We thank Joon Kim for expert flow cytometry assistance. Research in the authors&#8217; labs is supported by the SCORE program of the National Institutes of Health (grant SC1-GM095402 to B.D.O) and the Canadian Institutes of Health Research (to J.C.Z.P.). J.C.Z.P. is supported by a Canada Research Chair in Developmental Immunology. The biomedical research infrastructure of Hunter College is supported in part by the NIH Research Centers in Minority Institutions (RCMI) program via grant MD007599. We also acknowledge the New York State Stem Cell Science Program (NYSTEM) for its support of the initiation of stem cell research at Hunter College via grant C023048.

1，制备培养基，细胞因子和糊化板的<ol><li>用鹰的培养基（DMEM）的贝科的修改与高糖和丙酮酸钠准备胚胎干细胞培养基。添加20％ESC合格的胎牛血清（FBS），1％青霉素/链霉素，1％L-谷氨酰胺，1％HEPES缓冲液，1％非必需氨基酸，0.1％庆大霉素（50毫克/毫升）和0.1％（ 55μM）β-巯基乙醇。经过滤消毒胚胎干细胞培养基。 </li><li>通过根据制造商的说明制备1升阿尔法最低必需培养基（α-MEM）中从粉末制备OP9媒体。添加20％胎牛血清（FBS）和1％青霉素/链霉素。颠倒混匀。经过滤消毒成2个500毫升分装。 注意:使用的由粉末制成的介质，推荐和可能产生改进的OP9细胞的维持和体外分化的结果。这种方法已经成为我们的标准程序。然而，我们也注意到塔难道我们在某些时候使用预制的液体α-MEM足够的成功。 </li><li>制备通过加入10％二甲亚砜（DMSO）中，以90％FBS的冷冻介质。轻轻摇动，通过过滤消毒。保存在4℃。 </li><li>通过在完整OP9介质中溶解的人类重组受体Flt-3配体（FLT-3L），以10微克/毫升制备2000倍的Flt-3配体（10微克/毫升）。分装成的1.5 ml离心管中，并储存在-80℃。按照供应商的建议对稳定性和储存。 注:FLT-3L的稳定性是短（2个月，在4℃或3个月，在-80℃）。 </li><li>通过使用完整OP9媒体准备1000倍的IL-7（1微克/毫升）。分装成的1.5 ml离心管中，并储存在-80℃。按照稳定性和存储供应商的建议（IL-7在-80℃可稳定12个月）。 </li><li>准备好1000倍，白血病抑制因子（LIF）（10微克/毫升）用1:10稀释10 7个单位的LIFíÑ​​胚胎干细胞培养基。店内分装在4℃。一定要注意通过对分装小瓶制造商提供的截止日期。 注:产品是稳定的浓缩或稀释的形式至少18自生产之日起二个月。 </li><li>通过制备糊化6孔板中添加毫升0.1％的1.5明胶溶液成一个6孔板的每个孔中。留在无菌罩在室温下用在盖子的菜肴，使至少30分钟的涂层。菜也可以留在潮湿的培养箱中过夜。细胞接种前才取出所有剩余的明胶溶液。不要让明胶溶液完全变干的好。 </li></ol> 2，准备工作和饲养细胞和小鼠胚胎干细胞的维护（MESC） 注:孵育在湿润的37℃培养箱中培养5％的CO 2的所有单元格。 <ol><li>解冻小鼠胚胎成纤维细胞（MEF） <ol><li>快速解冻冷冻瓶（〜5×10 6 </丝裂霉素C处理（或其他有丝分裂逮捕）的MEF SUP&gt;细胞/瓶），并将它们转移到一个15毫升的试管中。 </li><li>添加8毫升ES细胞培养基的解冻细胞中，逐滴的方式从冷冻介质逐渐稀释的DMSO。 </li><li>离心细胞，在400×g下在4℃下5分钟。小心取出上清液，重悬细胞沉淀在3毫升胚胎干细胞介质。 </li><li>准备50毫升离心管用33ml预热的ES细胞介质。添加3毫升再悬浮的MEF以使终体积到36毫升</li><li>分发3毫升再悬浮的MEF为两个糊化的6孔板的每个孔中。将平板放入培养箱中至少6小时，以使细胞附着并摊开播种mESCs分化上之前。这些有丝分裂被捕饲养层可以是用于初始接种后几天可用的，如果其介质是每2-3天更换。 注:刚被捕的MEF也可以使用。 </li> &lt;/ OL&gt; </li><li>播种mESCs分化<ol><li>快速解冻，在37℃水浴冻结卓制克隆小瓶并按照2.1.1-2.1.3介绍准备细胞。 注意:我们冻结2瓶卓制从6孔板的融合很好。 </li><li>从6孔板与被捕MEF细胞单层（在步骤2.1.5的方法制备）的一个孔中（每MESC克隆）除去介质并播种3毫升mESCs分化对MEF的单层的顶端。添加3微升1000倍的LIF（10毫微克/毫升）。返回餐具放入孵化器。 </li></ol></li><li>胚胎干细胞的维护和胰蛋白酶介导的通道 注:根据卓制合流每日更换介质，或分割。理想情况下，收获和分离/重制版的隔日细胞。 <ol><li>除去ES细胞培养基，并用2毫升的PBS洗涤mESCs分化。删除的PBS。加入1 ml的0.25％胰蛋白酶的孵育在37℃下进行3-5分钟。 </li><li>胰酶消化收集细胞，并将它们转移到15ml离心管中。大力吸管将细胞悬液分手的米团块胚胎干细胞。加入2 ml完全的ES细胞培养基中的细胞悬浮液中以中和胰蛋白酶。 </li><li>自旋细胞在400×g下5分钟，在4℃下。除去上清，重悬沉淀在3毫升的ES细胞培养基。 </li><li>从制备的含有被捕MEF细胞单层的6孔板中取出媒体。在3ml的ES细胞培养基中加入细胞悬浮液（根据所期望的分流比）的适当量向每孔中被接种。添加3微升1000倍LIF的。一个融合卓制以及分裂1:6将准备通过2天。 </li></ol></li><li> OP9 / OP9-DL1细胞维持<ol><li>解冻OP9细胞的小瓶（按照步骤2.1.1-2.1.3使用OP9媒体） </li><li>添加7毫升OP9媒体10厘米组织培养皿。添加3毫升悬浮细胞中逐滴的方式，在整个板块分布的细胞。将细胞在夜间的孵化器。 </li><li>检查OP9细胞汇合的第二天。如果菜中含有许多死细胞漂浮，字符型数据已经在媒体和加入10毫升新鲜OP9媒体。返回菜的孵化器，如果接近满汇合没有观察到。斯普利特（胰酶）1:4不久的融合OP9单层。 注:板材应在2天左右再次到达同一汇合水平。小心不要让OP9细胞变得过度融合。 </li><li>冻结OP9细胞的早期传代的扩张股票。 注意:我们冻结2瓶OP9细胞从一个附近汇合10cm皿。扩大股票每管可以进一步传播到创建工作的股票，在卓制共培养实验后使用。保持在液氮中冷冻所有OP9股票，而不是在-80℃的冰箱中，由于温度波动可以在冻结的质量产生负面影响。 </li></ol></li></ol> 3 OP9-DL1共培养过程<ol><li>共培养的准备<ol><li>约前一星期共培养开始，解冻的MEF，mESCs分化和OP9细胞申通快递工作中正。由于不需要OP9-DL1细胞直至共培养8天，在此期间共培养开始后的前三天解冻OP9-DL1细胞。维持或根据需要冻结所有细胞。 </li><li>确定为10厘米板的制备对共培养物达到80％汇合基于实验的规模0天OP9细胞单层的初始数量（ 例如 ，区分和卓制克隆的数目的共培养时间点的数目以进行分析）。为了准备共同培养，分裂近1间OP9细胞融合菜:4和1:领先时，单层，将需要6个两天。 注意:一个需要每ESC克隆至少有一个板块。典型地，一个单一ESC克隆接种于单个板（如下所述）在第0天应该产生足够的细胞播种6板在第5天通过。每天5板将使一个后续分析的时间点。 </li><li>由于OP9单层将不断需要共同培养通道，小心总是mainta在另外的OP9培养板中以共培养并行足够数量。 </li></ol></li><li>第0天:共培养物的起始<ol><li>收集卓制在2.3.1-2.3.4。计数细胞。 </li><li>对于每个卓制克隆制备5×10 4卓制在10毫升每板OP9媒体。 注意:虽然我们还没有使用它，我们注意到，另一种介质组成的α-MEM，10％胎牛血清和5×10 -5 Mβ-巯基乙醇还报道了在分化的共培养物用10。 </li><li>从OP9菜肴删除旧的媒体，并添加10毫升卓制悬液，以照顾到整个板均匀地分布在细胞的菜肴。将菜在37℃培养箱中培养。 </li></ol></li><li>第3天:媒体变革<ol><li>从孵化器中取出菜肴，在显微镜下观察。菌落开始失去光泽，并出现变平。 </li><li>从菜肴取出介质，轻轻地加入10毫升新鲜OP9媒体。 </li><li>返回共培养皿于培养箱中培养。目视监视胚层样细胞集落的形成每日通知OP9馈线单层制剂的定时为下通道（第5天），这不应该被执行，直到〜菌落80-90％可视地显示中胚层样形态。在第5天细胞转移的步骤，最大延迟为2天。 </li></ol></li><li>第5天:胰蛋白酶介导的通路与预镀。 注:提前准备10厘米的菜足够数量的具有最佳融合OP9细胞。下一个步骤继续进行只有当共培养物在视觉上显示在步骤3.3.3中描述的特征（参见代表结果）。 <ol><li>从共培养皿取出介质。加4毫升的PBS洗。摇动的PBS和删除。添加4毫升0.25％胰蛋白酶和放置碗碟孵化器〜5分钟。 </li><li>破坏胰蛋白酶消化细胞活力移液层，注意不要引入过多的气泡，直到均匀，多为单细胞悬浮液来实现的。 </li><li>添加4毫升完整OP9媒体和吹打混匀。转移细胞进入一个新的，空的10cm平皿并置于培养箱中30分钟，以允许OP9细胞附着在培养皿中。这种"预镀"步骤减小转移到共培养物中的下一个步骤OP9细胞的数目。 </li><li>准备50ml离心管中有40微米的细胞过滤器。 </li><li>收集非贴壁细胞从预镀盘，并通过细胞过滤器进入管传递它们。 6毫升的PBS轻轻洗的菜，并通过相同的过滤器通过它，而使附着OP9细胞落后。 </li><li>离心细胞，在400×g下5分钟，在4℃下。除去上清液，重悬细胞沉淀在3毫升完整OP9媒体。计数细胞。 </li><li>从10厘米板的最佳融合OP9细胞中取出介质。 </li><li>对于每个分析时间点，种子5×10 5个细胞在OP9单层在10毫升的最终体积。 </li><li>加入5毫微克/毫升重组人FLT-3L和放置碗碟的孵化器。 </li></ol></li><li>第8天:造血祖细胞（HPC）的集合 注:在时间提前准备的6孔板与OP9或OP9-DL1细胞单层有足够数量，以便它们将〜80％汇合的这一步。 <ol><li>准备50ml离心管中有40微米的过滤器。 </li><li>从孵化器中取出菜肴，在显微镜下观察。高性能计算机的闪亮集群应该被松散地连接到OP9单层。收集尽可能多的这些细胞成为可能通过洗涤（但不是过度破坏）的OP9单层用吸管和菜的现有媒体。为了防止起泡，总是留下至少为1毫升介质中的移液管的尖端的这段HPC收集/洗涤步骤。 </li><li>通过40微米过滤的细胞成管。加入8毫升的PBS以相同的板和显微镜下检查是否所有的HPC我们重新收集。