作者感谢珍妮特Rossant原协议。这项研究是由国家健康授予CA106308到WH研究所的支持。

在这个协议中，我们描述了小鼠囊胚的编制和TS细胞株的建立。一般的鼠标操作前囊胚的集合，包括设立自然交配和超排卵诱导，基本上是按照标准的协议，由纳吉等人说明。2003年（pp146 - 150）。 Ts细胞的推导和维护<ol><li>设置之间的利益老鼠繁殖的十字架。 </li><li>准备作为饲养层细胞的小鼠胚胎成纤维细胞（MEFs）。前两天收集囊胚，MEFs（2 × 10 6细胞）均镀在100毫米的菜肴。第二天，这些细胞是治疗用10毫升的TS培养基中含有丝裂霉素C（20毫克/毫升）在37℃，5％的CO 2为2个小时的孵化。其次是用PBS洗涤细胞两次。丝裂霉素C处理的细胞，然后接种于12孔板（1 × 10 5细胞/孔）。 </li><li>在囊胚收集（3.5 DPC，被称为第一天）一天，MEF培养液取代TS液加1X FGF4/Heparin（0.5毫升/孔）。 </li><li>制备方法：收集子宫安乐死鼠标，找到子宫（图1A）。 </li><li>通过抓钳在宫颈（位于膀胱后面）删除子宫和子宫和子宫颈（图1B）交界处的跨越。拉子宫，修剪膜和脂肪组织中，削减低于交界处的子宫输卵管（图1C）。小体积的TS培养基（0.2毫升）在60毫米的钢板放置在子宫（图1D）。 </li><li>每1毫升的TS使用26号针头的1 ml注射器介质冲洗子宫角。针插入子宫基地，并刷新到一个新的60毫米板1毫升的TS中等缓慢。子宫应在冲洗充分膨胀和扩展。 </li><li>仔细收集并传输到一个干净的12孔盘包含的TS媒介使用控制口吸管的囊胚。洗净囊胚在12孔板通过TS中期串行传输三次。放置一个囊胚每口井（图2A）在12孔，丝裂霉素C处理的MEF的馈线和文化板块在37 ° C，5％的CO 2。 </li><li>囊胚将孵化卵透明带和附加井在24至36小时。一个小的产物将随时观察3天。饲料用500μl新鲜的TS中加1X FGF4/Heparin文化。 </li><li>在4或5天，胚泡的产物，应该取决于其大小分类。 TS细胞分类的理想大小，如图3b所示。用0.5毫升PBS洗一次的文化。添加100毫升0.25％胰蛋白酶/ EDTA和孵育5分钟，在37 ° C，5％的CO 2。移液器上下同一个P100的pipetteman大力的产物，分解成小团块。停止加入1.5F4H TS - 70CM（30％TS中等，70％的MEF - CM加1.5 FGF4/Heparin）入井胰蛋白酶。更改中等8小时后分解，并继续喂养细胞，每2天。 </li><li>在6和10（高度可变）天之间，TS细胞克隆可观察到。他们有这样一个明确的殖民地边界（图2C）的形态，地势平坦，上皮表。继续喂的文化，每2天</li><li>当Ts细胞达到50％汇合（通常是15和20天之间），用PBS洗一次，加100毫升0.25％胰蛋白酶/ EDTA。移液器在胰蛋白酶的向上和向下，以确保附近的单细胞悬液。停止加入含有丝裂霉素C处理的MEF的馈线（2.5 × 10 5 cells/6-well或5 × 10 5 cells/60mm板），以新的6孔或60mm板的TS - 70CM - 1.5F4H和转让胰蛋白酶。 </li><li>更改中等8小时通过后，继续喂文化每2天。 </li><li>经过一个或两个以上的通道上的MEF馈线之间的通道（3-5天），Ts细胞可培养他们。由于MEFs速度远远高于胰蛋白酶消化后的TS细胞培养板的重视，在贴壁率的这种差异可以利用单独从MEF馈线Ts细胞。