Name: robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders
Uploaded: 2022-02-09T14:45:00
Description: Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

这项工作由卡塔尔国家研究基金（QNRF）资助（资助号NPRP10-1221-160041）。Maryam Aghadi得到了卡塔尔国家研究基金（QNRF）的GSRA奖学金的支持。

该研究已获得适当的机构研究伦理委员会的批准，并按照1964年《赫尔辛基宣言》及其后来的修正案或类似的伦理标准中规定的伦理标准进行。该协议已获得HMC（编号16260/16）和QBRI（编号：2016-003）的机构审查委员会（IRB）的批准。这项工作还针对H1和H9等hESC进行了优化。血液样本是在完全知情同意的情况下从健康个体获得的。iPSCs由健康个体的外周血单核细胞（PBMC）产生。
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该协议至关重要，因为它能够提供高产量和高效率的MSC。这种大规模生产MSCs是通过将iPSCs衍生的EB与10μM的RA14，15瞬时孵育来实现的。用 10 μM RA 瞬时处理将 MSC 产量提高了 11.2 至 1542 倍14，15，该方案适用于 iPSC 和 hPSC。在此剂量和治疗持续时间下，RA通过直接或间接调节参与细胞增殖，凋亡以及细胞间和ECM细?...

间充质干细胞（MSCs）是产生中胚层起源细胞（如脂肪细胞，骨细胞和软骨细胞）的有效短暂资源，可以进一步用于模拟其各自的遗传疾病。然而，以前的方法依赖于从成人组织1获得这些MSC，这带来了从供体获得大量MSC的挑战，并且限制了它们在次优体外培养条件下保持功能存活1，2。这些障碍产生了对体外产生MSCs的方案的巨大需求。人诱导多能干细胞（iPSCs）可用作MSC的宝贵来源，表现出MSC特征3，4，5。iPSCs衍生的间充质干细胞可用作多种疾病的治疗选择。此外，iPSCs衍生的MSCs产生脂肪细胞的能力使其成为研究人类脂肪生成，肥胖和脂肪细胞相关疾病的有价值的体外人类模型。
目前脂肪细胞的分化方案可分为两组，一组涉及使用化学或基于蛋白质的混合物分化脂肪细胞，结果产量为30%-60%6，7，8，9，而另一组涉及遗传操作以稳健地诱导控制脂肪细胞发育的关键转录因子，以产生80%-90%10的产量，11.然而，基因操作并不能概括脂肪细胞分化的自然过程，并且经常掩盖脂肪生成过程中到达的微妙范式，使其对疾病建模目的无效12，13。因此，我们提出了一种通过使用尼罗河红荧光标记含脂脂肪细胞来将化学衍生的成熟脂肪细胞与未成熟脂肪细胞进行分类的方法。
在这里，我们提出了一种协议，涉及将iPSC衍生的拟胚体（EB）与全反式 维甲酸瞬时孵育以产生大量快速增殖的MSC，这可以进一步用于产生脂肪细胞14。我们还提出了一种通过使用亲脂性染料荧光标记其脂滴来从异质分化池中分选化学衍生的成熟脂肪细胞的方法;尼罗河红。这将允许产生具有增强功能的成熟脂肪细胞的纯群体，以准确模拟脂肪细胞相关的代谢紊乱。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adiponectin</td>
			<td>Abcam</td>
			<td>ab22554</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>anti-CD105</td>
			<td>BD Pharmingen</td>
			<td>560839</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD14</td>
			<td>BD Pharmingen</td>
			<td>561712</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD19</td>
			<td>BD Pharmingen</td>
			<td>555415</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD34</td>
			<td>BD Pharmingen</td>
			<td>555824</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD44</td>
			<td>abcam</td>
			<td>ab93758</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD45</td>
			<td>BD Pharmingen</td>
			<td> 
			560975</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD73</td>
			<td>BD Pharmingen</td>
			<td>550256</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>anti-CD90</td>
			<td>BD Pharmingen</td>
			<td>555596</td>
			<td>MSC differentiation marker</td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D</td>
			<td>233-FP</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>C/EBPA</td>
			<td>Abcam</td>
			<td>ab40761</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Torics</td>
			<td>1126</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>FABP4</td>
			<td>Abcam</td>
			<td>ab93945</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ThermoFisher</td>
			<td>10082147</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>ThermoFisher</td>
			<td>35050-061</td>
			<td>MSC culture media supplement</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma Aldrich</td>
			<td>I5879</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma Aldrich</td>
			<td>I7378</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma Aldrich</td>
			<td>91077C</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>ThermoFisher</td>
			<td>12660012</td>
			<td>Basal media for preparing matrigel</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>ThermoFisher</td>
			<td>11885084</td>
			<td>MSC culturing media</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Coating matrix</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>ThermoFisher</td>
			<td>12561056</td>
			<td>Adipocyte differentiation media</td>
		</tr>
		<tr>
			<td>Nilered</td>
			<td>Sigma Aldrich</td>
			<td>19123</td>
			<td>Sorting marker for adipocyte</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td>MSC/Adipocyte media supplement</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>Pierce&#8482; 20X TBS Buffer</td>
			<td>Thermo Fisher</td>
			<td>28358</td>
			<td>wash buffer</td>
		</tr>
		<tr>
			<td>PPARG</td>
			<td>Cell Signaling Technology</td>
			<td>2443</td>
			<td>Adipocyte maturation marker</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>5872</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625</td>
			<td>MSC differentiation media supplement</td>
		</tr>
		<tr>
			<td>Rock inhibitor</td>
			<td>Tocris</td>
			<td>1254/10</td>
			<td>hPSC culture media supplement</td>
		</tr>
		<tr>
			<td>Roziglitazone</td>
			<td>Sigma Aldrich</td>
			<td>R2408</td>
			<td>Adipocyte differentiation media supplement</td>
		</tr>
		<tr>
			<td>StemFlex</td>
			<td>ThermoFisher</td>
			<td>A334901</td>
			<td>hPSC culture media</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Thermo Fisher</td>
			<td>28314</td>
			<td>Permebealization reagent</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher</td>
			<td>25200072</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P7942</td>
			<td>Wash buffer</td>
		</tr>
	</tbody>
</table>

