This work was supported in part by the NIH/NHBI (U01 HL099776-5), the NIH Director&#8217;s New Innovator Award (DP2 OD004411-2), the California Institute of Regenerative Medicine (RB3-05129), the American Heart Association (14GRNT18630016) and the Endowed Faculty Scholar Award from the Lucile Packard Foundation for Children and the Child Health Research Institute at Stanford (to SMW). We also acknowledge funding support from the American Heart Association Predoctoral Fellowship 13PRE15770000, and National Science Foundation Graduate Research Fellowship Program DGE-114747 (AS).

注：供应商信息，在这个协议中使用的所有试剂已列于表1和 材料清单。所有解决方案和设备进入与细胞接触必须是无菌的，并且无菌技术应相应使用。除非另有规定执行所有培养孵育在加湿37℃，5％CO 2的培养箱中培养。在这个协议中，所有的分化在6孔板中进行，其中的人iPS细胞是种子。以下分化和纯化，细胞可被解离和再接种供下游使用。 1.准备中<ol><li>为E8介质，使原液的每个介质组分（ 碳酸氢钠 ，L-抗坏血酸-2-磷酸，亚硒酸钠，转铁蛋白，胰岛素，FGF2，TGFB1）和存储溶液在-20℃。加入储备溶液适量至瓶子的DMEM / F12，并过滤灭菌。该解决方案精矿ATION和反应设置为库存组分和E8介质可以在表1中找到。 </li><li>对于细胞外基质溶液中，解冻基底膜基质（10毫克/毫升通常供应）在4℃CO / N。基质胶是常用的多能性干细胞培养，因为人多能干细胞可以使用的α-6-β-1整联9很好地粘附到该材料。一旦解冻，使使用冰冷的枪头和管冰上2.25毫克（250微升）等分并储存在等分-80℃。 <ol><li>当ECMS预涂板，解冻和分装冰的细胞外基质溶液（ECMS）。在冰上200：混合ECM和冰冷的DMEM / F12培养基中的比例为1。保持冷静，以防止过早凝固ECMS。 </li></ol></li><li>对于RPMI / B27无胰岛素中，加10减B27胰岛素毫升到500ml RPMI媒体。 </li><li>对于RPMI / B27（胰岛素）中，加入10 mL B27补充（带insulin）的和5ml青霉素 - 链霉素抗生素到500ml RPMI培养基中。 </li><li>对低葡萄糖培养基中，加入10 mL B27添加剂和5ml的青霉素 - 链霉素抗生素到500ml葡萄糖的RPMI介质。 </li><li>用于冷冻/冷冻保存介质中，一起混合胎牛血清用二甲基亚砜（DMSO）以9：1的比例。冷冻介质可以存储长达1个月，在4℃。 注意：所有媒体应该被过滤并存储在4℃下并且除非另有说明在2周内使用。下面所用的所有试剂/溶液体积是供单个井在一6孔板，除非另有规定。 </li></ol> 2.预涂覆的6孔板的ECMS <ol><li>使ECMS通过混合基底膜基质（ 例如 ，基质胶）用冰冷的DMEM / F12培养基中的比例为1：200，然后应用2毫升新鲜制成ECM的到各孔为一个6孔板中。每个孔在6孔板将获得约90毫克ECMS。 </li><li>孵育ECMS上37℃下包被板1小时。之前立即镀细胞每孔，吸出DMEM / F12溶液。那么板块将准备接收细胞。 </li></ol> 3.解冻冰封人iPS细胞注：紧跟此过程，因为它可能导致最佳的培养人iPS细胞和成hiPSC-CMS的以下的冷冻/解冻循环下游分化。 <ol><li>人iPS细胞解冻一小瓶的6孔板的每个孔中。制备的15毫升锥形管中9ml冷E8介质与ROCK抑制剂（10μM）对每个小瓶。 ROCK抑制剂提高了人iPS细胞的生存以下冷冻保存10。保持在37℃的水浴的小瓶解冻所述细胞直至约5毫米直径的冰晶体在左边。 </li><li>喷用70％乙醇小瓶的外部。将它们转移到无菌层流罩之前，先用纸巾小瓶。 </li><li>转移细胞进入准备锥形管。冲洗吨他小瓶一次用500μlE8培养基并传送包含该解冻细胞相同的锥形管的平台。离心机在室温下以200×g下4分钟。 </li><li>离心后吸出上清液，并轻轻重悬细胞沉淀用2ml E8培养基与ROCK抑制剂（10μM）。转移的悬浮细胞至一个6孔板预先涂覆有ECM的一个阱。 </li><li>培养24小时后更换而不ROCK抑制剂与E8介质的介质。细胞应该坚持这个时候。 </li></ol> 4.传代人iPS细胞的<ol><li>通常情况下，在一个6孔板达到75％-80％汇合后通道人iPS细胞。吸出培养基。用1毫升无菌，RT PBS快速洗井。吸PBS。 <ol><li>加入500微升0.5毫摩尔EDTA或1x细胞剥离溶液（ 如的Accutase），并孵育在室温下1-7分钟。适当的时候EDTA孵化随hiPSC线。期待看到明显的卷曲Ø边缘周围的菌落ř增稠，因为这将表明，EDTA或细胞剥离溶液准备好被除去。 </li></ol></li><li>吸出的EDTA或细胞分离溶液。加入1毫升E8培养基补充有10μM的ROCK抑制剂。