通过培养细胞后，在37℃，5％的CO 2为30分钟。大多数MEFs将附加到该板块的TS人口仍处于悬浮状态。将上清转移至一个新的板块。几个段落重复这一过程中获得一个纯粹的TS人口。 </li><li>保持TS - 70CM 1.5F4H Ts细胞，并通过他们每4天（1:10至1:20分割）或文化达到70％汇合时。通过高密度（高于1:5分割）或培养他们过于汇合时可能会导致其分化的细胞。 </li><li> Ts细胞的身份可以确认，由一个细胞标记，CDX2（图3）免疫。 </li></ol>冻结Ts细胞<ol><li>准备2X冻存液（50％FBS，30％的TS中期和20％二甲基亚砜）。 </li><li>合作胰蛋白酶llect Ts细胞，其次是在450克离心6分钟。 2X冻存液等体积重悬在TS培养基。 </li><li>冻结的细胞慢慢使用异丙醇室-70 ° C和24-48小时后转移到液氮。 </li><li> 2瓶通常保存在一个60毫米的钢板（70％合流）。 </li></ol>解冻的TS细胞<ol><li>快速解冻小瓶在37 ° C，收集他们在10毫升的TS媒体离心（450克，6分钟），在30％TS媒体，70CM - 1.5F4H悬浮。 </li><li>一小瓶细胞接种于60毫米板。 </li></ol> TS细胞分化成TGC 撤出MEF - CM，FGF4和肝素可诱导的Ts细胞分化为滋养层巨细胞（TGCs）。 TGC的表型（大核）可以被检测到四天后感应（Tanaka等人，1998年和 Chiu 等 。，2008年）。 p450scc免疫染色检测TGC这种分化的文化标记（图4A）的表达。流式细胞仪分析PI（碘化丙啶）染色细胞进一步分化的细胞中含有较高的DNA含量（图4B）。这些结果表明，TS细胞内复制，成为多倍体TGCs。 代表性的成果<img src="/files/ftp_upload/1964/1964fig1.jpg" alt="图1" /> 图1。小鼠子宫剥离。 （一）子宫的位置，用箭头表示。 （二）跨越子宫和宫颈交界处，如图所示。 （三）切子宫略低于下的卵巢（OV）输卵管（OD）。 （四）形象的子宫。 <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="图2" /> 图2。代表图片说明小鼠囊胚，囊胚生长和滋养的殖民地。 （A）在E3.5小鼠囊胚收集所示。 ICM，内细胞团;吨，壁画滋养层; PT，极滋养层。 （B）在MEF馈线滋养产物所示。 TS，滋养干细胞。 （C）滋养层干细胞集落边缘清楚显示了MEF的馈线。箭头指示的单元格边界。 <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="图3" /> 图3。Ts细胞免疫分析鉴定。 TS（公元）和ES（控制，EH）细胞标记鉴定。细胞是免疫染色认识到Oct4的（A，E）或CDX2（B，F）的抗体，由合并后的图像中提出的DAPI（C，G）（D，H）counterstained。比例尺，50微米。 <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="图4" /> 图4。Ts细胞在体外分化。 （一）未分化（Undiff）还是有区别的（DIFF）细胞培养是一个TGC标记的表达研究，p450scc（绿色），counterstained用DAPI（红色）。 （二）流量分化的细胞与PI染色，流式细胞仪分析，措施的DNA含量（货币供应量M1，两到四个副本; M2，超过四份）。 M2人口占多倍体的滋养细胞。

