这项工作是支持由来自加州再生医学研究所（RC1 - 00104），公共卫生服务津贴（HL085377）从NHLBI，以及从波林基金会的礼物，以恒生银行的综合研究资助

报告的作者，与一些修改，公布的参数是基于以下书面协议。该协议是由加州大学旧金山分校的机构动物护理和使用（IACUC）和干细胞研究（SCRO）监督委员会的批准。 一，文化的人类胚胎干细胞（胚胎干细胞） <ol><li>成长在标准6孔（3.5厘米直径的井）板的人类胚胎干细胞。 </li><li>至少12小时之前，传代细胞，种子小鼠胚胎成纤维细胞（MEF; CF1的一天获得E12.5定时的小鼠交配，在1000次照射）的饲养层细胞上板涂1％明胶（6,7）。 MEFs应镀在50-60％的汇合点，或4 × 10 5％以及3.5厘米直径的细胞（ 图1） 。 </li><li>通道殖民地时已达到〜70％汇合（ 图2）细胞。 </li><li>通行的细胞，吸KSR生长中期（6）从井和0.5毫升含1毫克/毫升的浓度胶原酶IV KSR介质取代。 </li><li>板与胶原酶在37 ° C / 5％的CO 2 5分钟，或直至菌落边缘开始从板抬起。 </li><li>胶原酶从盘子中取出，每孔中加入0.5毫升的KSR，手动刮细胞完全从板块中删除。 </li><li>从每口井的地方，在一个15毫升的锥形管，收集细胞悬液，使细胞受重力，5分钟解决。 </li><li>从试管中取出上清液，留在了〜300μL总量的细胞和介质。 </li><li>在全卷中使用200μL的微移液器设置，轻轻的吸管将细胞悬液的5-6倍，打破了殖民地。 </li><li>把总量的9％使用KSR中（ 也就是说 ，如果一个收获，管应包含9毫升）的原始细胞以及毫升细胞悬液。补充重组人碱性成纤维细胞生长因子（bFGF）的12.5纳克/毫升的浓度介质。 </li><li>洗衣机的贝科的磷酸盐的MEF细胞（见步骤2）新井后缓冲生理盐水（DPBS），以除去所有的血清，添加3毫升细胞悬液，每孔和孵化在37 °的C / 5％的CO 2板块。 </li><li> KSR媒介变化与碱性成纤维细胞生长因子通过后24小时，每24-48小时，直到细胞准备再次传代。 </li></ol>二。嫁接人类胚胎干细胞颗粒的制备<ol><li>在嫁接前24小时，加冰糖抑制剂KSR介质的终浓度为10μM增加细胞活力（8）。 〜70％汇合时（5 × 10 5细胞）的细胞之一，将创建两个小球，每肾囊中的两个颗粒。 </li><li>要准备细胞颗粒移植术（9），孵化的岩石5分钟胶原酶IV抑制剂治疗的文化，在37 ° C / 5％的CO 2。 </li><li>从井里吸胶原酶，替换10％的青霉素/链霉素（PEN /链球菌），并用1 ml DPBS。 </li><li>手动刮盘的殖民地，一个额外的1笔/链球菌毫升DPBS冲洗井，每个细胞悬液1毫升等分转移到1.5 ml离心管。 </li><li>简单地说，在8000 X克旋转的离心管吸出上清，重新悬浮颗粒，用钢笔/链球菌500μL DPBS。 </li><li>为了帮助结合在一起，供移植的细胞，每管加入100μL1毫克/毫升菜豆在DPBS凝集素（PHA）。 </li><li>细胞/ PHA的悬挂在室温孵育5分钟，然后分拆为2分钟在8000 X克。旋转180 °的管内的转子井和自旋为2分钟在8000 X克。 </li><li>小心吸出上清液，留下颗粒完好无损。 </li><li>打跑管细胞沉淀轻轻吹打200μLKSR颗粒边缘，直到它慢慢升起。 </li><li>一旦脱落，立即转移颗粒0.4μmMillicell小插入，放置在含3毫升KSR，直径为3.5厘米的井，并在37 ° C / 5％的CO 2为2小时至过夜孵育。 </li></ol>三。肾包膜移植术<ol><li>执行根据使用适当的无菌技术体制指引，所​​有的动物程序。 </li><li>麻醉的免疫缺陷米色小鼠（CB17.Cg Prkdc SCID Lyst BG / CRL），年龄在8-12周，使用吸入3％isoflurane/0.5％O2和腹腔tribromoethanol（Avertin; 25毫克/公斤新鲜配制）的组合。将嫁接使用技术（10）每鼠细胞颗粒。 </li><li>放在加热垫，以防止低温鼠标后，刮胡子的切口面积，然后用洗必泰消毒。在一个2.5厘米的切口，通过皮肤，但只是偏离中心脊柱平行。 </li><li>保持无菌盐度的切口面积潮湿E，通过肌肉的小切口，肋骨下方，从脊椎〜1厘米。切口应足够大，以拉肾脏通过，但足够小，没有力量，肾将不滑进入腹腔。 </li><li>通过肌肉的切口，用一个玻璃巴斯德吸管已拉成钝，略呈弧形钩的形状，轻轻地拉出肾脏。 </li><li>湿用无菌DPBS的肾脏，以防止晒出胶囊。重复整个过程中，胶囊是很脆弱的的，这可能是必要的。 </li><li>使用杜蒙＃5镊子，轻轻一拉胶囊从肾脏和一个2毫米的切口，用Vannas弹簧剪刀。 </li><li>已拉成很细的，迟钝的探头与另一玻璃吸管，胶囊和肾脏之间的一个小口袋。 </li><li>利用大孔放置在一次性移液管吸头，从Millicell小插入一个单细胞沉淀。请一定要采取尽可能一点额外的媒体，多余的介质可以干扰颗粒转移。 </li><li>按住切口创建口袋的边缘，轻轻地放在旁开的颗粒，并推入朝拉吸管肾脏的一极的口袋。 </li><li>重复第二个颗粒的过程。下放置相同的肾囊，而是朝着相反的一极。 </li><li>小心地将胶囊回落到颗粒。颗粒应留在口袋，没有缝线有必要关闭胶囊。 </li><li>轻轻推入肾，进入腹腔，关闭使用附加的反向切割针4-0丝缝线（或更小的）的肌壁。