更有力的洗涤重复用PBS洗涤/采集步骤和（如有必要）。菌株的细胞分化成已含有从同一平板上的细胞的试管中。 </li><li>离心细胞，在400×g下5分钟，在4℃下。 </li><li>制备的3毫升主混合物（每平板接种在第5天）OP9媒体与5毫微克/毫升的Flt-3L和1纳克/毫升的IL-7。 </li><li>在OP9媒体FLT-3L + IL-7预混液中取出上清液，重悬沉淀（3毫升处理的每个10cm皿）。从各板传送所收集的细胞于6孔板与OP9细胞的一个孔。 <ol><li>为了驱动细胞向单核细胞，B细胞或红细胞谱系的种子收集的细胞上OP9细胞。为了得到T细胞，种子细胞到OP9-DL1细胞单层。 </li></ol></li><li>将6孔板进入培养箱。 </li></ol></li><li> 10日:媒体变革<ol><li>准备15毫升离心管中每个不同的MESC克隆饲养细胞型combina灰中共同培养。 </li><li>准备OP9媒体的主结构与FLT-3L为5毫微克/毫升和IL-7的1纳克/毫升。制备3毫升为每个孔中。 </li><li>轻轻收集介质从6孔培养板从含有相同的ESC克隆饲养细胞类型组合多口井相结合的媒体的每个孔中。 </li><li>加入2mL OP9媒体主结构（参见3.6.2节）到每口井。 </li><li>离心机在400×g下5分钟，在4℃下将收集的介质。除去上清，悬浮最初收集步骤3.6.3每片（非常小的，如果可见）在1毫升OP9媒体主结构（参见3.6.2节）每口井。 </li><li>分发1毫升细胞放回各孔最初从中收集的媒体带来的体积在各孔中至3ml。返回菜的孵化器。 </li></ol></li><li> 12日:无胰蛋白酶通道 注:提前准备的6孔盘与OP9或OP9-DL1细胞提供足够数量，使他们以最佳汇合的时候这个STEP。 12日是理想的流式细胞仪开发红细胞，单核细胞和早期T细胞的分析。 <ol><li>制备主混合物（3毫升每孔中，将在共培养继续）OP9媒体与受体Flt-3L的5纳克/毫升和IL-7的1纳克/毫升。 </li><li>制备的50ml离心管中以40微米的过滤器（一个用于在共培养每个ESC克隆饲养细胞类型的组合）。 </li><li>大力吸取现有媒体在每个孔中，以分解所有细胞（包括单分子层）中的井。继续有力吹打，直至状态类似于单细胞悬液的实现。 </li><li>通过过滤器进入管程将收集的细胞。加入3毫升的PBS到每口井洗并收集所有剩余的细胞。通过相同的过滤器通过细胞。从含有相同的ESC克隆饲养细胞组合型多井组合的细胞。 </li><li>丢弃使用过的细胞过滤和除去等分试样相当于一个阱（〜6ml）中的任何期望的流式细胞仪（或其它）分析，以进行在该日。在使用之前储存在冰这些等份。 </li><li>离心细胞，在400×g下5分钟，在4℃下。取出上清液。重悬在50ml离心管中的沉淀物在3ml每孔预混重新播种。每增加每个细胞悬液以及3毫升到适当的饲养细胞类型，然后将菜在孵化器。 </li></ol></li><li>第14天:媒体变革<ol><li>按照3.6节所述的步骤。 </li></ol></li><li>第16天:没有胰蛋白酶通道 注意:预先准备最佳汇合OP9或OP9-DL1细胞的6孔培养皿中用于此步骤。 注意:16天是理想的流式细胞术分析的B淋巴细胞和中间阶段的T细胞。 <ol><li>按照3.7节所述的步骤。 </li></ol></li><li> 18日:媒体变革<ol><li>按照3.6节所述的步骤。 </li></ol></li><li>第20天:没有胰蛋白酶通道 注:当日20 IŠ理想的流式细胞仪的中期分析到后期开发的T细胞。 <ol><li>按照3.7节所述的步骤。如果需要的话，进行分化细胞过去20天的交替介质变化和没有胰蛋白酶通路每隔两天，每天监测淋巴细胞膨胀率。 </li></ol></li></ol>

