We thank Ms. Deborah Postiff and Ms. Jackline Barikdar in the Tissue Procurement Core and Dr. Cindy DeLong in the Pluripotent Stem Cell Core Laboratory at the University of Michigan for her technical assistance. This study was supported by University of Michigan startup funding and grants from the Central Surgical Association, American College of Surgeons, Melanoma Research Alliance, and NIH/NCI (1K08CA197966-01) to F. Ito.

注:患者应知情同意参加机构审查委员会和人类多能干细胞委员会批准研究。 1.分离肿瘤浸润淋巴细胞培养<ol><li>获得不需要从病理学服务/组织采购芯病理诊断肿瘤材料。放置20-100克肿瘤标本，在50毫升试管用30ml肿瘤收集培养基（ 表1）。 </li><li>解剖坚实牢固，使用剪刀脆弱和/或血性坏死区肿瘤标本的正常组织。去除坏死组织后，用剪刀把剁碎的样本尽可能小。 </li><li>解离切碎样品成单细胞悬浮液使用根据制造商的说明一个离解和肿瘤的离解试剂盒（人）。过滤该悬浮液与被在50毫升管设置一个70微米的细胞过滤并用洗涤过滤器2次2毫升RPMI 1640。 </li><li>在200 xg离心离心在室温下5分钟。弃上清，重悬细胞在10ml的T细胞培养基（ 表1）。 </li><li>制备的两步梯度溶液如聚蔗糖（梯度溶液）在50毫升的管中，用10毫升100％梯度溶液低级步骤和用Dulbecco磷酸盐稀释30毫升的75％梯度溶液的中间步骤缓冲盐水，无钙，无镁（D-PBS（ - ））。 </li><li>层细胞悬浮液（来自步骤1.4）的梯度溶液（来自步骤1.5）。离心在20°C 400 XG 45分钟。 </li><li>收集含富集的TIL在100％梯度溶液和在50毫升的管中的75％的梯度溶液之间的界面层。通过加入20-30毫升的D-PBS中稀释的富集的TIL溶液的层（ - ）。 </li><li>在200 xg离心离心在室温下5分钟。弃上清，悬浮TIL浓缩部分在10毫升T细胞介质。 </li><li>培养馏分（来自步骤1.8）与2ml的T细胞培养基和6000单位/ ml重组人（rh的），IL-2在一个6孔板，在37℃，5％ 的 CO 2。 </li><li>更改一半的媒体第5天的文化开始后，此后每2-3天。 </li><li>分裂细胞以1:2的比例没有胰蛋白酶消化后轻轻悬浮，达到80-90％汇合时加倍井的数量。添加新鲜T细胞培养基和重组人IL-2的一半体积（1毫升）中的6000国际单位/毫升。 </li></ol> 2.丝裂霉素C处理的SNL饲养细胞板的制作<ol><li>达到在0.1％明胶包被的10cm皿10毫升SNL饲养细胞培养基（ 表1）的直至80-90％汇合（3-4×10 6细胞）培养SNL饲养细胞。 </li><li>添加310微升的0.4毫克/毫升丝裂霉素C溶液直接进入SNL饲养细胞培养皿，并在37℃，5％CO 2的2小时和15分钟孵育。吸媒体一次用5毫升的D-PBS洗涤细胞两次（ - ）。 </li><li>加入0.5毫升0.25％胰蛋白酶/ 1mM EDTA中并在室温下孵育1分钟以解离细胞。添加4.5毫升SNL饲养细胞培养基中，以中和重新悬浮细胞成一个单一的悬浮液中。 </li><li>转移细胞入15ml管中并离心，在200×g离心5分钟。吸出介质并在10ml SNL饲养细胞培养基的再悬浮后计数与血球细胞。 </li><li>板每SNL饲养细胞的孔1.5×10 6个细胞于10cm培养皿，在37℃下孵育，5％ 的 CO 2。使用3天之内的SNL饲养细胞。 </li></ol> 3.使用iPS细胞的仙台病毒的产生（SEV）的矢量<ol><li>收获肿瘤浸润淋巴细胞（来自步骤1.11），并激活的TIL的5×10 5个细胞与板结合的小鼠抗人CD3（1微克/毫升）和可溶性小鼠抗人CD28（5微克/毫升），用重组人IL-2在一个6孔板（6000国际单位/毫升）在2毫升的T细胞培养基中的37℃，5％CO 2的5天。 </li><li>收获肿瘤浸润淋巴细胞（来自步骤3.1）使用移液管在一个15毫升管和计数与血球细胞数。 </li><li>激活的TIL与板结合的小鼠抗人CD3（1微克/ ml）的1×10 5个细胞和可溶性小鼠抗人CD28（5微克/毫升），其中的rhIL-2（60单位/毫升）的500微升每孔重新编程介质（ 表1）在一个24孔板在37℃，24小时，5％CO 2。准备3孔为SeV载体感染:OCT3 / 4，SOX2，KLF4和c-MYC（1孔）; SeV载体感染（GFP:绿色荧光蛋白）（1井）;和细胞计数（1孔）。 </li><li> 24小时后（从步骤3.3），计数与血球细胞数量和确定每个病毒感染的各个（3-20）多重性（MOI）的体积。从-80℃贮存取出一组仙台病毒载体管。