Name: chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency
Uploaded: 2018-06-10T15:00:00
Description: Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÂ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

这项工作得到了 nih/内 (R01EY023962)、nih/发育研究院 (R01HD082098)、RPB 斯坦创新奖、马里兰干细胞研究基金 (2018-MSCRFV-4048、2014-MSCRFE-0742)、新德科学论坛奖和莫斯利基金会赠款的支持。

所有动物程序都是按照约翰霍普金斯医学院动物保育和使用委员会 (IACUC) 批准的动物护理准则和议定书进行的。
1. 为依赖于饲养者的常规 (人类胚胎干细胞中/mef) 或天真恢复的 (LIF-3i 中/mef) hPSC 培养小鼠胚胎成纤维细胞 (mef) 的制备
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购买或准备从 CF1 或 CF1 x DR4 混合 E13.5 小鼠胚胎的 MEF 馈线的内部低通道供应在发布的协议4之后。
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<li>Cry...

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LIF-3i 系统将经典小鼠2i 天真逆转鸡尾酒1的改良版应用于人类多潜能干细胞。hPSC 的自我更新 (不能单独扩展 2i) 在 LIF-2i 通过补充这鸡尾酒与 tankyrase 抑制剂 XAV939 稳定。LIF-3i 文化允许大量和有效地逆转传统 hPSC 到一个多能状态类似于人类植入前啮鼠动物3。虽然 XAV939 在 hPSC 中的作用机制很可能是复杂的, 并且与2i 协同, 但它们可能至少?...