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<li>Holmes, R., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+OP9-DL1+system%3A+generation+of+T-lymphocytes+from+embryonic+or+hematopoietic+stem+cells+in+vitro%5BTitle%5D)+AND+%22Cold+Spring+Harb+Protoc%22%5BJournal%5D)">The OP9-DL1 system: generation of T-lymphocytes from embryonic or hematopoietic stem cells in vitro.</a> Cold Spring Harb Protoc. 2009, (2009).</li>
<li>Arsov, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+autophagic+protein+beclin+1+early+in+lymphocyte+development%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">A role for autophagic protein beclin 1 early in lymphocyte development.</a> J Immunol. 186, 2201-2209 (2011).</li>
<li>Lahiji, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complete+TCR-alpha+gene+locus+control+region+activity+in+T+cells+derived+in+vitro+from+embryonic+stem+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Complete TCR-alpha gene locus control region activity in T cells derived in vitro from embryonic stem cells.</a> J Immunol. 191, 472-479 (2013).</li>
<li>Hirashima, M., Kataoka, H., Nishikawa, S., Matsuyoshi, N., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maturation+of+embryonic+stem+cells+into+endothelial+cells+in+an+in+vitro+model+of+vasculogenesis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis.</a> Blood. 93, 1253-1263 (1999).</li>
<li>Nishikawa, S. I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., Kodama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Progressive+lineage+analysis+by+cell+sorting+and+culture+identifies+FLK1%2BVE-cadherin%2B+cells+at+a+diverging+point+of+endothelial+and+hemopoietic+lineages%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages.</a> Development. 125, 1747-1757 (1998).</li>
<li>de Pooter, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Notch+signaling+requires+GATA-2+to+inhibit+myelopoiesis+from+embryonic+stem+cells+and+primary+hemopoietic+progenitors%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Notch signaling requires GATA-2 to inhibit myelopoiesis from embryonic stem cells and primary hemopoietic progenitors.</a> J Immunol. 176, 5267-5275 (2006).</li>
<li>Watarai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+functional+NKT+cells+in+vitro+from+embryonic+stem+cells+bearing+rearranged+invariant+Valpha14-Jalpha18+TCRalpha%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Generation of functional NKT cells in vitro from embryonic stem cells bearing rearranged invariant Valpha14-Jalpha18 TCRalpha.</a> 115, 230-237 (2010).</li>
<li>Pipkin, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+transfer+activates+and+delineates+a+locus+control+region+for+perforin%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Chromosome transfer activates and delineates a locus control region for perforin.</a> Immunity. 26, 29-41 (2007).</li>
</ol>