置换hiPSC菌落在井通过反复上下吹吸，并与一个P1000移液器喷雾菌落关闭。菌落被分成完成后5-10细胞团块。 </li><li>传送被在1ml E8介质与ROCK抑制剂的悬浮入15ml锥形管中的菌落。 </li><li>破坏大细胞团块成在E8中小团块。与抑制剂添加E8媒体的适当体积的细胞悬浮液稀释细胞。媒体量添加稀释取决于细胞密度，也可以接种井的数量。理想的情况下，人iPS细胞的一个单一的，80％的汇合以及应稀释1:12 <ol><li>添加11毫升E8介质ROCK抑制剂现有1毫升的CE在上述的15毫升锥形管LL溶液中以获得1:12的稀释。 </li></ol></li><li>进一步磨碎的细胞团块成小的细胞碎片（约50-200细胞在每个片段中是最好的）。避免过度捣碎，因为这减少了细胞的存活。 </li><li>吸ECMS来自预包被的板并加2ml悬浮细胞向每个孔中。每一个6孔板的约100,000个细胞是理想的。旨在均匀地分配围绕井的细胞，以避免在井的中心小区的聚类。 </li><li>经过培养24小时，更换E8介质中无ROCK抑制剂。改变培养基每24小时，直到细胞成为80％汇合。然后，将细胞准备通道一次。它通常需要段落之间的3-6天达到80％汇合。 </li></ol> 5.冻人iPS细胞注：紧跟这一程序，因为它可能导致最佳的培养和髋部的下游分化雪旺成hiPSC-CMS以下的冷冻/解冻循环。 <ol><li>标号低温管与所述细胞系的名称，细胞类型，通道号，以及冷冻时间。作为一般准则，用一个小瓶的6孔板的每个孔中。制备的15毫升锥形管中填充用9ml E8介质与ROCK抑制剂（10μM）。制备冷冻培养基用90％FBS和10％DMSO中，并保持该介质在4℃下，直到准备使用。 </li><li>吸出培养基，加入500微升0.5毫摩尔EDTA或1x细胞分离液，孵育在室温下2-7分钟。的EDTA或细胞剥离溶液接触的持续时间从细胞系而异的细胞系。 </li><li>吸EDTA或细胞分离液。加入1毫升E8介质。 </li><li>置换在阱hiPSC菌落通过上下抽吸并用P1000移液器喷雾菌落关闭。菌落不应被破碎成小于100的细胞团块。转移悬浮的细胞，以制备锥形管含有E8。离心机为RT代表在200×g下4分钟。 </li><li>吸出上清液，并添加500μl的冷冻介质中的颗粒。吹打的一至两倍重悬沉淀。细胞存活是通过保持细胞大团块改善。 </li><li>转移悬浮细胞成标低温管。快速移动的小瓶进入冷冻含有异丙醇的容器中，这将允许为逐步冷却。保持容器与细胞在-80℃下放置24小时，然后将细胞转移到液氮中用于长期贮存。 </li></ol>人类的iPSC 6.心脏分化注：加入时，所有的媒体应该至少在RT。 <ol><li>离解用EDTA或1x细胞剥离溶液后，种子就ECMS涂覆的6孔培养板中进行分化大约100,000人类iPSC（相同的步骤，细胞传代过程）。当细胞达到85％汇合，改变介质为RPMI / B27没有与6中胰岛素μMGSK3-β抑制剂CHIR99021（CHIR）并保持48小时。 </li><li> 48小时后，更换RPMI / B27含CHIR培养基无胰岛素中，独自离开24小时（到3天）。 </li><li>在第3天，无胰岛素与5μM的Wnt抑制剂IWR1改变媒体RPMI / B27并保持48小时（直到第5天）。 注：的Wnt抑制还可以使用其它小分子化合物尝试，如在早先的研究11说明。 IWR1被选择比其它小分子Wnt信号的抑制剂，由于在它已被证明可有效地抑制Wnt信号11的上升范围。 </li><li>第5天，改变介质回RPMI / B27无胰岛素中，留下48小时（到7天）。 </li><li>在第7天，更换用R​​PMI / B27培养基（胰岛素）培养基中，并以同样的培养基其后每3天更换培养基。心肌细胞的自律搏动首先应该是可见约8日复一日10。 </li></ol>经过葡萄糖饥饿7.净化人类心肌细胞的<ol><li>在第10天的后分化，改变在6孔板至2ml低葡萄糖培养基的每孔中的培养基，并维持细胞在该培养基中3天（直至第13天）。 </li><li>在第13天，恢复细胞的RPMI / B27培养基（胰岛素）。 </li><li>任选地，replate之前第二轮葡萄糖饥饿，以帮助放松从培养板的非心肌细胞，允许对非心肌细胞中葡萄糖饥饿容易解离的心肌细胞。 <ol><li>在第13天，吸出培养基，用PBS洗一次，并用500微升的细胞解离的酶进行5分钟，在37℃下解离的细胞分化成单细胞。具体地讲，5分钟酶处理后，可以使用一个1000微升吸管反复拉起细胞离解酶和喷涂它靠在cardiomyo手动解离从6孔板心肌CYTE单层。高达30移液重复可能需要解离的心肌细胞为单细胞。 </li><li>后的细胞被离解，并在单细胞形式，收集所有细胞入15ml锥形管中填充用5ml RPMI / B27培养基的胰岛素来稀释出细胞离解酶和离心机在200×g下4分钟。吸并丢弃上清液。 </li><li>重新悬浮细胞用2ml的RPMI / B27培养基和板到一个新ECMS被覆6孔板中。通常情况下，心肌更高的融合有助于细胞存活replating期间。旨在replate每个新的6孔盘以获得最佳的生存2000000细胞replating期间。 </li></ol></li><li>在第14天，改变介质回〜2毫升的低血糖中的第二个缺糖周期。培养的细胞在这种低血糖状态持续3天以上。大多数非心肌细胞会死在这种低血糖培养条件。 </li><li>在17天，改变R的中型〜2毫升PMI / B27介质胰岛素。剩余的细胞将是高度纯化心肌细胞。这些心肌细胞可用于基因表达分析，药物筛选，代谢分析，以及其他各种下游测定。 </li></ol>