迅速解冻SeV载体在水浴（37℃），将它们放在冰上。 病毒的体积（微升）= MOI（CIU /细胞）细胞的x个病毒/滴度（CIU /毫升）×10-3（微升/毫升） </li><li>通过加入SeV载体的混合物在10-20的MOI添加的每个单独地进行OCT3 / 4，SOX2，KLF4和c-Myc的四个仙台病毒载体管的计算出的卷到媒体，包括活化的TIL。 </li><li>为了确定转导效率，添加的SeV-GFP的计算的体积成活化的TIL的阱。 </li><li>放置盘（来自步骤3.5和步骤3.6）在培养箱中在37℃，5％CO 2的24小时。 24小时后，估计使用荧光显微镜下感染的SeV-GFP的TIL感染效率。 </li><li>收集在15ml试管中的细胞（来自步骤3.5）。在200 xg离心离心在室温下5分钟。 </li><li>弃上清，悬浮颗粒在0.5与灵长类ES细胞培养基和碱性成纤维细胞生长因子（bFGF，4纳克/毫升）胚胎干细胞媒体毫升。 </li><li>转移悬浮液到一个10cm皿该CONTA插件丝裂霉素C处理的SNL饲养细胞在10毫升人类胚胎干细胞媒体。更改胚胎干细胞媒体隔日，直到菌落生长。 </li><li>约18天到21，在净化台使用10微升末端除去在显微镜下菌落周围的SNL饲养细胞。 </li><li>使用10微升尖端刮落，并用200微升尖成每96孔培养板的孔100微升iPS细胞介质的转让。吸管上下在2-3次打破菌落成小团块具有50-100微米的平均尺寸。 </li><li>转移小块到6孔组织培养板用丝裂霉素C处理的SNL饲养细胞（来自步骤2）在2毫升每iPSCs的媒体以及碱性成纤维细胞生长因子（bFGF，4纳克/毫升）。每天换iPSCs的媒体。 </li><li>用1mg / ml胶原酶IV传送的iPSC到6孔组织培养板中的新鲜丝裂霉素C处理的SNL饲养细胞每5-6天。每次准备每个克隆的与免疫的板2口井ostaining。 </li><li>由多潜能标志物（SSEA3，SSEA4，TRA-1-60，TRA-1-81，和OCT3 / 4）1的免疫组织化学染色测定各克隆的完整重新编程。 注:多能性潜力的进一步评估，确认完全重新编程iPSC系可以由胚体的形成和分化测定或畸胎瘤形成分析16分化成三个胚层。 </li><li>确认完全重新编程iPSC系通过展示细胞遗传学分析正常核型。 </li><li>畸胎瘤的形成，注入未分化的TIL衍生的iPSC（1×10 6）悬浮在100μl含有10％FCS入6-8周龄雌性NOD / SCID小鼠的皮下组织的DMEM。染色畸胎瘤切片行HE。 </li></ol> 4.为iPS细胞SSEA3，SSEA4，TRA1-60和TRA1-81免疫荧光染色<ol><li>洗的iPSC用1毫升的PBS并用1ml 4％每年的固定iPSCs的10分钟raformaldehyde溶液。去除4％多聚甲醛溶液，并用1毫升的PBS洗涤3次的iPSCs的。 注:使用多聚甲醛的时候，因为它是有毒的注意。 </li><li>预处理用30分钟封闭缓冲液（ 表1）的iPSC。同时，稀释用PBS的TritonX稀释第一抗体（大鼠抗人SSEA3，小鼠抗人SSEA4，小鼠抗人TRA1-60和小鼠抗人TRA1-81）1:50 1.5％山羊血清溶液-100（总体积:1ml）中。 </li><li>除去封闭缓冲液（ 见表1）。添加稀释的初级抗体溶液孵育在室温下1小时。同时，稀释的第二抗体（抗小鼠IgG和IgM或抗大鼠IgM）1:50 1.5％山羊血清溶液。 </li><li>除去稀释的初级抗体溶液，并用1毫升的PBS 3次，每次洗的iPSC 5分钟。添加稀释的二级抗体溶液孵育在室温下在暗室中45分钟。 </l我&gt; <li>除去稀释二级抗体溶液并用1ml的PBS中的3次，每次洗的iPSC 5分钟。观察与免疫荧光显微镜的iPS细胞。 </li></ol> 5. iPS细胞的免疫荧光染色进行的Oct3 / 4 <ol><li>洗的iPSC用1毫升的PBS并用1ml的4％低聚甲醛溶液中10分钟固定的iPSC。去除4％多聚甲醛溶液，并用PBS洗涤3次的iPSCs的。 </li><li>添加透化缓冲液（ 表1）并在室温下孵育10分钟。除去透化缓冲液，并添加封闭缓冲液（ 表1）。 </li><li>在室温下孵育30分钟。同时，稀释第一抗体（小鼠抗人的Oct3 / 4）1:50在封闭缓冲液。 </li><li>添加稀释的初级抗体溶液中并在4℃孵育过夜。 </li><li>除去稀释的初级抗体溶液并用PBS 1ml的洗涤iPSCs的3次，每次5分钟。与此同时，二在封闭缓冲液100:琵琶二次抗体（山羊抗小鼠IgG / IgM抗体）1。 </li><li>添加稀释的第二抗体溶液并在黑暗的房间45分钟，在室温下孵育。 </li><li>除去稀释次级溶液并用1ml的PBS中的3次，每次洗的iPSC 5分钟。根据观察免疫荧光显微镜的iPS细胞。 </li></ol>

<ol>