The authors have nothing to disclose.

在OP9-DL1共培养系统已经利用血细胞类型的干细胞在发育过程中，研究了不同的基因产物的作用。8,12,13这也证明研究的基因调节DNA的功能的有效模式9,14中的细胞分化。使用这种方法作为一种替代整个小鼠模型所用的时间和实验，解决在造血许多基本问题，成本得到相当大的节约。然而，该协议用于这些目的的适应性要求，将在这些过程中使用卓制克隆中的电位变化的认定。公布协...

A cell culture system has been established in which mouse embryonic stem cells (mESC) are differentiated to T cells in vitro.1 This system exploits the ability of Notch signaling to drive T cell differentiation.2 The OP9-DL1 cell line was created by transducing bone marrow-derived OP9 cells3 with a Notch ligand, Delta-like 1 (DL1).4 Activation of the Notch signaling cascade in vitro facilitates T cell development to the exclusion of other cell lineages. With the inclusion of appropriate cytokines, this system provides a cell culture &#8220;microenvironment&#8221; that supports the sequential advancement of mESC toward hematopoietic and ultimately T cell lineages. This system supports the flow cytometric identification of T cells at the various developmental stages seen during normal T cell ontogeny in the thymus. For investigating selected questions relating to T cell development, this procedure has become an attractive alternative to in vivo whole mouse models5 and in vitro fetal thymic organ culture methods used to elicit T cell development from mouse embryonic stem cell derived hematopoietic precursors.6 The major advantage of the OP9 co-culture system is that it involves standard and straightforward cell culture techniques and does not depend on the continual use of experimental animals. 
We follow a detailed, previously published protocol in our experiments using this approach.7 We have utilized this technology to examine the hematopoietic differentiation products of non-manipulated mESC clones, high quality mESC clones handpicked to make chimeric embryos8 and stably-transfected ESC clones coming directly out of drug selection.9 We have noted that the temporal kinetics of initial in vitro differentiation from mESC to mesoderm-like colonies in this model can be variable among individual clones. The mESC-OP9 co-cultures can be visually assessed for progression to mesoderm. While this will usually be completed by the fifth day of co-culture, among individual clones, completion can be delayed for one or two days. Quantitative (~80-90%) mesoderm formation must be achieved prior to transfer in order to obtain optimal hematopoietic progenitor cell (HPC) formation and robust lymphopoiesis. Thus, when working with multiple mESC clones, this &#8220;day 5&#8221; passage is best delayed until all clones complete the transition to mesoderm-like colonies. This enables synchrony of subsequent development among the clones after their transfer into hematopoietic differentiation conditions. Three days after the passaging of the 80-90% mesodermal formations, HPCs are collected from the OP9 monolayers. HPCs can be seeded on new OP9 cells to allow differentiation of monocytic, erythroid and B cell lineages. Alternatively, HPCs can be seeded on OP9-DL1 cells and driven towards T cell development. All in vitro differentiation cultures are provided Flt-3L beginning at day 5, with further addition of IL-7 beginning at day 8. Flow cytometry analyses performed at various time points during the experiment enable monitoring of progress through the stages and lineages of hematopoietic differentiation and T cell development. CD4/CD8 double positive (DP) T cells begin emerging by day 16 of the co-culture, and both DP and CD8 single positive (SP) cells are abundant by day 20. The general outcome and robustness of co-culture is greatly dependent on the ability to visually ascertain the completion of the significant developmental turning points that occur. This protocol aims to be a guide to the recognition of these milestones, as well as the other critical parameters, that are key to successful differentiation.

<table><tbody><tr><td>DMEM</td><td>Corning</td><td>15-013-CV</td><td></td></tr><tr><td>Stem cell qualified FBS</td><td>Gemini</td><td>100-125</td><td>Heat inactivated</td></tr><tr><td>Penicillin/Streptomycin</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>L-alanyl L-glutamine</td><td>Corning</td><td>25-015-CI</td><td></td></tr><tr><td>HEPES buffer&amp;nbsp;</td><td>Millipore</td><td>TMS-003-C</td><td></td></tr><tr><td>Non-Essential Amino Acids</td><td>ThermoScientific</td><td>SH30853.01</td><td></td></tr><tr><td>Gentamicin Regent Solution (50 mg/ml)</td><td>Life Technologies&amp;nbsp;</td><td>15750-060</td><td></td></tr><tr><td>&amp;beta;-mercaptoethanol (55 mM) in DPBS</td><td>Life Technologies&amp;nbsp;</td><td>21985-023</td><td></td></tr><tr><td>Filter Unit</td><td>Millipore</td><td>SCGPU05RE</td><td>0.22 &amp;mu;m PES membrane</td></tr><tr><td>Cell Culture Grade Water</td><td>Corning</td><td>25-055-CM</td><td></td></tr><tr><td>&amp;alpha;-MEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>12000-022</td><td>Powder, reconstitute per manufacture recommendation</td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma</td><td>S5761-500G</td><td></td></tr><tr><td>FBS</td><td>ThermoScientific</td><td>SH 30396.03</td><td>Testing of individual lots required&amp;nbsp;</td></tr><tr><td>Dimethyl Sulphoxide&amp;nbsp;</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Recombinant Human Flt-3 Ligand</td><td>R&amp;amp;D Systems</td><td>308-FK</td><td></td></tr><tr><td>Recombinant Murine IL-7</td><td>PeproTech</td><td>217-17</td><td></td></tr><tr><td>LIF&amp;nbsp;</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Utrapure water with 0.1% gelatin</td><td>Millipore</td><td>ES-006-B</td><td></td></tr><tr><td>MEFs mitomycin C treated</td><td>Millipore</td><td>PMEF-CF</td><td>Any mitotically arresteded MEFs can be used</td></tr><tr><td>DPBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Cell strainer (40 &amp;mu;m)</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Tissue culture dish 100 x 20 mm</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>Multiwell 6-well</td><td>BD Falcon</td><td>353046</td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>5 ml Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td><td>BD Falcon</td><td>352235</td><td>Tubes for FACS&amp;nbsp;</td></tr><tr><td>ES R1 cells</td><td>ATCC</td><td>SCRC-1011</td><td></td></tr><tr><td>OP9 cells</td><td></td><td></td><td>Cells can be obtained from the Riken Laboratory Cell Repository (Japan).</td></tr><tr><td>OP9-DL1 cells</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td>Cells can be requested from the Z&amp;uacute;&amp;ntilde;iga-Pfl&amp;uuml;cker laboratory.</td></tr><tr><td>FlowJo software&amp;nbsp;</td><td>Tree Star</td><td></td><td>FACS data analyses</td></tr><tr><td>Flow Cytometer</td><td>BD</td><td></td><td>FACScan, FACSCalibur and FACSVantage have been used in our lab</td></tr></tbody></table>

