 细胞连接。在37和 #176 中孵育细胞; C 5% CO 2 5% O 2 气体孵化器, 直到造血细胞可见 (通常为5-7 天; 图 2D )。使用光学显微镜在100X 放大倍率下可视化. </li>
	<li> 通过轻轻地将介质从细胞中除去, 从而获得最终的造血祖。通过一个40和 #181; m 细胞过滤器和收集通过这个媒体。在井的黏附细胞, 增加0.5 毫升0.25% 胰蛋白酶-EDTA 对细胞和孵育在37和 #176; C 为 5 min. 在每个井中添加0.5 毫升停止解决方案。通过相同的40和 #181 的细胞; m 细胞过滤器汇集所有黏附和换药细胞一起。在 330 x g 上旋转5分钟. 
	<ol> <li> 若要评估此批量区域性的 CFC 潜能, 请继续执行6节. </li>
	</ol> </li> <li> 在内存中的缓冲区中清洗两次单元格。在 330 x g 上旋转5分钟. </li>
	<li> 重在 5 x 10 6 单元/mL 中的单元格的缓冲区中。在黑暗中用 anti-CD45 和 anti-CD34 抗体将细胞染色30分钟 (建议的荧光在 材料表 中). </li>
	<li> 在外地资产缓冲区中清洗两次单元格。在 330 x g 上旋转5分钟. </li>
	<li> 将 CD34 + CD45 + 单元格由资产管制署隔离 ( 图 2E )。将单元格分类为冷的 "资产管制" 缓冲区. </li>
	<li> 根据需要评估确定的 CD34 + CD45 + 造血祖细胞 (6 节中的氟氯化碳检测, 7 节中的淋巴潜能, 或其他调查员发起的化验). </li>
</ol> 6。氟氯化碳测定方法 <ol> <li> 对用于氟氯化碳测定的每个样品的等分进行解冻. </li>
	<li> 添加要检测的单元格 (步骤4.5、4.7、5.5 或 5.9)。彻底的涡旋, 让我们的文化 5-10 分钟, 直到气泡消散, 根据制造商和 #39 的指示。使用16和 #189; 在3毫升注射器上用量规针, 小心地移除2毫升. 
	注: 典型的电镀密度范围从 1000-2万细胞/毫升, 根据预期的祖频率. </li>
	<li> 小心, 在两个 35 mm 的组织培养皿中分别分配1毫升的细胞混合物。通过轻轻地敲击和旋转盘子, 在 35 mm 的盘子里均匀地分布着。将上述每个菜肴放入15厘米的组织培养皿中, 盛满水。孵育在37和 #176; C 5% CO 2 孵化器. </li>
	<li> 使用40X 和 #160 的光学显微镜来可视化微型显示器; 放大。量化所获得的各种氟氯化碳。分析原始 CFC 检测 8-10 天后电镀 ( 图 2C )。在电镀后14天内分析最终的氟氯化碳化验。典型的氟氯化碳检测菌落形态可以在 图 2F 中看到. </li>
</ol> 7。T 细胞检测建立最终造血电位 <ol> <li> 生长 OP9-DL4 细胞直到融合在一个100毫米组织培养皿中 30 , 31 , 32 . <ol> <li> 在电镀 T 细胞化验前解冻 OP9-DL4 细胞 72-96 h。重10毫升 OP9 介质中的 OP9-DL4 细胞, 并分配到10厘米的板。生长在37和 #176; C 5% CO 2 孵化器直到 70-90% 汇合. </li>
		<li> 从100毫米的盘子中采集 OP9-DL4 细胞, 取出培养基, 加入5毫升0.25% 胰蛋白酶-EDTA 5 分钟, 停止胰蛋白酶活性与5毫升 OP9 介质。旋转 330 x g 的细胞5分钟, 重1毫升 OP9 培养基中的细胞并计数细胞. 
		注: 一个10厘米的汇合 OP9-DL4 细胞将产生约 10 x 10 6 OP9-DL4 细胞. </li>
		<li> 48 小时之前的 T 细胞检测, 板 2 x 10 6 在12毫升 OP9 介质中的 24-井盘 (0.5 毫升/井) 的细胞。在37和 #176 中成长; C 5% CO 2 孵化器. </li>
	</ol> </li> <li> 添加要检测的 2000-1万单元格 (在步骤4.5 中获得), 4.7、5.5 或 5.9) 至1井中的 OP9-DL4 细胞在 OP9 介质中补充:30 ng/毫升 (只6天), 5 ng/毫升 IL-7, 和 5 ng/毫升 FLT3-L. 孵育在37和 #176; C 5% CO 2 公司ubator在此步骤中不发生任何媒体更改. </li>
	<li> 每 4-5 天, 通过 T 细胞祖细胞到新鲜的 OP9-DL4 基质干细胞在一个6井盘。研制1毫升吸管的细胞, 通过一个40和 #181; m 过滤器, 以消除结块。在 330 x g 旋转5分钟。重2毫升新鲜 OP9 培养基补充 5 ng/毫升 IL-7 和 5 ng/毫升 Flt3-L, 并放置在新鲜 OP9-DL4 细胞. 
	注:48 小时前传代, 板 2 x 10 6 新鲜 OP9-DL4 细胞在12毫升 OP9 介质, 并分发2毫升/井到 6-好菜. </li>
	<li> 经过至少21天的共培养, 研制细胞, 并通过一个40和 #181; m 过滤器, 以消除细胞碎片。在 330 x g 旋转5分钟, 吸气上清液. </li>
	<li> 在冷的缓冲区中清洗两次单元格。在 330 x g 上旋转5分钟. </li>
	<li> 重在 5 x 10 6 单元/mL 中的单元格的缓冲区中。染色细胞与 anti-CD45, anti-CD56, anti-CD4 和 anti-CD8 抗体30分钟冰在黑暗中 (建议的荧光组合在 材料表 )。应用活/死细胞染色 (DAPI, 等 ) 去除分析中的碎片. </li>
	<li> 在外地资产缓冲区中清洗两次单元格。在 330 x g 上旋转5分钟. </li>
	<li> 使用流式细胞仪分析单元格以评估 T 淋巴细胞祖群的存在 ( 图 3 ). </li>
</ol>