	<li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Wernig, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li><li>Maherali, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+reprogrammed+fibroblasts+show+global+epigenetic+remodeling+and+widespread+tissue+contribution.">Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Okita, K., Ichisaka, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+germline-competent+induced+pluripotent+stem+cells.">Generation of germline-competent induced pluripotent stem cells.</a> Nature. 448, 313-317 (2007).</li><li>Loh, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+T+cells+from+human+peripheral+blood.">Reprogramming of T cells from human peripheral blood.</a> Cell Stem Cell. 7, 15-19 (2010).</li><li>Seki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+from+human+terminally+differentiated+circulating+T+cells.">Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.</a> Cell Stem Cell. 7, 11-14 (2010).</li><li>Staerk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+human+peripheral+blood+cells+to+induced+pluripotent+stem+cells.">Reprogramming of human peripheral blood cells to induced pluripotent stem cells.</a> Cell Stem Cell. 7, 20-24 (2010).</li><li>Nishimura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+rejuvenated+antigen-specific+T+cells+by+reprogramming+to+pluripotency+and+redifferentiation.">Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation.</a> Cell Stem Cell. 12, 114-126 (2013).</li><li>Vizcardo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+Human+Tumor+Antigen-Specific+T+Cells+from+iPSCs+Derived+from+Mature+CD8(+)+T+Cells.">Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8(+) T Cells.</a> Cell Stem Cell. 12, 31-36 (2013).</li><li>Wakao, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+functional+human+mucosal-associated+invariant+T+cells+via+reprogramming+to+pluripotency+and+redifferentiation.">Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation.</a> Cell Stem Cell. 12, 546-558 (2013).</li><li>Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tumor-infiltrating+lymphocyte+cultures+for+use+in+adoptive+transfer+therapy+for+melanoma+patients.">Generation of tumor-infiltrating lymphocyte cultures for use in adoptive transfer therapy for melanoma patients.</a> J Immunother. 26, 332-342 (2003).