作者想感谢淳哲红（医学博士，副教授，妇产科学系，医学院高丽大学）提供胎盘组织。这项工作是由来自韩国的韩国国家研究基金会（NRF），补助（R1211902）的部分资助。

伦理声明：人类胎盘的研究进行前瞻性，与机构审查委员会对人类的高丽大学（AN09085-001）研究批准。所有的实验都是在无尘无菌室设施，在韩国大学医学中心进行。实验设计，并使用hPSCs程序批准了韩国大学医学中心的机构审查委员会（AN12277-003）。 1.准备仪器，文化传媒，和菜名<ol><li>涂层的所有培养皿用0.1％明胶。高压釜的磷酸盐缓冲盐水（PBS）和钳子。下15磅psi的消毒手术剪，微量离心管，并纱布在121℃下为15分钟以上。 </li><li>制备的预热0.25％胰蛋白酶 - 乙烯胺四乙酸（胰蛋白酶-EDTA），并含有20％胎牛血清（FBS），100U / ml青霉素预热过滤培养培养基（DMEM），和100微克/毫升streptomyc在开始操作前。 </li></ol>从人胎盘2.细胞分离<ol><li>获得胎盘组织，后剖腹产分娩，从健康的孕妇获得书面知情同意后进行正常或治疗性流产在7-32孕周。 </li><li>有从胎盘产科专科手术分离人胎盘绒毛膜板（高性能计算机）;在37℃下在0.25％胰蛋白酶-EDTA孵育这些20分钟，然后冲洗，用5ml PBS中。 </li><li>隔离用剪刀高性能计算机绒毛;剁碎的绒毛和用1ml PBS冲洗三次。切碎后，将绒毛成用枪头一个离心管中;洗用500μl的PBS将样品通过上下抽吸，并离心管120×g离心3分钟。用500μl预热的培养基洗两次组织。 </li><li>培养的细胞在明胶包被的平板培养基在37℃下，在5％CO 2 </子&gt;和下95％的湿度。预涂层在37℃下所有平板用0.1％明胶1小时。成纤维细胞样细胞的集落将形成并在相同的培养基中开始培养后约5-7天贴壁生长。 </li><li>交换介质每2-3天，直到第三通道。在培养期间，除去细胞碎片漂浮在培养基中;不断增长的胎盘成纤维细胞会附着到板上。培养物从HPC到第 10位的通道，然后在成纤维细胞样细胞后胰蛋白酶消化和丝裂霉素-C（10微克/毫升）处理收获它们用于人类胚胎干细胞培养物。 </li><li>之前冻结高性能计算机作为股票样本，使用逆转录聚合酶链反应（RT-PCR），以确认高性能计算机不沾染病原体通常感染胎盘，包括支原体（M. fermentans，M猪鼻，M精氨酸，男。ORALE，M. salivarium，人型支原体，M pulmonis，M. arthritidis，M. neurolyticum，猪肺炎支原体，和M.山羊 ）和细菌种类， 解脲脲原体 ，通过使用支原体检测试剂盒。 <ol><li>按照制造商的说明来进行RT-PCR，请使用套件中提供的所有引物和混合物。使用已培养了36天的支原体检测细胞的上清。加入3微升这些样品在PCR混合物并执行第1次的PCR程序为35个循环，每个包括94℃，30秒，55℃2分钟，72℃1分钟。 </li><li>小心加入0.5微升第1次PCR产物的PCR反应混合物，并执行第2 次的PCR程序，使用相同的条件前30个周期。分析扩增产物以1％琼脂糖凝胶电泳;申请10微升每个第1 和第 2 次 PCR产物向凝胶。 </li></ol></li><li>如果发现高性能计算机与病原体污染，消除这些使用抗生素的组合（BM-细胞周期蛋白），按照制造商的说明进行操作。添加含BM-周期蛋白1的10微克/毫升新培养基3天，然后处理细胞用5微克/毫升的BM-周期蛋白2另外4天。重复这个循环两次，然后检查是否有支原体污染了。 </li></ol> 3.收获来自人胎盘的细胞条件培养基（hPCCM） <ol><li>不含污染后，处理的贴壁细胞（85-90％汇合;（1.5×10 7个细胞/瓶）用丝裂霉素C（10微克/毫升）处理2小时和30分钟，并洗涤所述细胞三次，用10ml的PBS孵育细胞用预热的介质而不丝裂霉素-C 24小时。 </li><li>处理后一天，孵育细胞在10ml培养基（DMEM-F12补充20％敲除血清替代品[KOSR]，0.1毫β巯基乙醇，1％NEAA，和1％青霉素 - 链霉素）。经过24小时培养的，收获的CM在一个15毫升锥形管中，并使用0.22微米的注射器过滤器过滤的CM。 </li><li>添加另一个10毫升新媒体和孵化。每天收集所有上清液从培养1周和冻结所收获的培养基中于-80℃。避免反复冻融。 </li></ol> 4.文化人类胚胎干细胞（胚胎干细胞）和多能干细胞诱导（iPS细胞） <ol><li>获得人胚胎干细胞系，H1和H9细胞（分别在名义WA01和WA09美国国立卫生研究院的hESC注册表中列出），并诱导多能干细胞iPSC的1：的iPS（包皮）-1和iPSC的2（IISH1i- BM）从WiCell研究所（麦迪逊，威斯康星州，美国）。