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作者声明他们没有竞争的金融利益。

本议定书描述一个快速, 无血清, 无基质的方法分化的原始或最终造血祖细胞。胚层规范的原始或最终造血祖可以可靠地实现使用我们的协议, 这唯一的利用小分子抑制剂的规范 WNT 信号。阶段特异的 WNT 激活由 GSK3β抑制剂 CHIR9902133产生最终的造血胚层, 而 WNT 抑制剂 PORCN 抑制的 IWP234指定原始的造血胚层26。值得注意的是, 这种方法并不会产生 eng...

人类多能干细胞 (hPSCs) 被定义为包括人类胚胎干细胞 (干细胞) 和人类诱导多能干细胞 (hiPSCs), 并具有独特的能力, 不仅在适当的生长条件下进行自我更新,而且, 可以区分为从三种细菌层派生的所有单元类型: 内胚层、胚层和外胚层1。由于这些独特的能力, hPSCs 的再生医学, 疾病建模和细胞疗法的巨大希望2。虽然多个细胞类型已成功地区分了 hPSCs, 一个重要的挑战是体外规范的完全成人样 hPSC 衍生造血干细胞 (干细胞) 和最终造血祖细胞。
一个可能的障碍, 发展的人类干细胞从 hPSCs 是存在的多个造血程序在人类胚胎的3。第一个程序出现, 称为 "原始造血", 起源于外卵黄囊组织内, 最主要的特点是其瞬态生产的 erythroblast 祖 (EryP-氟氯化碳), 巨噬细胞, 和巨。值得注意的是, 这个程序并没有引起干细胞, 也没有引起 T 和 B 淋巴祖细胞。然而, 卵黄囊确实瞬时产生限制性的造血祖细胞, 如赤髓祖 (EMP4,5,6,7,8和红缺乏的淋巴引物多能祖 (LMPP9)。然而, 无论是 EMPs 还是 LMPPs 都是完全多能的, 或者能够在成人接受者身上植入。相比之下, 后来在发展, 经典定义的 "决定性" 的造血程序是指定的主动脉-性腺-肾区的胚胎正常, 导致所有成年造血血统, 包括 HSC。这些 intra-embryonic 明确造血细胞的规格发生在一个缺口依赖性的方式, 通过 endothelial-to-hematopoietic 过渡从 hemogenic 内皮 (他)3,10,11 ,12,13,14。除了重建能力, 这些细胞的多向电位和缺口依赖性可以用来区分这些最终的造血祖与 EMP 和 LMPP (在参考文献3,13).
了解 hPSCs 的原始和明确的造血规范的机制, 可能对在各种 hPSC 线上的最终造血祖细胞的再生生产至关重要。直到最近, hPSC 分化的协议, 可以分离多能原始和最终造血祖细胞不存在15,16,17,18, 19,20,21,22,23,24,25。许多方法使用胎牛血清 (FBS) 和/或基质共培养首先概述了 hPSC 分化的造血潜能, 与原始和最终造血电位的混合物15,16, 17,19,22,23,25。此外, 许多无血清造血协议描述的信号要求的规格胚层从 hPSCs 的海港造血电位18,20,21,24. 然而, 由于这些方法仍然产生了两种程序的异构混合物, 所以它们在临床应用和理解发展机制上的应用可能是有限的。
我们最近建立了这些研究, 概述了激活/节和 WNT 信号的阶段特定的信号要求的原始和最终的造血规范从 hPSC 派生的胚层18,26.后者是特别独特的, 因为它的使用阶段特定的 WNT 信号操作允许指定完全原始或完全明确造血祖细胞26。在胚层规范中, 标准 WNT 信号与 PORCN 抑制剂 IWP2 的抑制作用导致 CD43+ EryP-氟氯化碳和髓祖细胞的规范, 没有可检测到的淋巴潜能。与 GSK3β抑制剂 (CHIR99021) 在同一阶段的分化过程中, 对标准 WNT 信号的刺激形成鲜明对比, 导致完全缺乏可检测的 CD43+ EryP-CFC, 同时导致CD34+CD43−的规范。该种群具有髓样、 HBG表达红和 T 淋巴细胞电位。随后的分析发现, 他缺乏 CD7327、28和 CD18428的表达式, 其造血电位与缺口相关的28。此外, 单细胞克隆分析表明, 这些最终的造血谱系可以从多能单细胞28。在一起, 这些研究表明, 阶段特定的 WNT 信号操纵可以指定纯原始造血祖, 或多能缺口依赖的最终造血祖细胞。
在这里, 我们概述了我们的差异化策略, 产生完全原始或最终造血祖, 通过操纵规范 WNT 信号在胚层模式, 以及他们的下游造血谱系化验。这项协议是非常有价值的调查谁对生产的原始或最终造血祖 hPSCs 的再生医学应用。

