Name: large-scale production of cardiomyocytes from human pluripotent stem cells using a highly reproducible small molecule-based differentiation protocol
Uploaded: 2016-07-25T14:00:00
Description: Watch this Scientific Journal Video about Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol at JoVE.c

This study was funded by grants provided from Royan Institute, Iranian Council of Stem Cell Research and Technology, the Iran National Science Foundation (INSF), the National Health and Medical Research Council of Australia (NHMRC; 354400), the National Heart Foundation of Australia/Heart Kids Australia (G11S5629), and the New South Wales Cardiovascular Research Network. HF was supported by a University International Postgraduate Scholarship from the University of New South Wales, Australia. RPH was supported by a NHMRC Australia Fellowship. The authors express their gratitude to the human subjects who participated in this research.

1，文化传媒，细胞培养板的涂层和维护未分化hPSCs的制备<ol><li> 媒体准备 注意：使用消毒用0.22微米的过滤装置和存储介质在避光长达4周5℃。试剂的名称，供应商和目录编号列在材料表 。 <ol><li>对于小鼠胚胎成纤维（MEF）中，结合445毫升DMEM 50毫升胎牛血清（FBS）和5 ml细胞培养基（ 如 Glutamax的）。 </li><li>对于人类胚胎干细胞中，结合390毫升敲...

<ol>
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从hPSCs衍生的心肌细胞是人类疾病模型，药物筛选/毒性试验使用一个非常有吸引力的源，也许在将来，再生疗法。一个主要的障碍然而使用这些细胞，是为他们的有效利用提供足够高的品质的材料的能力。使用我们描述的协议，我们提供克服了这一限制的方法。 最近，针对参与心脏发生特定信号传导途径合成的小分子已被描述为增强心肌分化16,17,22,23。这些现在正在?...

人多能干细胞（hPSCs），包括人类胚胎干细胞（胚胎干细胞）和诱导多能干细胞（人iPS细胞），具有自我更新和能力的分化成三个胚层1,2的细胞的能力。由于这些特点，hPSCs人类疾病3-5，用于高通量药物筛选和毒性测定6,7-建模为临床应用8提供用于产生和可扩展的生产疾病相关的细胞类型的有价值的和无限制的源和潜在。从hPSCs心肌细胞的产生提供了机会，专门研究复杂的人类心血管疾病的机制及其可能的治疗方法，以前超出了我们的能力范围内，由于缺乏相关的动物模型和/或受影响的主要组织的可用性。 所有hPSCs n的上述申请的ecessitate生产高度浓缩的和功能性心肌细胞的大量数字。因此，可用一个高效的，可重复和可扩展的体外心脏分化协议适用于多种HPSC行是至关重要的。传统的心肌细胞的分化协议使用了不同的策略，如胚体的形成9，共培养技术10，诱导细胞因子11和蛋白转导方法12鸡尾酒。尽管这些技术的进步，大多数仍然效率低下苦，需要昂贵的生长因子，或试图使用多个HPSC线路时，提供有限的普遍性。迄今为止，这些挑战设置限制以生产用于在动物模型中的细胞疗法的研究HPSC衍生的心肌的，以及在制药工业中用于药物发现13。因此，对于大型强大且经济技术的发展进制生产的可伸缩的培养体系功能HPSC衍生的心肌将在很大程度上促进他们的商业和临床应用。 在这个手稿，我们报告具有成本效益和综合心脏分化系统具有疗效高，再现性和适用性从各种来源和培养方法，包括用于大规模生产的高度的方法产生的胚胎干细胞和人iPS细胞的发展使用生物反应器HPSC衍生的心肌的富集群体。此外，我们已经优化了这个协议并不适用于无饲养和/或单细胞培养HPSC线，如新建立的iPS细胞或相关疾病机理分析HPSC线大同伙。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Knockout DMEM</td>
			<td>Life Technologies</td>
			<td>10829018</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement (KO-SR)</td>
			<td>Life Technologies</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Fibroblast Growth Factor (bFGF)</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-843</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Life Technologies</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14287072</td>
			<td></td>
		</tr>
		<tr>
			<td>Attachment Factor (AF)</td>
			<td>Life Technologies</td>
			<td>S006100</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Gel</td>
			<td>Sigma-Aldrich</td>
			<td>E1270</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16140-071</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 minus insulin</td>
			<td>Gibco</td>
			<td>A18956-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Life Technologies</td>
			<td>17140-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Bioaustralis</td>
			<td>BIA-M1183</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-172</td>
			<td></td>
		</tr>
		<tr>
			<td>IWP2</td>
			<td>Miltenyi Biotec</td>
			<td>130-105-335</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-561</td>
			<td></td>
		</tr>
		<tr>
			<td>Purmorphamine</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-465</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-169</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly Vinyl Alcohol (PVA)</td>
			<td>Sigma-Aldrich</td>
			<td>363073</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Bio-Rad</td>
			<td>145-0013</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax&#160;</td>
			<td>Innovative Cell Technologies Inc.</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SL2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSPIN</td>
			<td>Integra Biosciences</td>
			<td>183 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flask with 1 pendulum, 100 ml&#160;</td>
			<td>Integra Biosciences</td>
			<td>182 023</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblasts (MEF)</td>
			<td>Prepared in-house (or commercially available)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human pluripotent stem cell (hPSC) lines</td>
			<td>Prepared in-house (or commercially available)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale production of cardiomyocytes from human pluripotent stem cells using a highly reproducible small molecule-based differentiation protocol

