pwd是支持由加拿大研究院（CIHR）健康研究奖学金和CIHR资助在西方大学的杰拉尔德·基德。笔者也想承认巨浪李圭尔夫大学的帮助，在这里提出的协议的发展。

1。媒体准备<ol><li>使用和实验将会发生的细胞培养孵化器松开帽提前准备媒体2-3小时。在本协议中使用的媒体： <ol><li>准备由DMEM/F12辅以1个B27，40 ng / ml的碱性成纤维细胞生长因子和20 ng / ml的EGF干细胞培养基。 </li><li>准备生殖细胞分化培养基由M199辅以0.05 IU FSH，LH 0.03 IU，3毫克/毫升BSA，10微升/毫升，0.23 mM的丙酮酸钠，1毫克/毫升胎球蛋白，和1 ng / ml的EGF。一个基本介质，包括所有添加剂，但省略FSH，LH和EGF可以准备并保存在4℃下长达一个星期。 </li></ol></li></ol> 2。干细胞培养制备<ol><li>分离干细胞从新生小鼠幼崽如前所述12。利用干细胞在体外生殖细胞分化2-4代。的效率OLC形成直接相关的起始干细胞的质量。这是最好的，只要人口清洁和健康的出现通常是在2代左右开始分化。进一步培养干细胞产生负面影响过去的第2代的OLC形成效率（结果未显示）。 </li><li> 48小时前开始分化干细胞亚文化。除去悬浮的球形细胞聚集和失效的介质从培养皿中，用血清吸管，在15毫升管。重要的是要只收集悬浮细胞球形聚集体，而不是附着在盘子的底部，都自发地分化细胞。 </li><li>颗粒的细胞以500×g，5分钟，弃上清，重悬在500μl的新鲜干细胞介质加热到37℃。轻轻吸取部分游离于细胞聚集。不要积极吸取细胞作为这会降低细胞活力。清洗部分分离的细胞为9.5毫升新鲜预热干细胞培养基上低附着10厘米的细胞培养皿。 </li><li>返回的细胞在37℃，5％CO 2细胞培养箱内培养48小时，在开始分化之前。 </li></ol> 3。 OLC分化<ol><li>使用血清吸管从培养皿中并置于15毫升管中取出干细胞，留下任何贴壁细胞。颗粒细胞在500 XG重悬在500微升无菌PBS。蓬勃吸管细胞使用宽孔1000微升尖不大于10-20细胞成团块。定期除去少量的细胞，在离解过程中，一个白色的光显微镜下检查，以确认凝集的细胞是在10-20个别细胞团块。功能分离，会导致细胞活力下降。 </li><li>添加9.5毫升PBS分离的细胞，并添加1此细胞悬浮液5μl的血球细胞计数。沉淀细胞以500×g，5分钟。 </li><li> 1.32X10 6细胞/ ml在分化培养基中的浓度为重悬细胞。 </li><li>板的细胞加入500微升，每到一个平底24孔悬挂菜。 </li><li>在5％CO 2的24孔板中，细胞培养孵化成在37℃下放置48小时。取出250微升培养基从各孔中的相应标记的1.5毫升管中。 ，每孔加入250μl新鲜分化培养基。离心使用了介质，在500×g离心5分钟。弃上清。在50μl新鲜的分化培养基中重悬细胞沉淀，并返回到相应的培养和分化板。 </li><li>每48小时改变介质的一半长达12天的分化。 </li></ol>