derivation of t cells in vitro from mouse embryonic stem cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

当在LIF存在下生长在MEF中，mESCs分化可以保持在未分化状态。在理想条件下，它们显示为细胞通过一个闪亮卤素在相差显微镜包围的紧凑集落（ 图1）。这些培养物必须每天监测。根据细胞的汇合，媒体可以被改变或细胞可以分裂。相邻卓制菌落不应该来彼此接触的点。未分化mESCs分化的正常，健康的文化是一个重要的起点， 在体外 T细胞分化。在起始的共培养，则建议的细胞?...

Watch this Scientific Journal Video about Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells at JoVE.com

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

对T细胞推导<em&gt;在体外</em&gt;从小鼠胚胎干细胞

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

derivation-of-t-cells-in-vitro-from-mouse-embryonic-stem-cells

Research

JoVE Journal

Immunology and Infection

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

11.0K 次观看.  City University of New York. Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

视频: Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫与感染

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

发育生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

癌症研究

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

鼠标朴素CD4<sup&gt; +</sup&gt; T细胞分离和<em&gt;在体外</em&gt;分化为T细胞亚群

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

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 ...

<meta charset="utf8"></head><body>使用流式细胞术从初始 CD4+ T 细胞中分化 Th17 细胞

In Vitro Differentiation of Naive CD4+ T Cells into Pathogenic Th17 Cells in Mouse

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have been linked to a wide range of diseases in recent times, including multiple sclerosis (MS), rheumatoid arthritis, acute respiratory distress syndrome (ARDS), sepsis, and other autoimmune disorders. However, the currently known in vitro differentiation protocols have difficulty achieving high purity of pathogenic Th17 cells, with the induction efficiency often below 50%, which is a key challenge in in vitro experiments. In this protocol, we propose an enhanced in vitro culture and differentiation protocol for pathogenic Th17 cells, which is used to directly differentiate naive CD4+ T cells isolated from mouse spleens into pathogenic Th17 cells. This protocol provides detailed instructions on splenocyte isolation, purification of naive CD4+ T cells, and differentiation of pathogenic Th17 cells. Through this protocol, we can achieve a differentiation purity of approximately 90% for pathogenic Th17 cells, which meets the basic needs of many cellular experiments.

<meta charset="utf8"></head><body>作者聚焦：使用细胞因子浓度调节实现 Th17 细胞的高纯度体外分化

This protocol describes the differentiation of na&#239;ve CD4+ T cells into pathogenic Th17 cells in vitro. Specifically, when combined with a multi-parameter flow cytometry-based approach, 90% purity of pathogenic Th17 cells can be obtained from na&#239;ve CD4+ T cells using this differentiation method.

小鼠幼稚 CD4+ T 细胞体外分化为致病性 Th17 细胞

In vitro T cell differentiation techniques are essential for both functional and mechanistic investigations of CD4+ T cells. Pathogenic Th17 cells have ...

对T细胞推导

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