作者们大大赞赏细胞分离技术帮助，为当前的电影和卡尔霍顿先生和乔纳森卢卡斯先生为他们的幻灯片编辑上的帮助海鹰潘女士和先生，Ian Bellayr。 DRS：涉案人员已与过去几年的发展，修改preplate技术下面的列表非常感谢。 BURHAN Gharaibeh宝红曹迪希布里奇特，托马斯R ·佩恩，张爱萍陆，海荣鹏大岛，东条英博，曾晓东，木，Kimimasa飞田，罗恩Jankowski，Makato Ikezawa。我们是从詹姆斯H康明斯，瑞安Pruchnic，约瑟夫Feduska，丽贝卡C. Schugar，海鹰潘和杰西卡Tebbets技术援助表示感谢。笔者也将像到承认的肌肉萎缩症协会（MDA）的，：美国国立卫生研究院研究院（NIH），国防部（DOD），儿童匹兹堡医院UPMC的，部对这项工作的资助矫形外科杂志，和美国匹兹堡大学。

在一般情况下，修改后的完整preplate过程需要1-2天的准备，1周执行的技术过程，细胞与免疫细胞化学鉴定2-3天，和一个额外的2-3周干细胞的人口膨胀。干细胞培养所需的设备与其他细胞培养系统，其中包括台式离心机， 二氧化碳培养箱，层流组织培养罩和倒置显微镜配备荧光和一台数码相机。分离出干细胞也可以被克隆，并可以储存在液氮目前或未来的研究1,2长远。此过程中使用的所有试剂和材料必须是无菌的无菌处理。 步骤1：准备<ol><li>胶原蛋白外衣组织培养皿和培养瓶：I型胶原小腿皮肤（1公升0.1克; Sigma - Aldrich公司）所得。外套塑料制品包括：T - 25瓶，6或12孔板，菜（60和100毫米，屋宇署猎鹰）前1,2所述。轻轻摇动塑料制品每隔半小时，4小时，以确保涂层的胶原蛋白。胶原蛋白溶液，然后删除。涂塑料制品，然后让其干燥，在组织培养罩无论在紫外灯和引擎盖，以保证无菌，终于扼或覆盖以备后用。 </li><li>准备细胞培养液：400毫升DMEM培养基（DMEM培养液，高血糖; Invitrogen公司），毫升热灭活胎牛血清（FBS，Invitrogen公司），50毫升马血清（HS，Invitrogen公司），和5 mL鸡胚提取物（CEE，准确化工有限公司）将使用一次性的0.22微米500毫升无菌过滤系统（康宁）的真空过滤。无菌青霉素/链霉素溶液（Invitrogen公司）将被添加最后100单位/ mL。制备的溶液可以保存在4 ° C，至少一个月。使用这个解决方案是从烧瓶中分离的细胞分裂的文化时，或准备用于移植的细胞。 </li><li>暖起来细胞分离的解决方案：下列试剂到被温暖最多至37 °彗星之前他们利用细胞的分离：汉克的缓冲盐溶液的HBSS，Invitrogen公司; 0.2％（重量/卷）胶原酶的类型第十一（西格玛Aldrich公司），与3200胶原蛋白消化每毫升的HBSS单位的平均水平; Dispase 2.4 units/1mL（Invitrogen公司）;胰蛋白酶（Invitrogen）的0.5％（WT /卷）。 </li><li>消毒，准备手术设备：手术过程需要包括无菌手术器械：不同大小的钳，微型剪刀，虹膜剪刀和/或手术刀片。此外，15日和50 mL聚丙烯离心管（BD猎鹰）;细胞过滤器（孔径70米）（BD猎鹰），18 - ，23 - 和27号针（BD PrecisionGlide）和10 - 20毫升（BD）的无菌注射器。 </li></ol>第2步：组织切片，隔离和机械分离1-4 组织切片进行下一个无菌培养罩，包括新鲜收获的肌腱和骨骼肌肉获得的野生型小鼠的后肢（女C57BL/6J，年龄4-5周或更年轻，杰克逊的胫骨或比目鱼肌实验室）。任何可见的结缔组织，血管，脂肪组织，然后从活检样品中删除。肌腱和肌肉组织，然后仔细辨认解剖显微镜下手术，以消除皮肤和骨骼的残余，并放置在一个包含冷（4 ° C）的HBSS（添加5％FBS的补充）。