作者想承认了宝贵的帮助：尼尔第一手稿的编辑和Kourtney威尔金森博士;露西信通博士，博士Brigit Willeford和迈克Basett在密歇根州立大学ALAC的认可鼠标设施，培训和动物服务的援助;德韦恩明智博士为他的援助显微镜和图像采集;塞萨尔蒙罗伊，汉娜Swoope，协助他们在密歇根州立大学与视频生产和Bobbie赫德尔斯顿。这项研究是由研究机构和密西西比州立大学生物科学系办公室。

第1部分：孕鼠剖腹<ol><li>使用颈椎脱位，安乐死在10.5-11.5 DPC怀孕C57BL6J雌性小鼠。 </li><li>抗菌肥皂与清洁腹部，然后，刮胡子。 </li><li>剃须后，用生理盐水溶液冲洗腹部。 </li><li>然后，用无菌纱布擦干腹部。 </li><li>盖鼠标与一个新的无菌领域。 </li><li>制作腹切口，使用镊子和解剖剪刀。 </li><li>识别和清除腹腔整个子宫。小鼠胚胎将子宫内可见。 </li><li>转移到培养皿中弥漫着的D - PBS的子宫，并保持它在冰上。 </li><li>使用无菌手术刀和镊子，取出每个胚胎的胎盘和多余的胚胎组织。 </li><li>转移到培养皿填充的D - PBS新鲜菜的胚胎。 </li><li>测量长度的小鼠胚胎。我们发现，根据胚胎的年龄大小不同。例如8.5 DPC测量平均为6mm，10.5 DPC测定平均为11mm，平均为16毫米测量和12.5 DPC。 </li><li>然后，去除DNA的提取它们的尾巴。 </li></ol>第2部分：性腺岭夹层在体视显微镜和光源肖特Fostec <ol><li>放置在培养皿滤纸。然后，将滤纸上的小鼠胚胎干的胚胎和固定解剖。 </li><li>用消毒的手术刀，使以上横向切断脐带来源的小鼠胚胎。 </li><li>立体显微镜下，用无菌的罚款钳和无菌罚款戏弄针，去除肠子和肝脏，泌尿系统是可见的。 在腹腔，肾脏位于横向比较肾脏和膀胱位于内侧和尾鳍。胚胎可以被认定为男性，因为性腺脊位于膀胱两侧。在女性，性腺脊牢固地附着在肾脏尾外侧端。 </li><li>剥离性腺脊出它背后的滑动针。削减了支持它的组织的性腺脊被删除。 </li><li>性腺脊转移到一个新的充满新鲜的D - PBS的培养皿。 显微镜下，男性性腺脊应剥离，大和椭圆形。相比之下，女性性腺脊应被发现，有长形和男性性腺脊比较小。 </li></ol>第3部分：性腺岭消化在体视显微镜： <ol><li>中肾解剖：分开，性腺中肾和使用罚款和尖锐的针脊。 <ul><li>这一点很重要，因为它是一个体组织在PGC的推导过程，将长满删除从性腺嵴中肾。 </li></ul></li><li>性腺岭消化：0.5ul新鲜的D - PBS下​​降收集在一个干净的性腺脊。添加20毫升胶原酶/ Dispase溶液（1mg/ml的最终浓度）。 <ul><li>胚胎应单独处理。每个胚胎应该有自己的独立和标记的培养皿。 </li></ul></li><li>性腺岭切碎：使用消毒针头和无菌细镊子4号，剪切成小块性腺脊。 </li><li>孵化：培养皿中转移到孵化器在37 ° 15分钟。 <ul><li>潜伏期的时间可能会有所不同组织之间。 </li></ul></li><li>移液：15分钟后，组织可以通过移液分离。使用直径40 -100毫米之间，突破拉升的玻璃毛细管移液器吹打向上和向下的。这将形成小团块。将团块一个前预热的D - PBS作最后洗的下降0.5毫升。 </li><li>洗涤后，转移到Eppendorf管的团块。 <ul><li>不要过分暴露的组织，以胶原酶。 </li><li>不要做一个单细胞悬液。这是很重要的的集落形成。如果它是一个单细胞悬液，PGCS倾向于分化和迁移。 </li><li>这个过程应该不会超过20分钟，或你的细胞就会失去生存能力。 </li></ul></li><li>在室温下5分钟在2000转离心。 </li><li>离心后，取出上清液，重悬在0.5毫升补充敲了媒体（在37 ° C预热）沉淀。 </li></ol>第4部分：元文化的生殖细胞 “你应该迅速执行下一个步骤，以维护隔离和推导PGCS的可行性。 <ol><li> 24小时之前，这个过程中，你要准备一个分裂灭活MEF - CF1的饲养层，J.等2008张以下的协议。 <ul><li>准备之一孕20菜NT雌性小鼠（6-8胚胎）。 </li><li> MEFs失去的能力是一个饲养层后5-6代（促进经济增长和防止分化）。 </li><li>不要使用MEFs超过48小时以上。 </li></ul></li><li>电镀前立即PGCS，MEF的介质必须慢慢从MEF饲养层。 </li><li>然后，添加0.5毫升/孔的预热补充到MEF饲养层的淘汰赛介质。 <ul><li> MEF媒体含有胎牛血清诱导分化的PGCS。 </li><li>淘汰赛介质必须辅以实物特异性生长因子之前使用，以确保生长因子的活性。 </li></ul></li><li>使用对称的分布在MEF饲养层，板PGCS。 <ul><li>如果这些作品的距离太近，他们往往聚集密集菌落附着不佳或开始区分。 </li><li>培养皿必须标有胚胎生发细胞系的名称，传代次数，和日期。 </li></ul></li><li>小心地转移到孵化器在37 ° C和5％的CO 2的培养皿。 </li><li>每24小时监控的细胞。 </li><li> 48小时后，取出250毫升的媒体，从文化的最上方，以避免令人不安的殖民地的附件。然后，添加250毫升的新鲜补充淘汰媒体（在37 ° C预热）。 </li><li>第7步后，再等待48小时，然后，彻底清除所有媒体和更换新鲜补充淘汰的媒体（在37 ° C预热）。这样做，每2天为8-10天，直到PGC的殖民地形式。 </li></ol>第5部分：胚胎生殖细胞<ol><li> PGC的隔离10天之后，胚胎生发细胞集落形成。 </li><li>胚胎生发细胞株是由手动通道，每8-10天保持活着。他们继续提出的未分化的形态及表达多能性标志物，如alkalyne磷酸，10月4，SSEA - 1，和SOX - 2 </li></ol>注释<ul><li>无菌/无菌条件下，在文化室至关重要。 </li><li>推导和文化，PGCS在净化器II级生物安全柜处理。 </li><li>孵化在加湿37 ° C和5％ 二氧化碳培养箱。 </li><li>被过滤掉所有的媒体和试剂，使用前先在一个0.2微米的过滤器保存在4 ° C，并预热，使用前在37 ° C。 </li><li>在清扫的步骤，所有的胚胎和组织必须保持在冰上。 </li><li>所有的试剂瓶中，放置在柜前用乙醇净化。 </li><li>手套，白大褂和护士帽的使用是强制性的。 </li></ul>结论我们已经提供了一个视频将告诉您，如何隔离，推导，文化10.5-11.5 DPC小鼠胚胎性腺脊的胚胎生殖细胞。胚胎生发细胞系的重现性隔离和长期的文化，提供了一个研究早期胚胎发育，遗传和表观遗传的生发模式，性腺形成的关键基础，在配子发生在男性和女性胚胎发育过程中，周围脏器的环境影响确定的途径，导致癌症和不育症。 <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="图1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

