分化的胚胎干细胞<ol><li>通过用ESC胰蛋白酶处理的胚胎干细胞（ESC）上收集的附近汇合瓶中。 </li><li>重悬细胞分化媒体5毫升，并转移到非明胶涂层的T25。 37 ° 45分钟的彗星。检索上清，通过离心收集细胞。 注：小鼠胚胎成纤维细胞从ESC文化（MEFs）容易附着到非明胶涂层的T25，从而从文化耗尽在此期间电镀步骤。 如果你不培养你的人事编制小组委员会上MEFs，你可以跳过前板 。 </li><li>悬浮MEF耗尽ESC 333333 cells/50毫升分化媒体。这种集中，结果在100 ESC/15毫升。 </li><li>使用多通道移液器，板100 cells/15毫升下降15厘米2培养皿板ESC。 8通道移液器，你应该能够适合滴每盘约18-22行（约每盘5毫升）。 注：此协议，2-5 15厘米2滴的菜就绰绰有余了，应该产生2-5 6 × 10 6天的EB -源性细胞 。 </li><li>轻轻翻转板倒置滴。孵育于37℃5％CO 2的 48小时。 </li><li>池滴，轻轻摇动板和转移至15 mL锥形管。 4-6毫升的PBS冲洗板，并添加到相同的15 mL锥形。 注意：您可能池五个悬滴板。 EBS将增长，因为他们继续分化，如果他们过于集中，他们往往形成大的团块。 </li><li>允许汇集胚比重（约10分钟）定居。不扰民胚吸上清。轻轻地悬浮在10毫升分化媒体转移到非贴壁10厘米的培养皿板。 </li><li>置于培养皿板板振动筛在50 rpm和孵化，在37℃，5％的CO 2 。 </li><li> 24小时后（分化），旋流板集中在培养皿中心胚。仔细交流媒体的50％（5毫升）5 mL新鲜分化媒体。返回板孵化器为两天。 </li></ol> EB分离和MSCV HOXB4 - IRES - GFP的感染<ol><li>分化6天，胚转移到15 mL锥形管。允许通过重力来解决。 </li><li>吸媒体和重新悬浮于10 mL PBS。允许胚，以安置受重力。吸PBS。 </li><li>加入250毫升分解酶的结构和1毫升PBS。在37℃水浴孵育20分钟，偶尔旋流管混合酶和EBS。 </li><li>添加8 mL的酶分解缓冲区。 </li><li>磨碎混合使用5毫升吸管，直到胚是完全分离的（约10倍;过度移液器将增加内的EB -派生的细胞制备的死亡）。 <ol><li>混合物，将成为与细胞分化游离于多云。 </li><li>配售期间trituration吸管针尖对锥管底部，将创建一个剪切力，这将大大方便分解。 </li></ol></li><li>通过离心收集细胞。 </li><li>悬浮在5毫升10％IMDM派生的EB -细胞计数采用台盼蓝拒 注：至第四天和分化的第六天，分化气蚀而导致在大量的细胞凋亡和细胞death.Thus，在第六天，超过30％的EB -源性细胞可能与台盼蓝染色 。 </li><li>悬浮的EB -派生的细胞，在10万细胞/ 2 mL的10％IMDM +病毒上清这样一个MOI 5-10 achieved.Add硫酸鱼精蛋白的终浓度为8毫克/毫升。 注：准备足够的EB /病毒上清混合板4个6孔板（假设2毫升/）。 </li><li>板2毫升EB /病毒上清混合，每孔4个6孔板OP9基质细胞（见下文）镀前。 </li><li>板在室温下在2500 RPM离心90分钟。传输板，以孵化器在37℃5％的CO 2。 </li><li> 4-6小时后，收获板上清，留在暂停通过离心收集任何潜在的细胞。 </li><li>悬浮颗粒（可能是小）在48 mL 10％的IMDM +细胞因子。分配2毫升/到原来的4个6，以及在其中进行感染，并收集上清板块的再悬浮。 注意：请务必准备10％IMDM +细胞因子在使用时的新鲜。 </li><li>在37℃，5％的CO 2七天板。菌落应明显第四天感染后，大和良好的第七天。一个强大的扩展应产量/七天40-60殖民地。 </li><li>在七天后感染，收集和池与胰蛋白酶处理所有井的所有细胞（包括OP9基质）。 注意：不要丢弃在胰蛋白酶treatment.At这一点在文化聘用的上清或PBS清洗，有些殖民地可能只是笼统地贴壁，并有许多细胞floasuspension.Collect和池所有的清洗和上清婷，以确保没有潜在的有价值的的细胞被丢弃。 </li><li>悬浮细胞，在8毫升新鲜的10％IMDM +因子。分配到四个T75烧瓶2毫升/烧瓶。到每个烧瓶中加入13毫升10％IMDM +的细胞因子。在37 ° C 5％CO 2为3天（共10天感染）。 </li></ol> OP9基质的文化和电镀<ol><li>根据20％的A - MEM标准协议，维护OP9基质。 OP9细胞生长时会发生变化，以汇合的属性。始终在80％左右，不超过1:3分割汇合分裂。 </li><li> 24小时前一天六个派生的EB - MSCV - HOXB4 - IRES - GFP的，板块25,000 OP9细胞/以及4个6孔板在20％A - MEM细胞的感染。 </li></ol>收集，分离和移植<ol><li>收集扩大OP9基质细胞为10天通过与胰蛋白酶处理。计数细胞。 注意：不要丢弃上清液或PBS洗涤受雇期间胰蛋白酶处理。在这点文化，有些殖民地可能只是笼统地贴壁，并有许多细胞在suspension.Collect和池浮动所有的清洗和上清，以确保没有潜在的有价值的的细胞被丢弃。 注：一个应该产生良好的扩展40-50 × 10 6细胞/原来的6孔板感染之间，共为160-200 × 10 6细胞， 。 </li><li>细胞可能是通过磁珠选择或流式细胞仪，根据标准的技术，这一点在协议中分割。 </li><li>移植，提供细胞通过眼窝或尾静脉注射到一个分割剂量照射2.5小时的9.25 GY Rag-2/gc缺乏能力的受助人（体重15-22克之间）。除了。 注：关键是，照射时间在15-22克之间的动物体重下降。 ，如果他们都超过22克，9.25 GY不会是一个足够的照射剂量，以确保适当的消融和细胞可能无法调解致死量照射和/或嫁接的造血车厢救援 。 如果较大的动物移植，适当的照射剂量，必须确定实验 。 </li><li>最低剂量的2-5 × 10 6细胞/动物是必需的，以保证救援致死量照射。如果分馏细胞，注入2-5 × 10 6细胞现金等价物（ 例如 ，如果感兴趣的人口占10％的普通细胞池，注入2-5 × 10 5细胞/动物） 。 注意：如果注射&gt; 2 × 10 6细胞，经尾静脉注入，以避免形成畸胎瘤在轨道上，这可能会导致细胞高复古轨道交付。 </li><li>保持RAG - 2 - GC / - / - / -蒸压笼中的不足之处者。根据“清洁”的动物设施，移植后的动物，可能需要5周后致命的辐射酸化水或Trimethiprim Sulfasoxazole（Septra）的管理。 </li><li>期望在外周血移植后3周&gt; 90％的GFP + ES衍生的白细胞。植入应多血统，尽管淋巴细胞被称为逆转录病毒HOXB4 IRES - GFP的沉默。 </li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