盘胶原涂层的菜肴，包含冷的HBSS，然后分别置于孤立的组织将被剁碎成粗浆使用微型剪刀和/或手术刀片（&lt;1 × 1毫米3 ）。 第3步：酶消化组织： 剁碎的组织，然后通过一系列步骤2,4,5酶分离<ol><li>转移到单独的15毫升管和离心机在4 ° C 5分钟〜3500转的肉末组织泥浆。 </li><li>去除上清，用HBSS中，再次重复离心。 </li><li>移除上清液，加入10毫升预热0.2％胶原酶型第十一章（Sigma公司）和消化这些泥浆。孵育60分钟，在37 ° C的同时，不断轻轻摇晃管。另外，由专人每10分钟摇动。 </li><li>离心和重新悬浮于10 ml dispase（2.4单位/ ml，Gibco公司）解决方案，这些泥浆。在37 ° C孵育45分钟，双手颤抖，或轻摇每10分钟。 </li><li>离心机和重新挂起，这些泥浆在0.2％胰蛋白酶的HBSS溶液10毫升。孵化的基础上，以单细胞组织可以通过显微镜下观察暂停执行释放的程度会有所不同。这是不建议超过30分钟，在37 °反相管C或轻摇每10分钟。 </li><li>离心产生的细胞S和重新暂停在增殖培养基（下午）10毫升的细胞沉淀。 </li><li>通过一系列针提取分离的细胞悬液。细胞，然后通过一个18G的2倍，那么23G，和最后一个27G的针头。 </li><li>穿过70微米的细胞过滤器，细胞提取物。 </li></ol>第4步：Preplating来隔离不同的细胞对细胞粘附率1-3人口<ol><li>离心机和增殖培养基重悬细胞沉淀。 </li><li>板的细胞到胶原蛋白涂层的T - 25瓶（或60毫米盘）的混合物，如PP1的标志。应拥有大量的屈光细胞核和其他碎片，在显微镜下观察细胞悬液。 </li><li>在37 °，饱和湿度，5％的CO 2培养箱中培养2小时中的C。 </li><li> nonadherent细胞转移到新的胶原蛋白涂层的T - 25瓶（或60毫米盘）和标记为PP2。添加到标有PP1的板块下午5毫升。早期坚持细胞附着于烧瓶中大多是肌纤维母细胞3。 </li><li>返回烧瓶孵化器，另外24小时，nonadherent细胞转移到一个新的胶原蛋白涂层的T - 25瓶（或菜），它标记为PP3。添加到PP2板的5毫升增殖培养基。这些坚持细胞附着到这个烧瓶大多是成纤维细胞2,3。 </li><li>返回烧瓶孵化器和在另一个24小时内重复的创建过程，直到人口PP6。保持各组悬浮细胞的增殖，并定期检查他们使用倒置显微镜，并允许他们。 PP3 - PP4的大多是成肌细胞肌卫星细胞常常被看作主要PP5 2。 </li><li>慢慢坚持细胞通常由PP6重视。在这一阶段，细胞出现小而圆，光的折射率，在数量上很稀疏，被认为是干细胞的人口1,2。 </li></ol>第5步：识别和分离出干细胞扩张<ol><li>隔离PP6细胞是极少数，需要保持胎牛血清丰富的增殖培养基（20％在DMEM培养液胎牛血清）每天更换。大部分细胞在下面的1-2周的培养，但目前在PP6文化死通常是一个大型的，健康的PP6人口扩张的一个额外的1-2周后创建的。从小鼠的干细胞高效表达干细胞抗原1（SCA1），CD34，胎肝激酶1（的Flk1），及其他干标记1,2,6,7可通过免疫细胞化学染色观察。这些干细胞还表现出多种分化能力 3,6,8 。 </li><li>分离出的细胞是维持和扩大在文化的细胞密度低（12菜50-100个细胞/孔或&lt;20-30％汇合在烧瓶或菜），以避免分化 1,2 。极少数细胞形成过程中扩张的克隆，这些克隆的干细胞可以进行长时间扩散（LTP），任何其他能力的研究。克隆的干细胞也可以储存在液态氮使用一般的细胞存储程序，例如用10％DMSO下午中等。设为任何其他备份研究干细胞的低通过股票的数量。 </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