切口应足够小，只有一个单一的缝合是必要的。 </li><li>关闭了一系列4-5中断针的皮肤，使用相同的缝合材料。 </li><li>之前，它在笼中放置鼠标，作为短期和长期的止痛药0.05毫克/公斤丁丙诺啡（11,12）和5毫克/公斤酮基布洛芬（12,13），分别。受控物质只应按照机构指引。 </li><li>动物转移到笼子里，并允许它恢复从麻醉加热垫。这大约需要20-30分钟。 </li><li>一旦鼠标是完全卧床，它可以返回畜舍设施。 </li></ol>四。畸胎瘤的收获，固定和切片<ol><li>在手术后8-12周，应该有一个肾脏附近勉强扪畸胎瘤。大，充满液体的囊肿，往往会以及发展，在6-8个星期开始。如果他们造成困扰的动物，这可能需要一个早期收获。然而，由于增长速度比囊肿畸胎瘤，你会希望尽可能长时间等待来获得足够的规模进行分析（ 即至少8个星期）的畸胎瘤。 </li><li>取出的肾，畸胎瘤，并使用相同的手术方法，其中肾最初访问，从腹部腔作为一个组织块周围的任何囊肿（见第三节，步骤1-4以上）。 </li><li>整个切除组织块放入一个含有10％的福尔马林管。 </li><li>允许畸胎瘤固定在4℃过夜° C。 </li><li>次日，从管中删除的畸胎瘤。解剖，不破坏本身的畸胎瘤（ 图3）任何可能与尽可能多的肾囊肿。较大的畸胎瘤可能被削减了一半，以方便清扫和嵌入。 </li><li>使用标准的石蜡固定技术工艺的畸胎瘤。自动单位（ 例如 ，Shandon超中心）： <table border="0"><tbody><tr><td>一</td><td> 80％的Flex酒精</td><td> 2个小时</td></tr><tr><td>二。 </td><td> 80％的Flex酒精</td><td> 1小时</td></tr><tr><td>三。 </td><td> 85％的Flex酒精</td><td> 1.5小时</td></tr><tr><td>四。 </td><td> 95％的Flex酒精</td><td> 1小时</td></tr><tr><td>五</td><td> Flex的酒精100％ </td><td> 1小时</td></tr><tr><td>六。 </td><td> Flex的酒精100％ </td><td> 1小时</td></tr><tr><td>七。 </td><td> Flex的酒精100％ </td><td> 1小时</td></tr><tr><td>八。 </td><td>清除祭3 </td><td> 1小时</td></tr><tr><td>九。 </td><td>清除祭3 </td><td> 1小时</td></tr><tr><td> X。 </td><td>清除祭3 </td><td> 1小时</TD&gt; </tr><tr><td>十一。 </td><td>石蜡（TissuePrep） </td><td> 2个小时</td></tr><tr><td>十二。 </td><td>石蜡（TissuePrep） </td><td> 2个小时</td></tr></tbody></table></li></ol>五，免疫组织化学和分析<ol><li>一旦嵌入石蜡，第畸胎瘤在使用旋转式切片机5μm的串行片，幻灯片上的浮动部分。 </li><li>染色前，烘烤至少一小时的幻灯片。 </li><li>苏木精和曙红染色的幻灯片，使用标准的方法来识别每个三个胚层（ 图 4）的组织结构代表： <table border="0"><tbody><tr><td>一</td><td>二甲苯洗</td><td> 3-5分钟</td></tr><tr><td>二。 </td><td>二甲苯</td><td> 3-5分钟</td></tr><tr><td>三。 </td><td> 100％的酒精</td><td> 3-5分钟</td></tr><tr><td>四。 </td><td> 100％的酒精</td><td> 3-5分钟</td></tr><tr><td>五</td><td> 95％的酒精</td><td> 3-5分钟</td></tr><tr><td>六。 </td><td> 80％的酒精</td><td> 3-5分钟</td></tr><tr><td>七。 </td><td>水洗（一些变化） </td><td> 2分钟</td></tr><tr><td>八。 </td><td>吉尔的苏木＃3 </td><td> 2-5分钟</td></tr><tr><td>九。 </td><td>水洗（一些变化） </td><td>直到水运行明确</td></tr><tr><td> X。 </td><td>斯科特的水（蓝色细胞核） </td><td> 3分钟或更长时间</td></tr><tr><td>十一。 </td><td>水洗（一些变化） </td><td> 1-2分钟</td></tr><tr><td>十二。 </td><td>曙红Y </td><td> 1分钟</td></tr><tr><td>十三。 </td><td>水洗（一些变化） </td><td> 30秒</td></tr><tr><td>十四。 </td><td> 80％的酒精</td><td> 1分钟</td></tr><tr><td>十五。 </td><td> 95％的酒精</td><td> 2分钟</td></tr><tr><td>十六。 </td><td> 95％的酒精</td><td> 2分钟</td></tr><tr><td>十七。 </td><td> 100％的酒精</td><td> 3分钟</td></tr><tr><td>十八。 </td><td> 100％的酒精（清洁） </td><td> 3分钟</td></tr><tr><td>十九。 </td><td>二甲苯</td><td> 2分钟或更长时间</td></tr><tr><td>二十。 </td><td>二甲苯</td><td> 2分钟或更长时间</td></tr></tbody></table></li><li>山Permount与每个幻灯片上的盖玻片。 </li></ol>六。代表性的成果<img src="/files/ftp_upload/2036/2036fig1.jpg" alt="图1" /> 图1。电镀照射CF1的小鼠胚胎成纤维细胞作为饲养层未分化的胚胎干细胞培养。MEFs镀〜50％汇合，在这里显示，或4 × 10 5细胞，每3.5厘米，直径在6孔培养皿。酒吧，100微米。 <img src="/files/ftp_upload/2036/2036fig2.jpg" alt="图2" /> 图2。在MEF饲养层的未分化的胚胎干细胞subconfluent文化的例子。人类胚胎干细胞是生长在MEF饲养层〜70％汇合之前，如下所示，然后传所述。酒吧，100微米。 <img src="/files/ftp_upload/2036/2036fig3.jpg" alt="图3" /> 图3。 explanted畸胎瘤的范例 。explantation，畸胎瘤，肾，任何配件的组织固定在福尔马林块，然后肾和配套组织都经过精心修剪掉之前，石蜡包埋。 <img src="/files/ftp_upload/2036/2036fig4.jpg" alt="图4" /> 图4。未分化的胚胎干细胞衍生的畸胎瘤包含在体内的所有三个胚层组织的代表。一个畸胎瘤，如图3所示的切片，苏木精和曙红染色，以确定胚胎组织。人类胚胎干细胞会引起所有三个胚层（外胚层（A，B），中胚层（C）内胚层衍生组织（四）。 （一）新生的神经管结构。 （二）原始鳞状上皮。 （三）软骨所包围简明间质胶囊。 （四）肠腺结构。酒吧，100微米。图片作者许可转载。 <img src="/files/ftp_upload/2036/2036fig5.jpg" alt="图5" /> 图5。畸胎瘤分析可用于地图上的标签，未分化的胚胎干细胞的命运，正如先前公布（9），特别标记的未标记的胚胎干细胞的人类胚胎干细胞的混合物，可用于地图标记的细胞在畸胎瘤形成实验的命运。如上所示，未分化的胚胎干细胞表达的表面标志，CD133，和增强型绿色荧光蛋白（EGFP）标记，无标记的，未分化的胚胎干细胞混合。这些都是用于肾移植术胶囊形成的畸胎瘤。苏木精和曙红染色畸胎瘤部分抗- EGFP抗体的免疫组织化学分析表明神经外胚层的CD133 +的人类胚胎干细胞的命运。畸胎瘤进行了处理， 如图4和抗- EGFP抗体（ 棕色 ）counterstained本地化内组织的CD133 + GFP +细胞的衍生工具。观察从胚胎外胚层，特别是神经上皮（ 左 ）和新生的神经管样结构（ 右 ）所产生的组织的CD133 +源性细胞（箭头）。酒吧，100微米。图片作者许可转载。 明胶 <ul><li>猪明胶0.1％ </li><li> 99.9％DPBS <ul><li>添加明胶DPBS，在37 ° C的一个小时或直到所有的明胶成溶液，过滤和消毒（0.22μm的）在使用前孵育。 </li></ul></li></ul> MEF的介质 <ul><li> 10％胎牛血清（合格） </li><li> 90％贝科的修改必要的中等（D - MEM） </li><li> 1X大号-谷氨酰胺</li><li> 1X笔/链球菌<ul><li>过滤器在使用前消毒。 </li></ul></li></ul> KSR（淘汰赛血清替代）中型 <ul><li> 20％的淘汰赛血清替代</li><li> 80％的基因敲除的D - MEM </li><li> 1X大号-谷氨酰胺</li><li> 1X非必需氨基酸</li><li> 0.1毫米β-巯基乙醇/ </li><li> 12.5纳克/毫升碱性成纤维细胞生长因子<ul><li>过滤消毒。 </li><li>储存于4 ° C，但加热到37℃前加入到细胞。 </li><li>使用前添加碱性成纤维细胞生长因子。 </li></ul></li></ul> 胶原酶 <ul><li>配售为1-2小时或在37 ° C到溶液中去，直到所有粉末溶解在KSR介质1毫克/毫升。 </li><li>过滤消毒。 </li></ul> Avertin <ul><li> 2,2,2 - tribromoethanol 0.25克</li><li> 0.25毫升2 - 甲基-2 - 丁醇<ul><li>在37 ° C孵育过夜，以形成100％的解决方案。 </li><li>手术前的几个小时，稀释至2.5％的工作液使用DPBS和孵化，在37 ° C为至少1小时。 </li><li>过滤消毒和分装。 </li><li> Tribromoethanol光非常敏感，所以在编制和使用的各个阶段保护光。 </li><li>使用前准备不超过12-24小时。 </li></ul></li></ul> 丁丙诺啡 <ul><li> 1毫克/毫升原液甲醇<ul><li>稀释至0.015毫克/毫升工作液用无菌H 2 O。 </li><li>在-20 ° C存储长达1个月。 </li></ul></li></ul> 酮基布洛芬 <ul><li> 0.5 mg / ml的工作液在DPBS <ul><li>添加DPBS，孵化过夜，于37 ° C的1-2个小时，直到所有粉末成溶液。 </li><li>过滤消毒，并储存于4 ° C。 </li></ul></li></ul>