干细胞被定义为一个有能力的细胞自我更新和产生多个不同的后代。胚胎干细胞（ESC）上都来源于早期胚胎的囊胚differentiative能力的多能干。其巨大的differentiative潜力，使他们推断如何哄他们产生临床上有用的细胞类型为中心的许多研究的重点。成功，造血干细胞从小鼠ESC（HSC）的推导，最近已经完成，可以在这个视频协议的可视化。 HSC的，可以说是最临床上利用的细胞群，是用来治疗无数的造血系统恶性肿瘤和障碍。然而，许多患者可能会受益于HSC的治疗得不到合适的捐赠者。 ESC键可以为这些患者提供替代来源的HSC。以下协议，建立了从ESC - HSC，可以研究，并告知努力找出人类胚胎的HSC的基线。在这个协议中，ESC 6天市售的血清前筛选最佳的造血分化的胚状体（EBS）是有区别的。分化是再分解，与逆转录病毒HOXB4感染。感染的EB -源性细胞接种于OP9基质，派生从颅骨的M - CSF - / - 小鼠骨髓基质细胞系，并合作培养中存在的十天，促进细胞因子的造血。在这种合作文化，被感染的细胞大大扩大，在一代人造成了数百万个细胞100S异构池。然后，这些细胞可以用于救援和重建致死剂量照射的小鼠。

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

该协议的细节从小鼠胚胎干细胞（ESC），并随后注入到致死剂量照射的的受体小鼠的造血干细胞移植的推导。简言之，电子调速器是有区别的胚体，然后用逆转录病毒HOXB4 OP9基质细胞和造血细胞因子共同培养的感染。

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 ...

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K 次观看.  Harvard Medical School. 该协议的细节从小鼠胚胎干细胞（ESC），并随后注入到致死剂量照射的的受体小鼠的造血干细胞移植的推导。简言之，电子调速器是有区别的胚体，然后用逆转录病毒HOXB4 OP9基质细胞和造血细胞因子共同培养的感染。

视频: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

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 ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

小鼠卵黄囊和AGM早期造血干细胞的分离

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...