细胞应解冻和培养按照制造商的说明进行操作。 </li><li>为对照组，在广泛使用的无饲养和无血清定义培养基，在37℃基底膜基质被覆菜肴和在5％CO 2培养细胞。最初，种子80-100团块hPSCs的我ñ35毫米的菜肴。生长细胞的这些菜，直到70-80％汇合，并定期通过细胞传代培养每5-6天一次，使用机械或酶方法（中性蛋白酶）。与介质洗涤细胞两次和板其中以1：4的比例。每天更换新鲜培养基介质。 </li><li>对于实验组，培养细胞中hPCCM上碗碟预涂覆有0.1％明胶和执行例行传代，每5天一次，通过机械或酶。板的细胞在1的范围内的比率：6-1：10。 hPSCs转移后附着到明胶内2天。每天更换新鲜培养基介质。 </li></ol>人多能干细胞的5.表征注意：表征使用几种方法，诸如碱性磷酸酶染色，免疫细胞化学，定量实时PCR（QRT-PCR）和免疫印迹hPSCs。 <ol><li>免疫荧光染色<ol><li>对于immunofluorescencE染色，培养hPSCs和固定细胞在8孔载玻片室用4％（重量/体积）低聚甲醛10分钟;然后，透化的细胞用0.1％（体积/体积）的Triton-X100 15分钟，并阻断含有0.1％1小时的细胞用3％（体积/体积）正常马血清的PBS（体积/体积）吐温20。两次每一步之间的5分钟洗涤PBS。 </li><li>孵育细胞与第一抗体（OCT-4，SSEA4，TRA-1-81，TRA-1-60，稀释1：1000）O / N在4℃下，并与二级抗体。 </li><li>在安装前，孵育细胞4&#39;，6-二脒基-2-苯基吲哚（DAPI）中5分钟，在黑暗中。洗涤细胞严格三次，每次5分钟，并干细胞完全，在黑暗中。保存细胞荧光封固剂，观察他们在荧光显微镜下。 </li></ol></li><li>定量RT-PCR <ol><li>分离总RNA，从使用根据制造商的规格的RNeasy小型试剂盒的细胞和量化使用纳米掉落分光光度计提取的RNA。 </LI&gt; <li>合成cDNA通过添加2微克的总RNA，以含寡20微升的反应混合物（dT）的引物和标II逆转录酶;按照此过程中的制造商的说明。 </li><li>扩增合成的cDNA用Bio-Rad公司的iCycler iQ的系统中，采用的iQ SYBR绿定量PCR主混合物和补充表S1中所描述的引物序列。正常化的目的基因的循环阈值的那些的甘油醛-3-磷酸脱氢酶（GAPDH）。 </li></ol></li><li>碱性磷酸酶（AP）染色<ol><li>检测使用的ES细胞表征试剂盒AP活性，根据制造商的协议。 </li><li>在第5天，吸出介质并固定在PBS中的4％多聚甲醛的hPSCs 12分钟。不要overfix;定影单元的时间超过2分钟，将导致在碱性磷酸酶的失活。吸出固定液，并用清水冲洗冲洗1X缓冲。别让井步骤之间干燥。 </li><li>加入足够的染色溶液来覆盖每个孔（每35毫米培养皿的孔1ml）。孵育板在黑暗中在RT下15分钟。 </li><li>孵育后，清洗餐具用1X冲洗缓冲。盖细胞用1×PBS，以防止干燥和使用Olympus显微镜检查和图像的染色细胞。 </li></ol></li><li> Western印迹<ol><li>执行如先前在9中所述免疫印迹分析。简单地说，确定蛋白质浓度在细胞裂解物。解决蛋白上的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶等量的蛋白质转印到聚偏二氟乙烯（PVDF）膜。 </li><li>块印迹和不同的初级抗体探查，O / N在4℃;随后孵育用HRP缀合的二级抗体的印迹1小时。对于保温之间，每次10分钟洗膜三次。检测用ECL试剂的信号。 </li&gt; </ol></li><li>短串联重复序列（STR）分析<ol><li> hPSCs从对照组和实验组在同一通道收获。使用DNA微试剂盒提取基因组DNA，根据生产商的说明。 </li><li>放大用于使用AmpF / STR PCR扩增试剂盒16不同基因位点的基因组DNA，根据制造商说明和毛细管电泳是使用遗传分析仪进行的。 </li></ol></li></ol>

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作者指出无利益冲突。

该模型的开发是为了成功传播hPSCs，同时保持它们的特性，在人源化无饲养培养条件不加入外源重组生长因子，例如bFGF的或胰岛素，使hPSCs的操纵在人源化的微环境。外源性bFGF补充是常见和基质如基质胶或层粘连蛋白的应用是用于无饲养培养hPSCs 14是必不可少的。 此协议是简单，易于实现。然而，有些步骤是其成功的关键。最重要的步骤是从胎盘绒毛膜细胞的无菌分?...

