我们承认美国国立卫生研究院新的创新奖DP2 116520 ORAU拉尔夫·鲍威青年教师提升奖的支持。

1。细胞维持<ol><li> H1的人类胚胎干细胞（WiCell）均保持在合格的人类胚胎干细胞基底膜涂井mTeSR1媒体，与媒体每天都在变化。井被涂上稀释在25毫升的培养液中加入300μL的人类胚胎干细胞基底膜基底膜解决方案的准备，F12键。被添加到每个六孔板以及1毫升本matrigel的解决方案，或400μL加入到12孔板的每个井，并允许在室温下1小时大衣。机械代细胞刮，一旦他们达到了1至直径1.5mm的殖民地，在分流比为1:4。 </li><li> RHMVEC（血管内皮细胞技术）保持MCDB上-131媒体与媒体改变隔日。细胞分裂由胰蛋白酶，一旦达到80％汇合，分流比为1:3。通道3和7之间的内皮细胞被用于这项研究。 </li></ol> 2。储备溶液的制备<ol><li>激活素A改组为50微克/毫升无菌带够学士含0.1％牛血清白蛋白。解决的办法是分装并储存于-20°C </li><li>表皮生长因子在500微克/毫升无菌10毫米醋酸进行了改组。解决的办法是分装并储存于-20°C </li><li>在10毫克/毫升的胰岛素，重组加入酸化H2O（pH值≤2），由0.1毫升冰醋酸10毫升的水，除了准备。 </li><li> DAPT作用进行了改组在DMSO在18毫克/毫升。进一步稀释到300μM浓度的DMEM：F12键，分装和储存在-20°C </li><li>在DMSO在5mg/ml KAAD环杷被改组。进一步稀释到2μM浓度的DMEM：F12键，分装和储存在-20°C </li><li>全反式维甲酸在2.7毫克/毫升95％的乙醇进行了改组。它被进一步稀释至2MM浓度的DMEM：F12键，分装和储存在-80°C。 </li></ol> 3。明确的内胚层（DE）和胰腺祖（PP）的感应<ol><li>一旦ĤESC键殖民地达到1至直径1.5mm，加入DMEM培养液进行德感应：F12键补充B27的0.2％BSA的100毫微克/毫升，激活素A和1μM的4天渥曼青霉素（0天4日）与媒体每天都在变化。 </li><li>德感应完成后，诱导胰腺祖细胞诱导培养液通过改变媒体：B27的0.2％BSA和0.2μM浓度为24小时（日4日5）KAAD环杷明在F12键补充。 </li><li>媒体24小时后改为DMEM培养基：F12辅以B27的0.2％BSA，0.2μM的浓度，浓度在2μm的全反式维甲酸与媒体每天为3天（5天，8天）KAAD环杷。 </li></ol> 4。胰腺成熟<ol><li>聚丙烯诱导后，各组分别改为成熟媒体组成的DMEM：F12键补充B27的钠，0.2％BSA，10毫米的烟，25微克/毫升胰岛素，30纳米2 SEO 3和50微克/毫升转2天媒体ays改变每天（8天，10天）。 </li><li>经过2天的成熟媒体，最终成熟的第一步是进行并行使用几个不同的条件下进行比较。根据下列条件：（一）缺口抑制剂DAPT作用（二）使用联合培养媒体只，无内皮细胞作为对照组及（iii）使用RHMVEC细胞接触共培养诱导分化。细胞维持在一个星期（10日17日）与媒体每天DAPT作用或联合培养媒体。 <ol><li> DAPT作用媒体准备补充DAPT作用的成熟媒体30微米。第一次接触这个群体中的细胞24小时内完成MCDB131，然后暴露于DAPT作用媒体为6天（11天17天）。 </li><li>合作文化传媒准备补充MCDB上-131媒体与B27的0.2％BSA，10毫米的烟，10纳克/毫升表皮生长因子，1微克/毫升氢化可的松，10毫克EndoGro，90微克/毫升的肝素。对媒体的控制条件下的分化细胞暴露MCDB上完全培养基-131（10日11日）24小时后，媒体被改变为6天（11天17天）（内皮细胞）共培养媒体。 </li><li>接触共培养条件下，一百万RHMVEC添加到完整MCDB上131媒体24小时的分化细胞（血管内皮细胞技术）（10日11日）允许附件。 24小时后，媒体被改变为6天（11天17天），与媒体的合作，文化传媒每天都在变化。 </li></ol></li></ol> 5。 QRT-PCR分析<ol><li>在每个阶段的分化（内胚层，胰腺祖成熟胰岛）年底裂解和RNA的提取使用NucleoSpin RNA II提取试剂盒，根据制造商的指示。 </li><li> RNA的质量进行检查，随后通过RNA科学研究SmartSpec Plus分光光度计使用量化RNA的吸光度在260 nm和280 nm。 </li><li>逆转录使用ImProm II逆转录ķ它根据制造商的指示。所有的反应进行了100纳克的RNA。 </li><li>根据制造商的指示使用的Mx3005P QPCR系统（安捷伦）和辉煌II的SYBR Green QPCR的主混合QRT-PCR进行。表1列出所用的引物。初始化步骤进行，在50°C为10分钟，95℃2分钟。 50个扩增循环如下：95℃30秒，54°，30秒和72℃1分钟， </li><li>倍的目标mRNA表达的变化是未分化的细胞，从CT值使用下列公式计算： 的ΔCT（标记我 ）= CT诊断（标记我 ） - CT（GAPDH的） Δ的ΔCT（标记I）=的ΔCT（标记）（样本） -的ΔCT（标记）（未分化的细胞） 相对表达（标记）= 2-ΔΔCT（标记） </li></ol> 6。免疫组化<ol><li>细胞在分化的每个阶段结束（内胚层，胰腺祖成熟胰岛）被固定在4％多聚甲醛在室温为15分钟。 </li><li>透固定细胞，用0.25％的Triton X-100（TX）的15分钟。 </li><li>非特异性染色被挡在10％驴血清孵育30分钟TX的0.05％。 </li><li>初级抗体孵化一夜之间在4°C推荐抗体稀释（见表1）在阻塞缓冲区。 </li><li>细胞用0.05％的TX 3次5分钟。 </li><li>一小时，在黑暗与相应的抗体封闭液稀释的二级抗体在室温下进行孵化。 </li><li>细胞用0.05％的TX 3次5分钟。 </li><li>赫司特进行核染色染色在1:1000稀释在PBS孵育用PBS洗涤5分钟。 </li><li>固定和染色细胞成像使用奥林巴斯IX81倒置显微镜和Metamorph成像软件。 </li></ol> 7。代表结果此外激活和Wortmannin 4天的未分化的胚胎干细胞诱导明确的内胚层QRT-PCR分析证实“Sox17德标记”，CXCR4，和FOXA2 Sox17免疫如图2所示。胰腺祖细胞标记和免疫组织化学染色为图3所示为PDX1 QRT-PCR证实了胰腺祖细胞分化后，除了环杷和维甲酸。 在分化的最后阶段，联系RHMVEC强烈的人类胚胎干细胞衍生的胰腺祖细胞诱导胰岛素表达上调的细胞共培养。合作文化介导的分化效率，通过共同分析mparing与控制条件下使用DAPT作用。 DAPT作用是用来作为阳性对照，因为它是目前使用最广泛的实现胰腺成熟的方法之一。由于共同的文化支持因素，血管内皮细胞补充修改后的媒体，与媒体进行额外的控制，在内皮细胞的情况下，以验证媒体上分化的影响。这一分析的详情载于图4。 <img alt="图1。" src="/files/ftp_upload/3759/3759fig1.jpg" /> 图1。多阶段的人类胚胎干细胞分化为胰岛素表达细胞的协议。 <img alt="图2。" src="/files/ftp_upload/3759/3759fig2.jpg" /> 图2。明确的内胚层诱导人类胚胎干细胞。一）QRT-PCR分析代表性DE暴露激活素A和渥曼青霉素4天的胚胎干细胞的标记。结果AR<html> é正常化方面未分化的胚胎干细胞。乙）Sox17染色（绿）和DAPI（蓝色）显示Sox17核表达。比例尺：50微米。 <img alt="图3。" src="/files/ftp_upload/3759/3759fig3.jpg" /> 图3。胰源性内胚层细胞的胚胎干细胞的祖细胞诱导。一）在胰腺癌早期人类胚胎干细胞衍生的内胚层细胞暴露环杷和维甲酸4天德诱导后，标记的QRT-PCR分析。结果未分化的胚胎干细胞正常化。二）PDX1染色（紫色）和DAPI（蓝色）显示PDX1核表达。比例尺：50微米。 “&gt; <img alt="图4。" src="/files/ftp_upload/3759/3759fig4.jpg" /> 图4。胰腺成熟阶段）QRT-PCR分析人类胚胎干细胞的胰腺激素胰腺癌使用DAPT作用成熟后，与媒体联合培养或联合培养（对照组）。结果未分化的胚胎干细胞正常化。 P值获得学生t-检验。 B）与DIL-AC-LDL标记RHMVEC（红色）共培养内皮细胞附着板，如果有空白（顶部）显示区域，被发现在其他RHMVEC可以区分ESC（底部）直接接触。 c）C-肽（绿色）染色分化细胞共培养方法。比例尺：12.5微米（顶部）和50微米（底部）四）RHMVEC的免疫染色表明胰岛素表达没有在RHMVEC的。 <table border="1"><tbody><tr><td> 标记 </td><td> primer1（5&#39;至3&#39;）&lt;/ TD&gt; <td> primer2（5&#39;至3&#39;） </td><td> 价 </td></tr><tr><td> SOX17 </td><td> CTCTGCCTCCTCCACGAA </td><td> CAGAATCCAGACCTGCACAA </td><td> 12 </td></tr><tr><td> CXCR4的</td><td> CACCGCATCTGGAGAACCA </td><td> GCCCATTTCCTCGGTGTAGTT </td><td> 4 </td></tr><tr><td> FOXA2 </td><td> GGAGCGGTGAAGATGGAA </td><td> TACGTGTTCATGCCGTTCAT </td><td> 12 </td></tr><tr><td>含PDX1 </td><td> AAGTCTACCAAAGCTCACGCG </td><td> GTAGGCGCCGCCTGC </td><td> 4 </td></tr><tr><td> PTF1 </td><td> CATAGAGAACGAAACCACCCTTTGAG </td><td> GCACGGAGTTTCCTGGACAGAGTTC </td><td> 12 </td></tr><tr><td> HLXB9 </td><td> CACCGCGGGCATGATC </td><td> ACTTCCCCAGGAGGTTCGA </td><td> 4 </td></tr><tr><td> HNF6 </td><td> TGTGGAAGTGGCTGCAGGA </td><td> TGTGAAGACCAACCTGGGCT </td><td> 5 </td></tr><tr><td> PAX6的</td><td> CGAATTCTGCAGGTGTCCAA </td><td> ACAGACCCCCTCGGACAGTAAT </td><td> 5 </td></tr><tr><td> NKX6.1 </td><td> AGACCCACTTTTTCCGGACA </td><td> CCAACGAATAGGCCAAACGA </td><td> 5 </td></tr><tr><td> ISL1 </td><td> GATCTATGTCACCTCGCAAGG </td><td> TACAACCACCATTTCACTG </td><td> 12 </td></tr><tr><td>胰高血糖素</td><td> AGGCAGACCCACTCAGTGA </td><td> AACAATGGCGACCTCTTCTG </td><td> 4 </td></tr><tr><td>胰岛素</td><td> AAGAGGCCATCAAGCAGATCA </td><td> CAGGAGGCGCATCCACA </td><td> 12 </td></tr></tbody></table> 表1。用于定量PCR反应的引物。