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

在这个视频中，我们展示的过程来收集E3.5从子宫和实验程序的囊胚建立TS细胞株。我们还描述了条件，保持Ts细胞的干性和诱导分化的细胞，其分化。两个关键的步骤，以取得纯TS细胞株的产物分解的时间（步骤9）和加工的第一段（第11步）。据报道，原始内胚层来源的细胞可以在文化产生广泛的囊胚后在第11步（Kunath等人，2005年）在第9步或繁茂的TS殖民地（&gt; 50％汇合），长大了。虽然原始内?...

试剂 TS介质： RPMI - 1640年，20％胎牛血清，1毫米丙酮酸钠，100毫米β-巯基乙醇和100毫克/毫升的青霉素，链霉素。 丝裂霉素C： 2毫克丝裂霉素C（Sigma公司的M - 0503）2毫升PBS溶解，并加入到200毫升的TS中（10毫微克/毫升）2毫升的混合物。直到使用20等分（10毫升）并储存在-20 ° C。新增100毫米板的每一个等份。 小鼠胚胎成纤维细胞条件培养基（MEF - CM）： 板MEFs（1 × 10 6 /板）在100毫米的钢板。第二天，添加10毫升，每道菜的TS介质，并持续48小时的文化。收集培养液，过滤（0.45毫米），并储存在-20 ° C在35毫升分装。解冻每个等分需要时可在4 ° C储存MEFs可用于准备之前，他们成为融合两个批次的CM。 PBS / BSA： 通过0.45毫米的注射器过滤溶解BSA的PBS（10毫升），过滤器，分数在五（西格玛A3311，0.1％（W / V））和1毫升分装在1.5毫升管。贮存于-70℃，解冻一管时，需要准备FGF4。 FGF4（1000倍）： 悬浮冻干FGF4（西格玛F8424，25毫克），1毫升的PBS / BSA溶液。充分混匀，10等份（100毫升）1.5毫升管和存储，在-70 ° C。在需要时解冻每管和储存其余4 ° C时，不要再冻结。在TS液稀释1000倍获得1X FGF4（25毫微克/毫升）。 肝素（1000倍）： 悬浮肝素在PBS（Sigma公司H3149，10000台）至终浓度为1毫克/毫升（1000倍）。设为100毫升分装到1.5毫升管和储存在-70 ° C。解冻等份，当需要存储的其余4 ° C时，不重新冻结。在TS培养基稀释1000时间获得1X肝素（1毫微克/毫升）。

derivation of mouse trophoblast stem cells from blastocysts

滋养层的规范，是最早的哺乳动物发育分化事件之一。从滋养层介导植入派生和滋养细胞谱系产生胎儿胎盘的一部分。因此，本宗族的发展是必不可少的胚胎存活率。最初是由田中滋养干细胞（TS）从小鼠囊胚细胞的推导<em&gt;等。</em&gt; 1998年。 Ts细胞的能力，以保持适当的刺激后的滋养特定的属性和阶段和细胞类型特异性标志物的表达提供了一个有价值的模型系统研究滋养细胞谱系的发展，即扼要早期的胎盘事件。此外，滋养细胞，少数细胞发生的自然基因组扩增的类型之一。虽然底层滋养多倍体的分子途径已经开始显现出来，滋养基因组扩增的生理作用和优势在很大程度上仍然难以捉摸。多倍体的文化滋养细胞的干细胞二倍体的发展，使得这<em&gt;体外</em&gt;系统阐明基因组复制和不稳定的监管机制，在健康和疾病的优良工具。在这里，我们描述在邱修改以前发表的报告为基础的协议<em&gt;等。</em&gt; 2008年。

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

在这段视频中，我们展示了小鼠囊胚的隔离和滋养干细胞从胚泡的推导。我们还介绍了干细胞财产的维护，以及在文化的分化诱导条件。

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 ...

derivation-of-mouse-trophoblast-stem-cells-from-blastocysts

Research

JoVE Journal

Biology

18.1K 次观看.  University of Rochester. 在这段视频中，我们展示了小鼠囊胚的隔离和滋养干细胞从胚泡的推导。我们还介绍了干细胞财产的维护，以及在文化的分化诱导条件。

视频: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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 ...

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...