<ol>
	<li>van den Hurk, R., Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+mammalian+oocytes+and+their+growth,+differentiation+and+maturation+within+ovarian+follicles.">Formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.</a> Theriogenology. 63, 1717-1751 (2005).</li><li>Molyneaux, K. A., Stallock, J., Schaible, K., Wylie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+analysis+of+living+mouse+germ+cell+migration.">Time-lapse analysis of living mouse germ cell migration.</a> Dev. Biol. 240, 488-498 (2001).</li><li>Lawson, K. A., Hage, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+the+origin+of+primordial+germ+cells+in+the+mouse.">Clonal analysis of the origin of primordial germ cells in the mouse.</a> Ciba Found Symp. 182, 68-91 (1994).</li><li>Ginsburg, M., Snow, M. H., McLaren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cells+in+the+mouse+embryo+during+gastrulation.">Primordial germ cells in the mouse embryo during gastrulation.</a> Development. 110, 521-528 (1990).</li><li>Hubner, K., Fuhrmann, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocytes+from+mouse+embryonic+stem+cells.">Derivation of oocytes from mouse embryonic stem cells.</a> Science. 300, 1251-1256 (2003).</li><li>Toyooka, Y., Tsunekawa, N., Akasu, R., Noce, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+can+form+germ+cells+in+vitro.">Embryonic stem cells can form germ cells in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 11457-11462 (2003).</li><li>Wang, L., Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-like+Cells+Induced+from+Mouse+Spermatogonial+Stem+Cells.">Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells.</a> Cell Biosci. 2, 27 (2012).</li><li>Dyce, P. W., Wen, L., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+germline+potential+of+stem+cells+derived+from+fetal+porcine+skin.">In vitro germline potential of stem cells derived from fetal porcine skin.</a> Nat. Cell Biol. 8, 384-390 (2006).</li><li>Danner, S., Kajahn, J., Geismann, C., Klink, E., Kruse, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+oocyte-like+cells+from+a+clonal+pancreatic+stem+cell+line.">Derivation of oocyte-like cells from a clonal pancreatic stem cell line.</a> Mol. Hum. Reprod. 13, 11-20 (2007).</li><li>Cheng, X., Chen, S., Yu, X., Zheng, P., Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP15+gene+is+activated+during+human+amniotic+fluid+stem+cell+differentiation+into+oocyte-like+cells.">BMP15 gene is activated during human amniotic fluid stem cell differentiation into oocyte-like cells.</a> DNA Cell Biol. 31, 1198-1204 (2012).</li><li>Song, S. H., Kumar, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+porcine+multipotent+stem/stromal+cells+derived+from+skin,+adipose,+and+ovarian+tissues+and+their+differentiation+in+vitro+into+putative+oocyte-like+cells.">Characterization of porcine multipotent stem/stromal cells derived from skin, adipose, and ovarian tissues and their differentiation in vitro into putative oocyte-like cells.</a> Stem Cells Dev. 20, 1359-1370 (2011).</li><li>Dyce, P. W., Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+germ+line+potential+of+stem+cells+derived+from+newborn+mouse+skin.">In vitro and in vivo germ line potential of stem cells derived from newborn mouse skin.</a> PLoS One. 6, e20339 (2011).</li><li>Linher, K., Dyce, P., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primordial+germ+cell-like+cells+differentiated+in+vitro+from+skin-derived+stem+cells.">Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells.</a> PLoS One. 4, e8263 (2009).</li><li>Hayashi, K., Ogushi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Offspring+from+Oocytes+Derived+from+in+Vitro+Primordial+Germ+Cell-Like+Cells+in+Mice.">Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-Like Cells in Mice.</a> Science. , (2012).</li></ol>

没有利益冲突的声明。

迄今为止，功能运行限值和条件没有被开发使用一种完全的在体外测定。最近的一项研究由Hayashi 等。是能够产生后代从PGC细胞样细胞形成的体外和在体内进一步发展14传送。与ES细胞或诱导性多能干细胞，他们能够形成PGC类似细胞，当在体内移植后恢复开始成熟，受精，并用来产生后代14。这表明，从各种来源的干细胞有可能正确的条件下，形成?...

在早期胚胎发育，配子发生，通过一系列的阶段，包括原始生殖细胞（PGC）的形成，迁移，并最终定植生殖腺嵴。在这段时间内，原始生殖细胞进行增殖和分化到越来越成熟配子1。从尿囊胚胎肠的基础，并最终最终殖民性腺脊沿背墙，原始生殖细胞迁移的事实使得他们极其困难的研究2。尽管在该领域的进步，试图了解胆囊癌的形成和分化的研究已阻碍了其有限的数量，位置，和迁徙的性质3,4。 在最近几年，胚胎干细胞已被证明有可能形成生殖细胞在体外 5,6。同样地，一些成体干细胞也被证明有可能形成生殖细胞FOLL由于在体外培养7-11。最近分离到新生小鼠皮肤干细胞体外分化成生殖细胞和早期阶段的卵母细胞12。在分化过程中的干细胞表达Oct4的子集增加，形成类似卵丘 - 卵母细胞复合物的结构。卵母细胞样细胞（运行限值）可以捡到的文化和自然的卵母细胞相比。使用这种文化运行限值和测量方法40-45微米获得卵母细胞，如Gdf9b，瓦萨，DAZL表示了类似的标记。至目前为止，运行限值和条件仍然不能成熟或受精12。 虽然运行限值和条件仍无法运作，用于形成这些运行限值和条件的协议仍可能有实用作为检测研究早期生殖细胞的形成和发展，内封闭在体外系统。所以原来的出版物讨论形成的OLC马蒂奇利用干细胞作为干细胞来源8胎猪皮肤。扩大这项研究PGC-早期形成的细胞诱导分化过程中被证明是如OCT4运行限值和条件和快递等PGC标记的前体，VASA，STELLA，C-KIT，DAZL 13。此数据支持的潜在利用此系统来研究生殖细胞的形成和分化的影响 。从新生小鼠皮肤形成的OLC使用的协议将被证明在这个视频文章。该协议提供了一个体外模型来研究生殖细胞的发育，可能提供了一种手段，阐明其增殖和分化的重要因素。