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMD)</td><td>Corning</td><td>10-016</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), ES cell rated</td><td>Gemini Bioproducts</td><td>100-500</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SH30396.03</td><td></td></tr><tr><td>L-glutamine, 200 mM solution</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Life Technologies</td><td>25300054</td><td></td></tr><tr><td>Gelatin, porcine skin, Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Alpha-MEM</td><td>Life Technologies</td><td>12000-022</td><td></td></tr><tr><td>DMEM-F12</td><td>Corning</td><td>10-092-CV</td><td></td></tr><tr><td>Knock-out serum replacement</td><td>Life Technologies</td><td>10828028</td><td>&quot;KOSR&quot;</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Life Technologies</td><td>11140050</td><td></td></tr><tr><td>b-mercaptoethanol, 55 mM solution</td><td>Life Technologies</td><td>21985023</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td>H1758</td><td></td></tr><tr><td>Fraction V, Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP1605</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Corning</td><td>10-080</td><td></td></tr><tr><td>N2 supplement</td><td>Life Technologies</td><td>17502048</td><td></td></tr><tr><td>B27 supplement, no vitamin A</td><td>Life Technologies</td><td>12587010</td><td></td></tr><tr><td>Stempro-34 (SP34)</td><td>Life Technologies</td><td>10639011</td><td>&quot;SP34&quot;</td></tr><tr><td>Growth factor reduced Matrigel</td><td>Corning</td><td>354230</td><td>&quot;MAT&quot;</td></tr><tr><td>L-absorbic acid</td><td>Sigma-Aldrich</td><td>A4403</td><td></td></tr><tr><td>Human serum transferrin</td><td>Sigma-Aldrich</td><td>10652202001</td><td></td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma-Aldrich</td><td>M6145</td><td></td></tr><tr><td>Collagenase B</td><td>Roche</td><td>11088831001</td><td></td></tr><tr><td>Collagenase II</td><td>Life Technologies</td><td>17101015</td><td></td></tr><tr><td>DNaseI</td><td>Calbiochem</td><td>260913</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Life Technologies</td><td>14190144</td><td></td></tr><tr><td>bFGF</td><td>R&amp;amp;D Systems</td><td>233-FB</td><td></td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP</td><td></td></tr><tr><td>Activin A</td><td>R&amp;amp;D Systems</td><td>338-AC</td><td></td></tr><tr><td>VEGF</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td></td></tr><tr><td>SCF</td><td>R&amp;amp;D Systems</td><td>255-SC</td><td></td></tr><tr><td>IGF-1</td><td>R&amp;amp;D Systems</td><td>291-G1</td><td></td></tr><tr><td>IL-3</td><td>R&amp;amp;D Systems</td><td>203-IL</td><td></td></tr><tr><td>IL-6</td><td>R&amp;amp;D Systems</td><td>206-IL</td><td></td></tr><tr><td>IL-7</td><td>R&amp;amp;D Systems</td><td>207-IL</td><td></td></tr><tr><td>IL-11</td><td>R&amp;amp;D Systems</td><td>218-1L</td><td></td></tr><tr><td>TPO</td><td>R&amp;amp;D Systems</td><td>288-TP</td><td></td></tr><tr><td>EPO</td><td>Peprotech</td><td>100-64</td><td></td></tr><tr><td>Flt-3 ligand (FLT3-L)</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris</td><td>4423</td><td></td></tr><tr><td>IWP2</td><td>Tocris</td><td>3533</td><td></td></tr><tr><td>Angiotensin II</td><td>Sigma-Aldrich</td><td>A9525</td><td></td></tr><tr><td>Losartan Potassium</td><td>Tocris</td><td>3798</td><td></td></tr><tr><td>CD4 PerCP Cy5.5 Clone RPA-T4</td><td>BD Biosciences</td><td>560650</td><td>Dilution 1:100; T cell assay</td></tr><tr><td>CD8 PE Clone RPA-T8</td><td>BD Biosciences</td><td>561950</td><td>Dilution 1:10; T cell assay</td></tr><tr><td>CD34 APC Clone 8G12</td><td>BD Biosciences</td><td>340441</td><td>Dilution 1:100; EHT assay</td></tr><tr><td>CD34 