2i (甲酮/ERK 和 GSK3β抑制剂) 培养系统最初开发, 以改善异种血清基小鼠胚胎干细胞 (卓制) 的文化, 以统一的地面状态, 干细胞类似于老鼠植入前啮鼠动物1。然而, 2i 不支持人类多潜能干细胞 (hPSC)2线的稳定维护。最近据报道, 各种复杂的小分子、生长因子补充和转基因方法捕获认定类似人类天真的多能分子状态2。然而, 许多用这些方法创建的 "天真的" 状态也表现出核型不稳定, epigenomic 缺陷 (例如,全球父母基因印迹的丧失), 或分化潜能受损。
相比之下, 对 GSK3β、ERK 和 tankyrase 信号和白血病抑制因子 (LIF-3i) 的三重化学抑制的鸡尾酒, 足以满足常规 hPSC 线3的一种稳定幼稚的逆转。LIF-3i-reverted 天真的 hPSC (hPSC) 维持正常的核型, 并增加其表达的幼稚特定的人类植入前啮鼠动物基因 (如北美, KLF2, NR5A2, DNMT3L, HERVH, 斯特拉 (DPPA3), KLF17, TFCP2L1)。LIF-3i 逆转也授予 hPSC 与卓制相似的天真干细胞独特的分子和生物化学特征, 包括增加磷酸化 STAT3 信号, 减少 ERK 磷酸化, 全球 5-甲基胞嘧啶 CpG低甲基化, 全基因组的 CpG 去甲基化在胚胎干细胞 (ESC) 特定的基因启动器, 和显性远端 OCT4 增强剂的使用。此外, 与其他幼稚的逆转方法, 导致 aberrantly hypomethylated 印迹基因组的基因座, LIF-3i-reverted n-hPSC 没有系统性丧失印迹的 CpG 模式或丧失 DNA 甲基表达 (例如,DNMT1, DNMT3A, DNMT3B)3。
一种直接的 LIF-3i 培养, 广泛的常规人类胚胎干细胞 (人类胚胎干细胞) 和人类诱导的多能干细胞 (hiPSC) 生长在喂食器或 E8 无饲养条件获得迅速和大量逆转到一个天真的啮鼠动物样的状态。然而, 直接 LIF-3i 幼稚逆转可能是低效的一些不稳定的常规 hPSC 线由于固有的基因组和沿袭引多变性产生的基因多样性的捐助者的背景。
因此, 为了拓宽 LIF-3i 方法的效用, 本文提出了一种渐进优化, 它允许在喂食器上培养的几乎任何常规人类胚胎干细胞或无转基因 hiPSC 线的普遍幼稚逆转。这种普及天真的逆转方法在常规 hPSC 中采用了一个瞬态初始培养步骤, 补充了 LIF-3i 鸡尾酒与另外两个小分子 (LIF-5i), 事实蛋白激酶 a (佛司可林) 和声波刺猬 (嘘) (purmorphamine) 信号。在 LIF-5i 中, 常规 hPSC 的一个初始通道将它们适应随后的稳定 LIF-3i 批量回归。最初的 LIF-5i 适应显著增加了在 E8 或喂食器上生长的常规 hPSC 的初始单细胞克隆增殖 (在其随后稳定的、连续的 LIF-3i 单独的通道之前)。传统的 hPSC 线适应了第一个通道在 LIF-5i 容忍随后批量克隆传代的天真恢复细胞在 LIF-3i 条件, 这省却需要采摘和亚克隆的稀有稳定的殖民地, 或常规使用抗凋亡分子或相关蛋白激酶 (岩石) 抑制剂。
LIF-3i 方法已成功地用于稳定地扩展和维护 > 30 独立, 基因多样性的常规 hPSC 线 > 10–30通道使用非酶或酶解离方法, 并没有诱导染色体或 epigenomic 异常的证据, 包括印迹基因位点的异常。此外, 序列 LIF-5i/LIF-3i 文化是唯一的天真的逆转方法, 迄今已报告, 改善功能干细胞的常规 hPSC 线, 通过减少其谱系引种基因表达和大大提高了他们的多向分化效力。LIF-3i 天真回归方法消除了谱系引种、常规 hPSC 线分化的内在联运变异性, 将在再生医学和细胞治疗中有很大的应用前景。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- SSEA-1/CD15 antibody, APC conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">561716</td>
			<td style="width:371px;">use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti- TFCP2L1 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA029708</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-beta-Actin antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab6276</td>
			<td>use at 1:5000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD146 antibody,&#160; PE conjugated&#160;</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">550315</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 antibody, APC conjugated</td>
			<td style="width:161px;">eBioscience</td>
			<td style="width:119px;">17-0319-42</td>
			<td>use 2&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-CD31 microbead kit</td>
			<td style="width:161px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-091-935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NANOG antibody</td>
			<td style="width:161px;">Abcam</td>
			<td style="width:119px;">ab109250</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-NR5A2 antibody</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">HPA005455</td>
			<td>use at a 1:100 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-p44/42 MAPK (Erk1/2) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4695</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">4370</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-phospho-STAT3 (Tyr705) antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9145</td>
			<td>use at 1:1000 (Western blot)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-rabbit immunoglobulin antibody, biotinylated</td>
			<td style="width:161px;">Agilent</td>
			<td style="width:119px;">E0432</td>
			<td>use at a 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 antibody, APC conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">FAB1435A</td>
			<td>use 5&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-SSEA-4 GloLIVE antibody, NL493 conjugated</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC1435G</td>
			<td>use at 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STAT3 antibody</td>
			<td style="width:161px;">Cell Signaling</td>
			<td style="width:119px;">9139</td>
			<td>use at 1:1000 (Western blot), for detection of total protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-STELLA/DPPA3 antibody</td>
			<td style="width:161px;">Millipore</td>
			<td style="width:119px;">MAB4388</td>
			<td>use at a 1:50 dilution (immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA-1-60 GloLIVE antibody, NL557 conjugated&#160;</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">NLLC4770R</td>
			<td>use at&#160; a 1:50 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-60 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0068</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">anti-TRA-1-81 StainAlive Antibody, DyLight 488 conjugated</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">09-0069</td>
			<td>use at a 1:100 dilution (live and fixed immunostainings)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-60 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560193</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">anti-TRA1-81 antibody, PE conjugated</td>
			<td style="width:161px;">BD Biosciences</td>
			<td style="width:119px;">560161</td>
			<td>use 10&#181;L per assay (FACS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">APEL2-Li</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Bovine Serum Albumin</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CellAdhere dilution buffer</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7183</td>
			<td>dilutent for Vitronectin XF&#8482; matrix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CF1 mouse</td>
			<td style="width:161px;">Charles river</td>
			<td style="width:119px;">023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">CHIR99021</td>
			<td style="width:161px;">R&#38;D System</td>