最大化人多能干细胞（hPSCs）用于研究，疾病建模，药物和临床应用的利益需要用于大规模生产的功能细胞类型，包括心肌细胞的稳健的方法。在这里，我们证明了WNT，TGF-β，和SHH的时间操纵信号通路导致静态悬浮和搅拌生物反应器系统的单细胞​​的高效心肌细胞的分化传代HPSC线。采用这种策略导致〜100％打浆球体，始终如一地含有后培养15天&gt; 80％肌钙蛋白T阳性细胞，在多个HPSC行验证。我们也对这个协议与目前不适合于单细胞传代，成功其中已在42 HPSC线被验证的细胞系使用的变化情况。使用这些协议所产生的心肌细胞表达系特异性标志物和展预计electrophysiological功能。我们的协议提出了大规模生产人类心肌细胞的简单，高效，强大的平台。

为了建立一个简单的方法，用于从hPSCs心肌细胞的大规模分化，我们创造了一种细胞用WNT /β连环蛋白活化剂（CHIR99021）最初处理的协议16，随后用WNT抑制剂/β-连环蛋白和转化生长因子β（TGF-β）的途径（IWP2 16和SB431542 17，分别）和最后的音猬（SHH）途径（嘌吗啡胺）的活化剂17（ 图1A）。在我们的差异化策略，（175±25微米的球?...

Watch this Scientific Journal Video about Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol at JoVE.c

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

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 ...