robust differentiation of human ipscs into a pure population of adipocytes to study adipocyte-associated disorders

诱导多能干细胞（iPSC）技术的最新进展允许产生不同的细胞类型，包括脂肪细胞。然而，目前的分化方法效率低，不能产生均匀的脂肪细胞群。在这里，我们通过使用基于全反式视黄体的方法以高产量生产间充质干细胞（MSCs）来规避这个问题。通过调节控制细胞增殖、存活和粘附的途径，我们的分化策略允许有效生成分化成纯多能MSC群体的胚胎体（EB）。该方法产生的大量MSC为产生脂肪细胞提供了理想的来源。然而，脂肪细胞分化导致的样品异质性仍然是一个挑战。因此，我们使用基于尼罗河红的方法使用FACS纯化含脂成熟脂肪细胞。这种分选策略使我们能够建立一种可靠的方法来使用具有降低样品异质性和增强细胞功能的脂肪细胞池来模拟脂肪细胞相关的代谢紊乱。

间充质分化过程中细胞的示意图和形态：iPSC 分化为 MSC 涉及跨越 EB 形成、MSC 分化和 MSC 扩增的不同发育阶段（图 1）。在这些发育阶段，细胞由于受到不同的刺激化学物质而获得各种形态。在开始分化时，细胞被铺在悬浮液中，并且预期是圆形的，具有明确的细胞边界，同时直径为小到中等尺寸（图2）。在分化的初始阶段选择悬浮培养细胞使其与自?...

Watch this Scientific Journal Video about Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders at JoVE.com

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

该方案允许从诱导多能干细胞（iPSC）产生纯脂肪细胞群。维甲酸用于将iPSCs分化为间充质干细胞（MSC），用于产生脂肪细胞。然后，采用基于尼罗河红染色的分选方法获得纯脂肪细胞。