The goal of this protocol is the propagation of human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) in fully humanized ex vivo feeder-free conditions without requiring additional exogenous supplementation and synthetic substrates. To date, the development of ex vivo hPSC culture models, that enable the introduction of culture products to the clinic, has been a major concern in stem cell research. Specifically, two critical problems need to be addressed. First, a humanized ex vivo culture system for the propagation of hPSCs that obviates the risk of contamination by animal cell products is needed. Second, a feeder-free culture model is needed to facilitate easy and economic mass production of hPSCs. For therapeutic applications of hPSCs, it is necessary to identify the factors that regulate their self-renewal and differentiation.
Since Xu et al. initially reported the feasibility of using conditioned media (CM) derived from mouse embryonic fibroblasts (MEF) to grow hESCs on Matrigel1, many studies have examined optimal ex vivo propagation methods for hPSCs2-4. However, an ideal humanized ex vivo feeder-free hPSC propagation system has not been developed because current methods require additional exogenous substrates including bFGF and insulin, well-known hPSC-sustaining factors, in culture media5-7. Moreover, synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures.
The rationale behind the development and use of this protocol is based on our previous studies showing that human placenta chorion cells excellently support the propagation of hPSCs without bFGF supplementation8-11. This protocol has a number of advantages including its simplicity with respect to handling and its cost-effectiveness, and it is a near-perfect humanized culture that enables hPSC propagation without exogenous synthetic substrates. The application of human placenta-derived CM (hPCCM) for hPSCs involves 3 steps. First, chorion cells are isolated from the human placenta and cultured. Second, hPCCM is produced from cultured cells. Third, hPSCs are cultured using hPCCM and their characteristics are confirmed. 
This protocol will facilitate clinical applications of hPSCs and studies of the mechanisms of hPSC proliferation and attachment. In this paper, the protocol for the successful propagation of hPSCs in hPCCM on a gelatin-coated dish is presented.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mitomycin-C</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>M4287</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>Mycoplasma detection kit</td>
			<td>TaKaRa Bio Inc.</td>
			<td>#6601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>#354277</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>STEMCELL Technologies Inc.</td>
			<td>#05850</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS02100</td>
			<td>1mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich Corporation</td>
			<td>G2500</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>BM-Cyclin</td>
			<td>Roche</td>
			<td>799 050</td>
			<td>10ug/ml</td>
		</tr>
		<tr>
			<td>RNeasy mini kit&#160;</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano Drop Spectrophotometer</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iQ SYBR Green qPCR Master Mix&#160;</td>
			<td>Bio-Rad Laboratories</td>
			<td>#170-8882AP</td>
			<td></td>
		</tr>
		<tr>
			<td>ES Cell Characterization kit</td>
			<td>Chemicon International, Inc.,</td>
			<td>SCR001</td>
			<td></td>
		</tr>
		<tr>
			<td>Power cDNA Synthesis Kit&#160;</td>
			<td>iNtRON Biotechnology</td>
			<td>25011</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp&#174; DNA Micro kit</td>
			<td>Qiagen</td>
			<td>56304</td>
			<td></td>
		</tr>
		<tr>
			<td>AmpF/STR&#174; Identifiler&#174; PCR Amplification kit</td>
			<td>Applied Biosystems Inc.</td>
			<td>4322288</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#174; 3130xl Genetic Analyzer&#160;</td>
			<td>Applied Biosystems Inc.</td>
			<td></td>
			<td></td>
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a novel culture model for human pluripotent stem cell propagation on gelatin in placenta-conditioned media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

此HPSC传播方法的一个优点是，它使用了由人细胞分泌的成分。在协议A最ciritical步骤是人胎盘来源的细胞的分离和培养。这需要从HPC板绒毛的精确部分准确清扫。 图1示出了用于来自人胎盘的细胞分离的步骤。这个过程是简单的和方便的细胞的分离，并且可以很容易地在1小时内进行。如在图2中描述的，组织附着到发芽鲁棒明胶涂层盘和成纤维细胞样细胞。在传代通过胰蛋...

Watch this Scientific Journal Video about A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media at JoVE.com

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

一种新的文化模式的人多能干明胶胎盘条件培养基细胞增殖

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

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Research

JoVE Journal

Developmental Biology

7.8K 次观看.  Graduate School of Medicine, Korea University. This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

视频: A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

科研

发育生物学

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

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 ...

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 ...

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

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

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, ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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.