The overall goal of this experiment is to differentiate human pluripotent stem cells into cardiomyocytes with high efficiency and reproducibility. This protocol can be scaled up in a bioreactor or modified for differentiating cell lines, which are sensitive to single cell culture. This method can help answer key questions in cardiac disease modeling using human induced pluripotent stem cells.The main advantages of this technique are that it is cost-effective and has high efficiency, reproducibility, and applicability for both human embryonic and induced pluripotent stem cells. Our demonstrator today is Hananeh Fonoudi, currently a PhD student at the Victor Chang Cardiac Research Institute, coming to us from the Royan Institute. Before coating, use a cleaning brush and distilled water to thoroughly remove any dust or culture residue from a 100 milliliter spinner flask with a glass pendulum.Next, fill the flask with 70%ethanol. After 30 minutes, rinse the flask with distilled water, and fill it with five molar sodium hydroxide. The next morning, rinse the flash under running water for five minutes, and fill it with one molar hydrochloric acid.After 15 minutes, rinse the flask under running water for another five minutes, followed by two rinses with double-distilled water. Place the flask in a biosafety cabinet to dry. Then, add 1.5 milliliters of siliconizing solution to the flask, and rotate the flask horizontally to cover all of the surfaces with silicon.Heat the coated flask in a dry oven for one hour at 100 degrees Celsius. When the flask has cooled, rinse it three times for 15 minutes with deionized water. Then, autoclave sterilize the container at 121 degrees Celsius for 20 minutes.After passaging undifferentiated spheroids, using the single cell passaging method at least three times, use a five milliliter pipette to transfer all of the undifferentiated spheroids into a 15 milliliter conical tube. Wash the wells with one milliliter of human embryonic stem cell medium to collect any remaining spheroids, and pool the wash with the harvested spheroids. Allow the spheroids to sediment for five minutes to form a loose pellet.Then, aspirate the supernatant, taking care not remove any spheroids. Next, rinse the spheroids with one milliliter of PBS for five minutes, and incubate them in 5 milliliters of Trypsin EDTA at 37 degrees Celsius. After four to five minutes, stop the reaction with one milliliter of human embryonic stem cell medium, and use a P1000 pipette to dissociate the spheroids into a single cell suspension.After counting by trypan blue exclusion, resuspend the cells at a 200, 000 cell per milliliter concentration, in conditioned medium supplemented with basic fibroblast growth factor and ROCK inhibitor, and transfer 100 milliliters of the cell suspension into the siliconized bioreactor flask. Place the flask on the agitator at 35 RPM. After 24 hours, increase the agitation to 40 RPM.After 48 hours, stop the agitation for five to ten minutes until all of the reformed spheroids have settled to the bottom of the flask, and replace half of the medium with conditioned medium supplemented with basic fibroblast growth factor. Then, restart the agitator at 40 RPM for another 24 hours, replacing half of the medium by the same process every day for the next three days. On day five, stop the agitator for five to ten minutes.Then, transfer all of the spheroids to a 50 milliliter tube in 50 milliliters of medium or less. Discard any of the remaining medium in the flask, taking care that no spheroids remain in the vessel. Then, carefully remove the supernatant from the settled spheroids, and wash cell clusters in 25 milliliters of PBS with calcium and magnesium.After five to ten minutes, remove the wash, and resuspend the spheroids in 40 milliliters of RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, ROCK inhibitor, and polyvinyl alcohol. Transfer all of the spheroids back to the bioreactor flask. Then, add fresh RPMI-B27 medium to a total volume of 100 milliliters, and restart the agitation at 40 RPM.After 24 hours, follow the same process to change media from RPMI-B27 medium. After an additional 24 hours, follow the same process and change media with RPMI-B27 plus IWP2, purmorphamine, and SB43. Leave for 48 hours.First beating is usually observed after seven days. For the fast differentiation of human pluripotent stem cell lines that have not been adapted to single cell culture, first treat to 70 to 80%confluent human pluripotent stem cell culture, grown on mouse embryonic fibroblast cells, with 5 milliliters of dissociation solution for 30 to 60 seconds. Dissociation solution should only be left until the MEFs round up and hPSC colony edges become clear.If the cells are left too long, they will not differentiate correctly. At the appropriate time, quickly replace the dissociation solution with 75 milliliters of collagenase type IV, and incubate the cells for five to 15 minutes at 37 degrees Celsius, checking the culture under the microscope every few minutes. When whole colonies begin to detach, replace the collagenase with 1.5 milliliters of human embryonic stem cell medium, and use a P1000 pipette to gently detach whole cell colonies from the bottom of the plate.Cells should be carefully detached so that the colonies do not break up into small pieces. Then, use a five milliliter pipette to transfer the cells into a 15 milliliter conical tube, and wash the wells with one milliliter of human embryonic stem cell medium, pooling any remaining cells in the 15 milliliter tube. After five minutes of sedimentation, carefully replace the supernatant with two milliliters of fresh human embryonic stem cell medium supplemented with basic fibroblast growth factor.Using a new five milliliter pipette, transfer the cells into an ultra-low attachment plate, and incubate the colonies for six to 12 hours. At the end of the incubation, use a five milliliter pipette to carefully transfer the colony aggregates into a new 15 milliliter tube, and collect any remaining aggregates with an RPMI-B27 medium wash. When the colonies have settled, replace the supernatant with fresh RPMI-B27 medium supplemented with glycogen synthase kinase-3 inhibitor, and carefully transfer the colonies to laminin-coated plates for attachment at 37 degrees Celsius.After 24 hours, change the medium with RPMI-B27 medium. After another 24 hours, change the medium with RPMI-B27 containing IWP2, purmorphamine, and SB43. First beating is usually observed after seven days.To date, the static suspension differentiation protocol has been tested on five human embryonic stem cell and four human induced pluripotent stem cells lines resulting in more than 90%beating spheroids from each line, demonstrating the high reproducibility of this protocol among different human pluripotent stem cell lines. The differentiating spheroids exhibit a similar behavior in a stirred suspension bioreactor environment as in the static system, with approximately 100%of spheroids observed to beating at day 10. Indeed, beating spheroid sections collected at day 30 reveal the cytoplasmic and nuclear expression of cardiac troponin T and the cardiac transcription factor NKX2.5 respectively.Further, when undifferentiated spheroids are replaced with undifferentiated cell aggregates, beating clusters appear seven days after differentiation. With 42 tested human induced pluripotent stem cell lines generating on average more than 60%cardiac troponin T positive cell populations by day 15. The beating clusters also demonstrate a similar cardiac troponin T cytoplasmic and NKX2.5 nuclear expression as observed for the stirred suspension bioreactor-generated spheroids.While attempting this procedure, it is important to remember to use hPSCs which has been adapted to single cell culture for at least three passages prior to the differentiation in the bioreactor. This procedure can be combined with other methods for enriching cardiomyocyte to acquire a pure population of cells. This technique has paved the way for commercialization of hPSC-derived cardiomyocyte.It has the potential for use in clinical, pharmaceutical, tissue engineering, and organ or organoid development applications. After watching this video, you should have a good understanding of how to efficiently differentiate human pluripotent stem cells into cardiomyocyte and to scale up the culture in bioreactor.

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

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 ...

科研

发育生物学

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

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 ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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 ...

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 ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

多能干细胞半自动化生产肝样细胞的研究

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

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 ...