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The authors do not declare competing interests.

得到大量的高纯度的hiPSC衍生的心肌细胞是基本心脏研究以及临床和转化应用的关键。心脏分化协议已经在最近几年发生了巨大的改进，从利用心源性生长胚状体为基础的方法转变因子2，以矩阵夹心方法12，最后以小分子调制和单层为基础的方法5。上述协议，这里描述的方案中显示的最高和最可重复的心脏分化效率不同hiPSC细胞系通过使用小分子CHIR和IWR 5,7调制的W...

原代人心肌细胞是因为侵入性心脏活检，难以解离为单细胞的，而且由于差的长期存活的细胞在培养中的要求很难获得。鉴于这种缺乏原代人心肌细胞，针对具体患者的诱导的人多能干细胞衍生的心肌细胞的（hiPSC-CM）的技术已被认为是一个功能强大的替代来源的心肌细胞的基础研究以及临床和翻译的应用，如疾病建模和药物发现1。在分化的多能干细胞分化为心肌细胞的早期努力采用使用胚状体（EB）分化的协议，但这种方法是低效的生产心肌细胞，因为常常少于25％的细胞在使用EB均跳动的心肌细胞2,3。相比较而言，用激活素A和BMP4的细胞因子的单层基分化方案所显示的更高的效率比的EB，但该协议还比较低效率的，需要昂贵的生长因子，并在人多能干细胞系的4有限数量的唯一功能。近日，一个高效，hiPSC单层型心肌细胞分化协议是通过调控Wnt信号/β-catenin信号5开发。这些hiPSC-CM的表达肌钙蛋白T和α辅肌动蛋白，双肌蛋白是心肌6标准的标记。这里的协议描述是这样的小分子为基础，馈线无细胞，单层分化法5,7的适应。我们能够获得后7-10天（ 图1），从人iPS细胞跳动的心肌细胞。然而，下面的一个心肌细胞的分化导致50％的跳动细胞，始终如一的免疫染色显示了非心肌细胞是阴性的心肌特异性标记物，如心脏特异性肌钙蛋白T和α-辅肌动蛋白的群体的存在。来福rther纯化心肌细胞，并消除非心肌细胞，异质分化细胞群经受由具有非常低葡萄糖培养基中多天（ 图2）处理它们对葡萄糖饥饿。这种处理有选择地消除非心肌细胞由于心肌细胞的能力，但不是非心肌细胞，代谢乳酸作为主要能量来源，以便在一个低葡萄糖环境8生存。在此之后的纯化步骤中，在心肌细胞中，以非心肌细胞的比率增加了40％被观察到的，（ 图3，图4），并且这些细胞可以用于下游的基因表达分析，疾病建模和药物筛选试验。

