Name: derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation
Uploaded: 2015-03-18T15:00:00
Description: 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

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。 ...

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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 (Video) | JoVE

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 ...

Research

JoVE Journal

Developmental Biology

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

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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

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

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