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胰腺发育期间，胰腺细胞的分化是在靠近主动脉内皮细胞;此外，胰岛细胞密集的血管，促进血糖和胰岛激素的迅速交流。鉴于这些事实，这并不奇怪，内皮细胞在胰腺器官的过程中发挥重要作用。虽然胰腺发育过程中血管内皮细胞的重要性日益受到重视，及其在胚胎干细胞体外分化中的作用研究。在我们前面的报告中，我们建立了小鼠胚胎干细胞的13对胰腺癌成熟内皮细胞?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 浓度 </td></tr><tr><td> mTeSR1（与补充） </td><td>干细胞技术</td><td> 5850 </td><td></td></tr><tr><td>人类胚胎干细胞合格的基底膜</td><td> BD公司</td><td> 354277 </td><td></td></tr><tr><td> DMEM培养基：F12 </td><td> Invitrogen公司</td><td> 11330-032 </td><td></td></tr><tr><td> MCDB上的131 </td><td> Invitrogen公司</td><td> 10372019 </td><td></td></tr><tr><td> MCDB上-131（完整） </td><td>血管内皮细胞技术</td><td> MCDB上的131 </td><td></td></tr><tr><td> B27的补充</td><td> Invitrogen公司</td><td> 17504044 </td><td></td></tr><tr><td>激活素A </td><td>研发</td><td> <a href="http://www.rndsystems.com/pdf/338-ac.pdf">338-AC</a> </td><td> 100ng/ml的</td></tr><tr><td>渥曼青霉素</td><td> Invitrogen公司</td><td> W3144 </td><td> 1μM </td></tr><tr><td> KAAD-Cyclopamie </td><td> Sigma-Aldrich公司</td><td> C4116 </td><td> 0.2μm的</td></tr><tr><td>全反式维甲酸</td><td> Sigma-Aldrich公司</td><td> R2625 </td><td> 2μM </td></tr><tr><td> DAPT作用</td><td> Sigma-Aldrich公司</td><td> D5942 </td><td> 30μM </td></tr><tr><td>烟</td><td> Sigma-Aldrich公司</td><td> N0636 </td><td> 10毫米</td></tr><tr><td>亚硒酸钠</td><td> Sigma-Aldrich公司</td><td> S5261 </td><td> 30纳米</td></tr><tr><td>胰岛素</td><td> Sigma-Aldrich公司</td><td> I1882 </td><td> 25微克/毫升</td></tr><tr><td>转</td><td> Sigma-Aldrich公司</td><td> T8158 </td><td> 50微克/毫升</td></tr><tr><td>表皮生长因子</td><td>研发</td><td> <a href="http://www.rndsystems.com/pdf/236-eg.pdf">236 - EG</a> </td><td> 10ng/ml时</td></tr><tr><td> EndoGro </td><td>血管内皮细胞技术</td><td> ENDOGRO </td><td> 10毫克</td></tr><tr><td>肝素</td><td> Sigma-Aldrich公司</td><td> H3149 </td><td> 90μg/ml </td></tr><tr><td>氢化可的松</td><td> Sigma-Aldrich公司</td><td> H0888 </td><td> 1μg/ml </td></tr><tr><td> NucleoSpin RNA的II </td><td>马歇雷内格尔</td><td> 740955 </td><td></td></tr><tr><td> ImProm II反转录系统</td><td> Promega公司</td><td> A3800 </td><td></td></tr><tr><td>辉煌II的SYBR Green QPCR的主混合</td><td> straragene </td><td> 600548 </td><td></td></tr><tr><td> sox17山羊多克隆抗体</td><td>圣克鲁斯</td><td> SC-17355 </td><td> 1/500 </td></tr><tr><td>含PDX1山羊多克隆抗体</td><td>圣克鲁斯</td><td> SC-14662 </td><td> 1/500 </td></tr><tr><td> C-肽兔多克隆</td><td>细胞信号</td><td> 4593 </td><td> 1/500 </td></tr><tr><td>的Alexa Fluor 488驴抗兔IgG </td><td> Invitrogen公司</td><td> -21206 </td><td> 1/1000 </td></tr><tr><td>的Alexa Fluor 647驴抗山羊IgG </td><td> Invitrogen公司</td><td> -21447 </td><td> 1/1000 </td></tr></tbody></table>表2。试剂和试剂盒。