			<td style="width:119px;">L5283</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">confocal microscope system</td>
			<td style="width:161px;">Zeiss</td>
			<td style="width:119px;">LSM 510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">cord blood CD34+ derived iPSC line</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A18945</td>
			<td>also referred as 6.2 line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Corning Costar tissue culture-treated 6-well plates</td>
			<td style="width:161px;">Corning</td>
			<td style="width:119px;">3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Countess&#160; cell counting chamber slide</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">C10228</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Countess automated cell counter</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)&#160;</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11995-065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">DMEM-F12</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11330-032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DMSO (dimethyl sulfoxide)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">DR4 mouse</td>
			<td style="width:161px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">3208</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Essential 8 (E8) medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">5940</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Fetal bovin serum (FBS)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SH30071.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Forskolin</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0025</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Gelatin (porcine)</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>resuspend in water and sterilize with an autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">KnockOut Serum Replacement</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10828-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">L-Glutamine (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">MEM Non-essential amino acid (MEM NEAA) (100X)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">mTeSR1 medium</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">85850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Nalgene cryogenic vials</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5000-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Nunc Lab-Tek II Chamber Slide System</td>
			<td style="width:161px;">Fisher Scientific</td>
			<td style="width:119px;">154534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Paraformaldehyde (PFA) solution , 4% in PBS</td>
			<td style="width:161px;">USB Corporation&#160;</td>
			<td style="width:119px;">19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">PD0325901</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">PZ0162</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Penicillin/streptomycin (10,000 U/mL)</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Phosphate buffered saline (PBS)</td>
			<td style="width:161px;">Biological Industries</td>
			<td style="width:119px;">02-023-1A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Purmorphamine</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0009</td>
			<td>reconstitute at 10mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human Activin A</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">AF-120-14E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">recombinant human Bone morphogenetic protein (BMP)-4</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">120-05ET</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human FGF-basic (bFGF)</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">recombinant human LIF</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">300-05</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">SB431542&#160;</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0010-05</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Stemolecule Y27632 in Solution</td>
			<td style="width:161px;">Stemgent</td>
			<td style="width:119px;">04-0012-02</td>
			<td>ROCK inhibitor in solution (10mM)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">StemPro Accutase Cell Dissociation Reagent</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A11105-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Streptavidin-Cy3 conjugate</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">S6402</td>
			<td>use at 1:500-1:1000 dilution (immunostainings)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">Thermo Scientific Mr. Frosty Freezing Container</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">5100-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vascular endothelial growth factor (VEGF)-165</td>
			<td style="width:161px;">Peprotech</td>
			<td style="width:119px;">100-21</td>
			<td>resupend at 100ug/mL in 0.1% bovine serum albumin in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Vitronectin XF matrix</td>
			<td style="width:161px;">StemCell Technologies</td>
			<td style="width:119px;">7180</td>
			<td>dilute at 40&#181;L/mL in CellAdhere&#8482; dilution buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">XAV939</td>
			<td style="width:161px;">Sigma Aldrich</td>
			<td style="width:119px;">X3004</td>
			<td>reconstitute at 100mM in DMSO</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:373px;">&#946;-mercaptoethanol</td>
			<td style="width:161px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">21985-023</td>
			<td>light sensitive</td>
		</tr>
	</tbody>
</table>