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12500</td>
			<td></td>
		 </tr>
		 <tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>PHG0311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>bFGF</td>
			<td>Invitrogen</td>
			<td>PHG0266</td>
			<td></td>
		 </tr>
		 <tr>
			<td>ITS</td>
			<td>Invitrogen</td>
			<td>41400-045</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-6003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sodium Pyruvate</td>
			<td>Invitrogen</td>
			<td>11360-070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetuin</td>
			<td>Sigma</td>
			<td>F3385</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FSH</td>
			<td>Sioux Biochemicals</td>
			<td>915</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LH</td>
			<td>Sioux Biochemicals</td>
			<td>925</td>
			<td></td>
		 </tr>
		 <tr>
			<td>M199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td></td>
		 </tr>
		 <tr>
			<td>24 well plates</td>
			<td>Nunc</td>
			<td>144530</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 cm dishes</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		 </tr>
		 <tr>
			<td>15 ml tubes</td>
			<td>BD</td>
			<td>352095</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

differentiation of newborn mouse skin derived stem cells into germ-like cells in vitro

生殖细胞的形成和分化研究历来非常困难的，因为低的细胞的数量和它们的位置深在胚胎发育。一个“封闭” 在体外系统的可用性可能证明是无价的，我们理解配子。形成的卵母细胞样细胞（OLCS）从成体干细胞，分离新生小鼠的皮肤，已被证明可以可视化在这个视频协议。由此产生的运行限值表达与卵母细胞，如Oct4的，瓦萨，BMP15和SCP3一致的各种标记。然而，他们依然无法接受由于未能完成减数分裂成熟或施肥。此协议将提供一个系统，是用于研究早期形成和分化成更加成熟的配子生殖细胞。在早期分化表达Oct4的（潜在的生殖细胞样细胞）的数量达到〜5％，但是目前的f万传彪，乔秀云，孔惠运行限值和条件仍然相对低效的。该协议是相对简单的，但应特别注意采取措施，确保起始细胞群体是健康和早日通过。

最初的细胞附着到培养皿的底部，并传播出去。在42-72小时进入分化的微小悬浮OCT4阳性细胞形成和增殖（ 图1A）。多数会消失的可视化后不久，这些细胞和贴壁细胞会形成致密的殖民地文化底部聚集。经过几天的，这些聚集体分离从培养表面。在这些聚集体中，可以观察到一个大的细胞（ 图1b和1c）。在初始的可视化的细胞将〜35微米的直径。随着持续的文化...

Watch this Scientific Journal Video about Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro at JoVE.com

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

在最近几年使用几种不同类型的成体干细胞，生殖细胞的体外培养方法中使用的形成已被证明。本文将直观地呈现新生小鼠干细胞分化的早期卵母细胞样细胞。

新生小鼠皮肤干细胞分化成生殖细胞<em在体外</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

differentiation-newborn-mouse-skin-derived-stem-cells-into-germ-like

Research

JoVE Journal

Biology

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

9.2K 次观看.  The University of Western Ontario. 在最近几年使用几种不同类型的成体干细胞，生殖细胞的体外培养方法中使用的形成已被证明。本文将直观地呈现新生小鼠干细胞分化的早期卵母细胞样细胞。

视频: Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

在体外分化的小鼠胚胎干细胞（MES）使用悬滴法

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

科研

生物学

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 ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

高效的小鼠胚胎干细胞分化成运动神经元

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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 ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

恒河猴脂肪干细胞的分离、增殖和分化

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...