endothelial cell co-culture mediates maturation of human embryonic stem cell to pancreatic insulin producing cells in a directed differentiation approach

胚胎干细胞（ESC）上有两个主要特点是：它们可以无限期地在体外培植，在未分化的状态，他们是多能干细胞，因而有可能分化成多种细胞。这种特性使胚胎干细胞非常有吸引力的基于细胞治疗和再生治疗应用1。然而，对于充分发挥其潜力，实现细胞分化成成熟和功能的表型，这是一项艰巨的任务。诱导细胞分化的一个很有前途的方法是在体外设置紧密地模仿器官的路径。胰腺癌的发展被称为发生在特定的阶段，开始与内胚层，它可以发展成多个器官，包括肝脏和胰腺。内胚层诱导可以通过调制节点的通路，通过另外一个3激活素，多种生长因子结合4-7 </SUP&gt;。明确的内胚层细胞，然后经过刺猬抑制实现由8环杷此外，它可以在体外抑制胰腺癌的承诺。是由几个平行的事件，包括抑制Notch信号通路介导胰腺成熟;胰腺祖细胞聚集成3维簇;诱导的血管;仅举几例。远远的ESC源性胰岛祖细胞体外成熟的最成功的已实现通过抑制Notch信号DAPT作用补充9。虽然成功了，这个结果中低产减少功能的成熟型。研究较少的地方，是内皮细胞在胰腺成熟细胞信号，这是越来越多地被作为一个重要的因素在体内胰岛成熟10,11赞赏的效果。 目前的研究，探讨内皮素等的影响人类ESC源性胰腺祖细胞的成熟胰岛素的胰岛样细胞胶质细胞信号。我们报告一个多阶段的人类胚胎干细胞首次对胚层诱导激活素抑制PI3K通路的一个定向分化协议。胰腺内胚层细胞的规范实现随着维甲酸诱导此外维甲酸抑制环杷刺猬信号。成熟的最后阶段被诱导内皮细胞共培养的配置实现信号。虽然一些内皮细胞已在共同的文化测试，在此我们提出我们的数据与大鼠心脏微血管内皮细胞（RHMVEC），主要是为便于分析。

Watch this Scientific Journal Video about Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach at J

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

目前的研究，介绍了胰腺人类胚胎干细胞分化诱导定向分化的方法。具有重要意义的发现，血管内皮细胞共培养介导派生为胰岛素表达细胞的胰腺祖细胞的人类胚胎干细胞的成熟。

共培养内皮细胞介导成熟的人类胚胎干细胞向胰腺中胰岛素生成细胞在定向分化的方法

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

endothelial-cell-co-culture-mediates-maturation-human-embryonic-stem

Research

JoVE Journal

Biology

16.2K 次观看.  University of Pittsburgh. 目前的研究，介绍了胰腺人类胚胎干细胞分化诱导定向分化的方法。具有重要意义的发现，血管内皮细胞共培养介导派生为胰岛素表达细胞的胰腺祖细胞的人类胚胎干细胞的成熟。

视频: Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...