chemical reversion of conventional human pluripotent stem cells to a na&#239;ve-like state with improved multilineage differentiation potency

天真的人类多潜能干细胞 (n-hPSC) 具有改进的功能, 可能对再生医学产生广泛的影响。该协议的目标是有效地恢复沿袭的, 传统的人类多潜能干细胞 (hPSC) 保持在无馈线或馈线依赖的条件, 以一个天真的干细胞, 改善功能。这种化学幼稚的逆转方法采用了经典的白血病抑制因子 (LIF), GSK3β和甲酮/ERK 抑制鸡尾酒 (LIF-2i), 补充只有 tankyrase 抑制剂 XAV939 (LIF-3i)。LIF-3i 将传统的 hPSC 还原为一种稳定的多能态, 采用人类预植入啮鼠动物的生物化学、转录和表观遗传特征。这种 LIF-3i 方法需要最小的细胞培养操作, 并在广泛的人类胚胎干细胞 (人类胚胎干细胞) 和无转基因人类诱导的多潜能干细胞 (hiPSC) 线高度重现。LIF-3i 方法在分化之前不需要重新启动步骤;hPSC 可直接与极高的效率相区别, 维持核型和 epigenomic 的稳定性 (包括印迹位点)。为提高该方法的普遍性, 传统的 hPSC 首先培养在 LIF-3i 鸡尾酒补充两个额外的小分子, 事实蛋白激酶 A (佛司可林) 和声波刺猬 (嘘) (purmorphamine) 信号 (LIF-5i)。这一简短的 LIF-5i 适应步骤显著增强了传统 hPSC 的初始克隆扩展, 允许它们随后以 LIF-3i 的数量单独恢复, 从而不需要采摘/亚克隆稀有的 N hPSC殖民地以后。LIF-5i-stabilized hPSCs 随后在 LIF-3i 单独维持, 不需要抗凋亡分子。最重要的是, LIF-3i 逆转明显改善了常规 hPSC 的功能干细胞, 通过减少其谱系引种基因表达和清除联运变异的定向分化通常观察到独立的 hPSC 线。给出了 LIF-3i-reverted n-hPSC 的代表性特征, 并给出了等基因系 hPSC 在谱系引物与的功能比较的实验策略。概述了类似幼稚的国家。

该协议优化了在馈线依赖和馈线无关的传统 hPSC 文化 (图 1) 中 LIF-3i 的高效天真的回归。本文描述的详细协议概述了从馈线依赖或无馈线常规 hPSC 条件 (如E8 介质) 开始的 LIF-3i 的顺序适应。
图 1–3了几种常规人类胚胎干细胞和无转基因 hiPSC 线 LIF-3i 逆转的代表性结果。这些典型的结果?...

Watch this Scientific Journal Video about Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÂ¯ve-like State with Improved Multilineage Differentiation Potency at JoVE.com

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve-like State with Improved Multilineage Differentiation Potency

改良多向分化效力的常规人多能干细胞的化学逆转与幼稚样态

我们提出一项协议, 以有效, 散装, 快速化学逆转传统的谱系诱导人类多潜能干细胞 (hPSC) 成一个 epigenomically 稳定幼稚的植入啮鼠动物样多潜能状态。该方法在常规 hPSC 线的多向分化上降低了谱系引种基因的表达和显著改善。

Na&#239;ve human pluripotent stem cells (N-hPSC) with improved functionality may have a wide impact in regenerative medicine. The goal of this protocol is ...

Research

JoVE Journal

Developmental Biology

Chemical Reversion of Conventional Human Pluripotent Stem Cells to a Na&#239;ve-like State with Improved Multilineage Differentiation Potency

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

人类多能干细胞的模板微图案用于探测分化命运的空间组织

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

科研

发育生物学

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

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

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 ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

在人多能的体外干细胞向滋养层细胞

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

高效可扩展的人多潜能干细胞临床相容角膜缘上皮干细胞的定向分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

人类多能干细胞有效分化为肝细胞

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...