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B., Nishikawa, S., Muguruma, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat Biotechnol. 25, 681-686 (2007).</li><li>King, F. W., Ritner, C., Liszewski, W., Kwan, H. C., Pedersen, A., Leavitt, A. D., Bernstein, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subpopulations+of+human+embryonic+stem+cells+with+distinct+tissue-specific+fates+can+be+selected+from+pluripotent+cultures.">Subpopulations of human embryonic stem cells with distinct tissue-specific fates can be selected from pluripotent cultures.</a> Stem Cells Dev. 18, 1441-1450 (2009).</li><li>Cunha, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelio-mesenchymal+interactions+in+primordial+gland+structures+which+become+responsive+to+androgenic+stimulation.">Epithelio-mesenchymal interactions in primordial gland structures which become responsive to androgenic stimulation.</a> Anat Rec. 172, 179-195 (1972).</li><li>Kirsch, J. H., Klaus, J. A., Blizzard, K. K., Hurn, P. D., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain+evaluation+and+response+to+buprenorphine+in+rats+subjected+to+sham+middle+cerebral+artery+occlusion.">Pain evaluation and response to buprenorphine in rats subjected to sham middle cerebral artery occlusion.</a> Contemp Top Lab Anim Sci. 41, 9-14 (2002).</li><li>Lee-Parritz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesia+for+rodent+experimental+surgery.">Analgesia for rodent experimental surgery.</a> Isr J Vet Med. 62, 74-78 (2007).</li><li>Julou, L., Guyonnet, J. C., Ducrot, R., Pasquet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+pharmacological+and+toxicological+studies+on+ketoprofen.">Some pharmacological and toxicological studies on ketoprofen.</a> Rheumatol Rehabil. , (1976).</li></ol>