这项研究后，进行审查和机构动物护理和使用委员会（IACUC）我们的鼠标协议＃08030（活性氧物种的ROS在种系发育和肿瘤形成中的作用：控股和繁殖协议）批准在密西西比州立大学。

钳第14号和15日，手术刀35毫米，解剖剪刀，手术领域，抗菌肥皂，盐水0.9％，纱布，冰泡沫容器，刀片，手术刀35毫米，4号和5级罚款钳，细戏弄针处理，过滤纸，然后在拉玻璃吸液管。<br /<br /&gt;设备：解剖立体显微镜，光源肖特Fostec，倒置显微镜，微量移液器10和200ul，离心机，生物安全柜。

isolation and derivation of mouse embryonic germinal cells

胚胎生殖细胞的能力（例如），以区分在早期发育阶段，是一个完美的模型，以解决我们的假设，对癌症和不育症的原始生殖细胞（PGCS），后来进入配子。该协议显示了如何在10.5-11.5天coitum（DPC）的小鼠胚胎来自发展中国家性腺的原始生殖细胞分离。发展中国家从小鼠胚胎性腺脊（C57BL6J）分离机械破碎与胶原酶，然后镀上了一个小鼠胚胎成纤维细胞饲养层（MEF - CF1）以前分裂与丝裂霉素C灭活淘汰赛媒体的存在，并辅以白血病抑制，因子（LIF），碱性成纤维细胞生长因子（bFGF），干细胞因子（SCF）。使用这些优化方法盈科鉴定，隔离，并建立培养条件允许超过40天，EG细胞的长期培养。胚胎生发细胞株显示胚胎的表型多能状态的常用标记和表达。文化的生发细胞的分离和衍生提供了一个工具，以了解体外他们的发展提供了机会，在遗传和表观遗传一级监察累积损伤后接触氧化应激。

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

胚胎生殖细胞分化成原始生殖细胞在早期发展阶段的能力，是一个完美的模型，以解决我们的假设，对癌症和不育症。该协议显示了如何从发展中国家性腺的原始生殖细胞中分离出来后10.5-11.5天coitum小鼠胚胎。

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 ...

isolation-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.0K 次观看.  Mississippi State University. 胚胎生殖细胞分化成原始生殖细胞在早期发展阶段的能力，是一个完美的模型，以解决我们的假设，对癌症和不育症。该协议显示了如何从发展中国家性腺的原始生殖细胞中分离出来后10.5-11.5天coitum小鼠胚胎。

视频: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

禽流感胚胎瓣膜祖细胞的分离和培养

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

胚胎心室血管内皮细胞的分离

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...