PE-Cy7 Clone 8G12</td><td>BD Biosciences</td><td>348801</td><td>Dilution 1:100; Hemogenic endothelium</td></tr><tr><td>CD43 FITC Clone 1G10</td><td>BD Biosciences</td><td>555475</td><td>Dilution 1:10; Hemogenic endothelium</td></tr><tr><td>CD45 APC-Cy7 Clone 2D1</td><td>BD Biosciences</td><td>557833</td><td>Dilution 1:50; T cell assay</td></tr><tr><td>CD45 eFluor450 Clone 2D1</td><td>BD Biosciences</td><td>642284</td><td>Dilution 1:50; EHT assay</td></tr><tr><td>CD56 APC Clone B159</td><td>BD Biosciences</td><td>555518</td><td>Dilution 1:20; T cell assay</td></tr><tr><td>CD73 PE Clone AD2</td><td>BD Biosciences</td><td>550257</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>CD184 APC Clone 12G5</td><td>BD Biosciences</td><td>555976</td><td>Dilution 1:50; Hemogenic endothelium</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole (DAPI)</td><td>BD Biosciences</td><td>564907</td><td>Dilution 1:10,000; T cell assay</td></tr><tr><td>OP9 DL4 cells</td><td></td><td></td><td>Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156</td></tr><tr><td>MethoCult H4034</td><td>Stemcell Technologies</td><td>4034</td><td>&quot;MeC&quot;</td></tr><tr><td>Milli-Q water purification system</td><td>EMD Millipore</td><td></td><td></td></tr><tr><td>5% CO2 incubator</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>Multigas incubator</td><td></td><td></td><td>Set at 37 C, 5% CO2, 5% O2</td></tr><tr><td>6 well tissue culture plate</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>24 well tissue culture plate</td><td>Corning</td><td>353226</td><td></td></tr><tr><td>6 well low-adherence tissue culture plate</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>24 well low-adherence tissue culture plate</td><td>Corning</td><td>3473</td><td></td></tr><tr><td>35 mm tissue culture dishes</td><td>Corning</td><td>353001</td><td></td></tr><tr><td>Blunt-end needle, 16 gauge</td><td>Corning</td><td>305198</td><td></td></tr><tr><td>3 cc syringes</td><td>Corning</td><td>309657</td><td></td></tr><tr><td>5 mL polypropylene test tube</td><td>Corning</td><td>352063</td><td></td></tr><tr><td>5 mL polystyrene test tube</td><td>Corning</td><td>352058</td><td></td></tr><tr><td>15 mL polypropylene conical</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50 mL polypropylene conical</td><td>Corning</td><td>430921</td><td></td></tr><tr><td>2 mL serological pipette</td><td>Corning</td><td>357507</td><td></td></tr><tr><td>5 mL serological pipette</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>10 mL serological pipette</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Corning</td><td>4489</td><td></td></tr><tr><td>Cell scrapers</td><td>Corning</td><td>353085</td><td></td></tr><tr><td>2.0 mL cryovials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>5 mL test tube with 40 &amp;micro;M cell strainer</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>40 &amp;micro;M cell strainer</td><td>Corning</td><td>352340</td><td></td></tr><tr><td>Cell culture centrifuge</td><td></td><td></td><td></td></tr><tr><td>Biosafety hood</td><td></td><td></td><td></td></tr><tr><td>FACS AriaII or equivalent</td><td></td><td></td><td></td></tr><tr><td>LSRii or equivalent</td><td></td><td></td><td></td></tr><tr><td>FlowJo software</td><td>TreeStar</td><td></td><td></td></tr><tr><td>Water bath</td><td></td><td></td><td>Set at 37 C</td></tr><tr><td>0.22 &amp;micro;M filtration system</td><td>Corning</td><td></td><td></td></tr><tr><td>Autoclave</td><td></td><td></td><td></td></tr><tr><td>4 C refrigerator</td><td></td><td></td><td></td></tr><tr><td>-20 C Freezer</td><td></td><td></td><td></td></tr><tr><td>-80 C Freezer</td><td></td><td></td><td></td></tr></tbody></table>