我们已经适应了一个高效的映射在体内环境中的人类胚胎干细胞分化的命运的目的畸胎瘤形成实验。一个专门挑选的人类胚胎干细胞的数量少，混合的未分化的野生型胚胎干细胞和菜豆凝集素，形成一个细胞沉淀。这是嫁接在免疫缺陷小鼠的肾囊下方。最初选定的人类胚胎干细胞的命运，然后可以通过免疫组化（ 图 5）确定，如前呈报（9 ）。这种方法提供了有价值的工具，识别和生成组织特定试剂，细胞治疗。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2-Methyl-2-butanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>240486</td>
<td>Avertin</td>
</tr>
<tr>
<td>2,2,2-Tribromoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T48402</td>
<td>Avertin</td>
</tr>
<tr>
<td>4-0 silk sutures</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>641G</td>
<td>&nbsp;</td>
<tr>
<td>bFGF</td>
<td>&nbsp;</td>
<td>R&amp;D Systems</td>
<td>233FB</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>B7536</td>
<td>&nbsp;</td>
<tr>
<td>Clear-Rite 3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046-341</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase IV</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C5138</td>
<td>&nbsp;</td>
<tr>
<td>D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11965</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190</td>
<td>&nbsp;</td>
<tr>
<td>Dupont #5 forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>500233</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>Eosin Y</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>23-245-658</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum (Qualified)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Flex alcohol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-046344</td>
<td>&nbsp;</td>
<tr>
<td>Gill&#x2019;s Hematoxylin #3</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>22-050-204</td>
<td>&nbsp;</td>
<tr>
<td>Hemostat, straight</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501241</td>
<td>General surgery</td>
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<tr>
<td>Iris forceps</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>15914</td>
<td>General surgery</td>
</tr>
<tr>
<td>Ketoprofen</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>K2012</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut D-MEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10829</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25020-081</td>
<td>&nbsp;</td>
<tr>
<td>MILLICELL inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICM01250</td>
<td>&nbsp;</td>
<tr>
<td>Nonessential Amino Acids</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11140-050</td>
<td>&nbsp;</td>
<tr>
<td>Operating scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>501754</td>
<td>General surgery</td>
</tr>
<tr>
<td>Paraffin (TissuePrep)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>8002-74-02</td>
<td>&nbsp;</td>
<tr>
<td>Pen/Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Permount</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SP15-100</td>
<td>&nbsp;</td>
<tr>
<td>PHA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>L1668</td>
<td>&nbsp;</td>
<tr>
<td>Porcine gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G6144</td>
<td>&nbsp;</td>
<tr>
<td>ROCK inhibitor</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>Y-27632</td>
<td>&nbsp;</td>
<tr>
<td>SCID beige mice</td>
<td>&nbsp;</td>
<td>Charles River </td>
<td>250</td>
<td>&nbsp;</td>
<tr>
<td>Scott&#x2019;s Wash</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>6697-32</td>
<td>Ricca Chemicals</td>
</tr>
<tr>
<td>Vannas spring scissors</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>14003</td>
<td>Kidney capsule</td>
</tr>
<tr>
<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M7522</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fate mapping of human embryonic stem cells by teratoma formation