人们已经认识到，一些成人组织包含多个的干细胞。在过去几年的研究，干细胞已被发现在肌肉骨骼软组织，包括骨骼肌肉和肌腱。目前协议的细节一个过程，作为修改preplate技术，已成功在我们的实验室，从肌腱和骨骼肌肉中分离出成人干细胞。使用上面描述的细胞修改preplate技术可分为基于其粘附胶原涂层烧瓶（图1）的特点，几个人口。发现组织内的成纤维细胞和肌纤维母细胞快速坚持在24-48小时内的胶原蛋白涂层烧瓶，最初是从其他细胞分离，PP1 - PP2。坚持生肌或内皮细胞后一个较长的一段时间，在48-96小时内，（PP3 - PP5）的胶原蛋白涂层烧瓶，并能有所收获，其细胞表面标志确定。后来的预镀细胞，96小时或更高版本（PP6），被视为成人干细胞（图2）。后期的preplate细胞出现小的，圆形，并开始隔离半透明，并且可以进一步为他们的干细胞抗原1（SCA1），CD34 +，胎肝激酶1（的Flk1）的表达，流式细胞仪或免疫细胞化学的特点，并其他干细胞标记物（图3）。分离出干细胞，通过其分化成三个胚层自我更新的能力和实验研究已证明其多能：中胚层，外胚层，内胚层 9 。多个调查也显示，这些干细胞分化成的中胚层谱系细胞，包括骨细胞，脂肪细胞，软骨细胞和造血干细胞6,8。其他研究已经表明，成体干细胞分化成外胚层细胞谱系，通过他们的神经元和神经胶质细胞标记和他们的能力，提高肢体功能后的末梢神经已经损坏10的表达。这些孤立的成人干细胞修复受伤和患病的肌肉骨骼组织是有益的， 和其他体内研究有关的其他组织包括心脏，肝脏，脊髓以及特定的医疗条件，如小肠黏膜下层和治疗膀胱压力性尿失禁9。 <img src="/files/ftp_upload/2011/2011fig1.jpg" alt="图1" /> 图1。 <img src="/files/ftp_upload/2011/2011fig2.jpg" alt="图2" /> 图2。 <img src="/files/ftp_upload/2011/2011fig3.jpg" alt="图3" /> 图3。

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<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#11995-073</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#10437-028</td>
<td>&nbsp;</td>
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<td>Heat-inactivated horse serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#26050-088</td>
<td>&nbsp;</td>
<tr>
<td>Chick embryo extract</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>#CE650T-10</td>
<td>&nbsp;</td>
<tr>
<td>100U ml penicillin/streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15140-122</td>
<td>&nbsp;</td>
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<td>Dulbecco&#x2019;s PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#14190-250</td>
<td>&nbsp;</td>
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<td>Hank&#x2019;s Buffered Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#24020-117</td>
<td>&nbsp;</td>
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<td>Collagenase type XI 0.2%(wt/vol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C7657</td>
<td>&nbsp;</td>
<tr>
<td>Dispase2.4U ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#17105-041</td>
<td>&nbsp;</td>
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<td>Trypsin-EDTA0.5%(wt/vol) (10x;5g liter trypsin (1:250)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>#15400-054</td>
<td>&nbsp;</td>
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<td>Collagen for coating flask : Type I collagen derived from calf skin 0.1 g liter</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#C-9791</td>
<td>&nbsp;</td>
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<td>DMSO</td>
<td>&nbsp;</td>
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<td>#D-2650</td>
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isolating stem cells from soft musculoskeletal tissues

成人干细胞及其在再生医学中的应用方面一直讨论。成人干细胞产生了极大的兴奋治疗受伤和患病的组织，由于其令人印象深刻的功能，经过多系细胞分化和自我更新的能力。最重要的是，这些特质使他们在使用自体细胞移植治疗的优势。目前的协议将介绍读者修改preplate技术，软组织，如肌腱和肌肉，骨骼肌肉系统1<sup&gt; ST</sup&gt;酶分离，然后放置在中等胶原涂层烧瓶。每天新烧瓶上清，这是中和其余的浮动细胞组成的，是连续转移。干细胞是最慢的的要坚持烧瓶通常需要5-7天（串行传输或preplates）。通过使用这种技术，在这些组织的成人干细胞目前相当的非侵入性的程序，可以轻松地收获通过。

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

隔离肌肉骨骼软组织的成人干细胞的基础上坚持细胞的速度向烧瓶。

从软肌肉骨骼组织中分离干细胞

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Nanyang Technological University

Research

JoVE Journal

Biology

13.3K 次观看.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. 隔离肌肉骨骼软组织的成人干细胞的基础上坚持细胞的速度向烧瓶。

视频: Isolating Stem Cells from Soft Musculoskeletal Tissues

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

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 ...

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 ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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 ...