directed differentiation of primitive and definitive hematopoietic progenitors from human pluripotent stem cells

再生医学的主要目标之一是从人类多能干细胞 (hPSCs) 中产生和维持造血干细胞 (干细胞)。直到最近, 将 hPSCs 分化为干细胞的努力主要产生了缺乏 HSC 潜能的造血祖细胞, 而不是像卵黄囊造血。这些产生的造血祖细胞可能有有限的效用, 为体外疾病模型的各种成人造血疾病, 特别是那些淋巴谱系。然而, 我们最近描述的方法产生赤-骨髓淋巴多向确定造血祖细胞的 hPSCs 使用一个阶段特定的定向分化协议, 我们在这里概述。通过在基底膜 plasticware 上 hPSCs 酶解离, 形成了胚体 (EBs)。EBs 被区分对胚层由重组 BMP4, 随后被指定到明确的造血程序由 GSK3β抑制剂, CHIR99021。另外, 原始的造血是由 PORCN 抑制剂, IWP2 指定的。造血是进一步推动通过增加重组 VEGF 和支持造血细胞因子。使用这种方法产生的造血祖细胞有可能用于疾病和发育模型,体外。

在图 1中说明了从 hPSCs 中归纳出原始和最终造血祖细胞的示意图。胚层模式的规范 WNT 信号发生在 2-3 的差异, 其次是造血祖规范。
有代表性的流动细胞分析和菌落形成纤维素测定 hPSC 衍生的造血分化培养物, 如图 2所示。IWP2-treated 分化文化将产生一个独特的 CD34+CD43−</su...

Watch this Scientific Journal Video about Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells at JoVE.com

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

在这里, 我们提出了人类多能干细胞 (hPSC) 的文化协议, 用于区分 hPSCs 成 CD34 的+造血祖细胞。此方法使用特定于阶段的规范 WNT 信号的操作来指定单元格完全是最终的或原始的造血程序。

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

directed-differentiation-primitive-definitive-hematopoietic

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

10.9K 次观看.  Washington University in St. Louis. 在这里, 我们提出了人类多能干细胞 (hPSC) 的文化协议, 用于区分 hPSCs 成 CD34 的+造血祖细胞。此方法使用特定于阶段的规范 WNT 信号的操作来指定单元格完全是最终的或原始的造血程序。

视频: Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

科研

发育生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

免疫与感染

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 ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

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.

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.

从人多能干细胞定向多巴胺能神经元分化

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

神经科学

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

血源性内皮细胞与人多能干细胞的定向分化

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

破骨细胞与人诱导多能干细胞的分化和表征

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

使用定义的分化系统从多能干细胞的肝祖原体规范

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...