人类胚胎干细胞（胚胎干细胞）具有无限的自我更新能力，并能分化成所有三个胚层（1）衍生的细胞。导演的人类胚胎干细胞分化成特定类型的细胞在再生医学领域（如（2-5））产生了很大的兴趣，并确定方法<em&gt;在体内</em&gt;选择或操纵胚胎干细胞的命运，这种努力是必不可少的。我们已经适应了用于此目的的高效的畸胎瘤形成实验。一个专门挑选的人类胚胎干细胞的数量少，混合的未分化的野生型胚胎干细胞和菜豆凝集素，形成一个细胞沉淀。这是嫁接在免疫缺陷小鼠的肾囊下方。数为2.5 × 10<sup&gt; 5</sup&gt;人类胚胎干细胞都需要形成一个16厘米<sup&gt; 3</sup&gt;在8-12周的畸胎瘤。最初选定的人类胚胎干细胞的命运，然后可以由免疫组织化学。这种方法提供了一个有价值的工具，用于描述特定组织细胞治疗试剂。

Watch this Scientific Journal Video about Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation at JoVE.com

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

人类胚胎干细胞定向分化成特定的细胞在再生医学产生了很大的兴趣。我们提供一个简洁，一步一步确定协议<em&gt;在体内</em&gt;细胞治疗为特征的组织特异性试剂，提供了一个有价值的工具选定的人类胚胎干细胞的命运。

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

fate-mapping-of-human-embryonic-stem-cells-by-teratoma-formation

Research

JoVE Journal

Biology

17.1K 次观看.  University of California San Francisco. 人类胚胎干细胞定向分化成特定的细胞在再生医学产生了很大的兴趣。我们提供一个简洁，一步一步确定协议在体内细胞治疗为特征的组织特异性试剂，提供了一个有价值的工具选定的人类胚胎干细胞的命运。

视频: Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

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 ...

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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 ...