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

以前的方法依赖于在体外获得间充质干细胞的高侵入性方法，但这些间充质干细胞具有产生30%异质池的局限性，效率范围为30%至60%在这里，我们提出了一种用于产生大量快速增殖的间充质干细胞的方案，该干细胞可以通过使用尼罗河红进行分类来产生纯的成熟脂肪细胞群。当iPSCs达到80%汇合时，用DPBS洗涤细胞并加入含有EDTA的解离培养基。将细胞在 37 摄氏度下孵育一分钟。一分钟后，吸出解离试剂并将细胞置于37摄氏度下再放置一分钟。接下来，从培养箱中取出板。每孔加入一毫升StemFlex培养基，并小心地将细胞收集在15毫升锥形管中。离心细胞。除去上清液，并轻轻地将它们重悬于含有10微摩尔岩石抑制剂的3毫升MSC分化培养基中。混合细胞悬液，并在24孔超低附着板中每孔分配500微升。然后将板放入37摄氏度的培养箱中。24小时后，为了诱导获得的胚胎体，将它们收集在15毫升的管中并让它们沉淀下来。15分钟后，除去上清液，加入补充有10微摩尔维甲酸的MSC分化培养基。轻轻地重悬胚胎体，并在相同的24孔超低附着板中分配每孔500微升。然后将板放入培养箱中。48小时后，将胚胎体收集在15毫升的管中，让它们静置15分钟。然后除去上清液，加入补充有0.1微摩尔维甲酸的MSC分化培养基。重悬胚胎体后，在同一24孔板中每孔分配500微升，并将板放入培养箱中。最后一次视黄酸处理后48小时，收集胚胎体，除去上清液，加入不含细胞因子的DMEM低葡萄糖培养基。在同一 24 孔超低附着板中轻轻重悬并分配每孔 500 微升细胞悬液，然后在 37 摄氏度下孵育板。48小时后，将iPSC来源的胚胎小体铺板，将胚胎小体收集在15毫升管中，使其沉降，然后除去上清液，并重悬于两毫升新鲜MSC分化培养基中。接下来，将悬浮液转移到基底膜基质涂层的六孔板的两个孔中，并在37摄氏度下孵育细胞。五天后，用含有每毫升2.5纳克碱性成纤维细胞生长因子的新鲜MSC分化培养基替换用过的培养基，并继续MCS分化长达11天和15天。当铺好的胚胎体达到80%至90%汇合时，通过用DPBS洗涤，加入胰蛋白酶-EDTA，并在37摄氏度下孵育一分钟。从平板中取出胰蛋白酶-EDTA，并在37摄氏度下再次孵育一分钟。接下来，每孔添加一毫升培养基。三分钟后，使用MSC分化培养基在15毫升锥形管中收集细胞并离心细胞。然后将细胞重悬于含有碱性成纤维细胞生长因子的MSC分化培养基中，并以1：3的比例将细胞接种在基底膜基质包被的平板上。在MSCs达到90%汇合后，继续培养它们48小时，以使它们经历一段时间的生长停滞。然后取出培养基并用DBPS洗涤细胞。洗涤后，将完整的脂肪细胞分化培养基添加到平板中，并将细胞在37摄氏度下孵育。每隔一天更换培养基，持续 14 天。将 iPSC 来源的 MSC 分化为脂肪细胞。要使用尼罗河红对脂肪细胞进行分类，请按照文本手稿中的说明在DMSO中制备尼罗河红工作溶液。使用前，解冻储备溶液并将其在DPBS中复溶，以达到300纳摩尔的工作溶液浓度。在脂肪细胞分化的第14天或之后，从细胞中丢弃培养基并使用DPBS洗涤。然后加入尼罗河红工作溶液。盖上平板并在37摄氏度下孵育细胞。15分钟后，用胰蛋白酶-EDTA替换尼罗河红溶液，并将细胞在37摄氏度下孵育四分钟。然后使用含有5%FBS的DMEM在15毫升锥形管中收集细胞并离心细胞。除去上清液并以每毫升100万个细胞的密度将细胞重悬于DPBS中。然后使用 FACS 分选仪，使用 FL-1 通道分离尼罗河红阳性细胞。收集分选的细胞并在脂肪细胞分化培养基中重新培养它们，或进行RNA提取，然后对脂肪细胞分化标志物进行定量分析。在开始分化时，悬浮接种的细胞是圆形的，具有明确的细胞边界，直径为小到中等尺寸。胚胎体的生存能力是通过快速增殖行为来观察到的，从而产生更多的MSC。这种快速增殖行为以及它们奇特和细长的形态即使在将MSC传代到新鲜的基质包被板上后仍能保留。良好的分化可产生可靠的MSC，间充质表面标志物CD73、CD44和CD90的表达效率大于90%。此外，对细胞缺乏描述造血表型CD14，CD34和CD19的表面标志物的评估显示表达效率低于1%，FABP4（终末分化脂肪细胞的标志物）在细胞质分布中的高表达表明其发育成熟。此外，脂联素（脂肪细胞成熟的另一个标志物）的高表达表明脂肪细胞的功能足以响应葡萄糖信号传导进行脂质储存和脂肪生成。染色后，尼罗红仅与含脂成熟脂肪细胞结合，使其成为使用荧光激活流式细胞术分选成熟脂肪细胞的有效工具。与未分选的细胞相比，尼罗河红阳性细胞的成熟标志物显着上调至少两倍。在胚胎体形成和间充质细胞解离过程中收集细胞时，您必须非常温和，因为这会极大地影响间充质干细胞解离后的存活效率。从携带突变的患者获得的纯脂肪细胞群可以进行功能和全转录组测序，以确定受特定突变影响的调节和信号通路，而数据不受样品异质性的影响。该技术将允许在体外可扩展地生成间充质干细胞，无需从人类供体中收集它们，以便于将它们分化为脂肪细胞，软骨细胞和骨细胞，以了解与组织相关的疾病发病机制。

robust-differentiation-human-ipscs-into-pure-population-adipocytes-to

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

在SH-SY5Y人神经母细胞瘤分化

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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 various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

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 ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

将人诱导的多能干细胞分化为神经元和神经胶质的混合培养物进行神经毒性测试的方案

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

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.

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 ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

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 ...