<table border="0" cellpadding="0" cellspacing="0" width="550">
	<tbody>
		<tr height="18">
			<td height="18" style="height:18px;width:207px;">Name</td>
			<td style="width:199px;">Company</td>
			<td style="width:145px;">Catalog number</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Matrigel (9-12 mg/mL)</td>
			<td>BD Biosciences</td>
			<td>354277</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">RPMI media</td>
			<td>Invitrogen</td>
			<td>11835055</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Glucose free RPMI media</td>
			<td>Invitrogen</td>
			<td>11879-020</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">B27 Minus Insulin</td>
			<td>Invitrogen</td>
			<td>A1895601</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">B27 Supplement (w/ insulin)</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Pen-strep antibiotic</td>
			<td>Invitrogen</td>
			<td>15140122</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Fetal bovine serum</td>
			<td>BenchMark</td>
			<td>100-106</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">DMSO</td>
			<td>Sigma</td>
			<td>D-2650</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">ROCK inhibitor Y-27632</td>
			<td>EMD Millipore</td>
			<td>688000</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">CHIR99021</td>
			<td>Thermo Fisher</td>
			<td>508306</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">IWR1</td>
			<td>Sigma</td>
			<td>I0161</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Accutase</td>
			<td>Millipore</td>
			<td>SCR005</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Cell lifter</td>
			<td>Fisher</td>
			<td>08-100-240</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Cryovial</td>
			<td>Fisher (NUNC tubes)</td>
			<td>375418</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">TrypLE Select Enzyme</td>
			<td>Invitrogen</td>
			<td>12563-011</td>
		</tr>
	</tbody>
</table>

derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation

诱导的人多能干细胞衍生的心肌细胞（hiPSC-CMS）已成为一种重要的细胞源，以解决缺乏可用于基础研究和翻译的应用程序主心肌。分化人iPS细胞为心肌细胞，已经开发了各种协议，包括胚状体（EB）为基础的分化和生长因子诱导。然而，这些协议是低效，并在其产生纯化的心肌细胞的能力高度可变。最近，的Wnt /β-catenin信号的小分子为基础的协议利用调制被证明促进心脏分化效率高。与此协议中，分化的细胞大于50％-60％为心肌肌钙蛋白阳性心肌细胞持续观察。为了进一步增加心肌的纯度，分化的细胞进行葡萄糖饥饿基于代谢差异特异性消除非心肌细胞s心肌细胞和非心肌细胞之间。使用此选择策略，我们一致地得到增加心肌非心肌细胞的比率在分化细胞群一大于30％。这些高度纯化心肌要增强人类iPSC的基于体外疾病模型研究和药物筛选测定法的结果的可靠性。

在hiPSC分化的形态变化。 所述hiPSC在无饲养平板上培养生长为平坦的，二维的菌落。当达到约85％汇合，人iPS细胞分别用6微米的CHIR处理分化（ 图1A）。细胞死亡，一个正常的和普遍的现象，显着量的CHIR治疗的24小时后进行观察。后CHIR治疗两天，人iPS细胞持续分化朝向中胚层的命运。相较于细胞的hiPSC克隆，细胞大小，这些天2细胞增多。手机号码也可...

Watch this Scientific Journal Video about Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose S

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

从人类诱导多能干细胞使用小分子调制分化和随后的葡萄糖饥饿高度纯化心肌推导

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

derivation-highly-purified-cardiomyocytes-from-human-induced

Research

JoVE Journal

Developmental Biology

18.4K 次观看.  Stanford University School of Medicine. Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening. 

视频: Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

树突棘从锥体神经元的三维定量从人类诱导多能干细胞衍生

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

从多能干细胞使用BMP拮抗剂Grem2心房心肌细胞分化

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

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

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

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 ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

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) ...

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

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

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 Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.