</li><li>Gros, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+identifies+the+patient-specific+CD8(+)+tumor-reactive+repertoire+infiltrating+human+tumors.">PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors.</a> J Clin Invest. 124, 2246-2259 (2014).</li><li>Rosenberg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+Complete+Responses+in+Heavily+Pretreated+Patients+with+Metastatic+Melanoma+Using+T-Cell+Transfer+Immunotherapy.">Durable Complete Responses in Heavily Pretreated Patients with Metastatic Melanoma Using T-Cell Transfer Immunotherapy.</a> Clinical Cancer Research. 17, 4550-4557 (2011).</li><li>Restifo, N. P., Dudley, M. E., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adoptive+immunotherapy+for+cancer:+harnessing+the+T+cell+response.">Adoptive immunotherapy for cancer: harnessing the T cell response.</a> Nat Rev Immunol. 12, 269-281 (2012).</li><li>Saito, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+of+Melanoma+Tumor-Infiltrating+Lymphocytes+to+Induced+Pluripotent+Stem+Cells.">Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells.</a> Stem Cells International. 2016, 11 (2016).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-362 (2009).</li><li>Ban, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+transgene-free+human+induced+pluripotent+stem+cells+(iPSCs)+by+temperature-sensitive+Sendai+virus+vectors.">Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.</a> Proc Natl Acad Sci U S A. 108, 14234-14239 (2011).</li><li>Fujie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Type+of+Sendai+Virus+Vector+Provides+Transgene-Free+iPS+Cells+Derived+from+Chimpanzee+Blood.">New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood.</a> PLoS One. 9, e113052 (2014).</li></ol>

The authors declare that they have no competing financial interests.

在这里，我们证明了通过四个转录的SeV介导转导重新编程黑素瘤的TIL到的iPSC一个协议因素OCT3 / 4，SOX2，KLF4和c-MYC。这种方法，采用了SeV载体系统重新编程的T细胞，提供了一个非整合方法7的优点。 先前的研究表明，重编程的SeV体系是高效可靠的改编不仅成纤维细胞，而且外周血T细胞7,17。此外，我们最近表明，黑色素瘤的TIL是更加分化和表达更高水平的抑制?...

细胞重新编程的技术，通过一组已定义的转录因子的过表达允许产生诱导性多能干细胞（iPS细胞）的保持在基于细胞的疗法1,2-领域大有希望。这些iPS细胞表现出转录和后生特征和具有自我更新和多能性的能力，类似于胚胎干细胞（ESC）3-5。在重编程技术在过去十年取得了显着的进步，使我们能够产生甚至从终末分化细胞，如T细胞6-8人的iPS细胞。 T细胞来源的iPS细胞（TiPSCs）保留的T细胞受体（TCR）链基因作为原始T细胞相同的重排结构，它允许从TiPSCs 9-11抗原特异性T细胞的再生。 近80％的黑色素瘤浸润淋巴细胞（的TIL）中获得来自病人的肿瘤特异性识别肿瘤相关抗原的ND保持对原有的肿瘤细胞杀伤12。值得注意的是，在的TIL细胞程序性死亡蛋白-1（PD-1）的表达被发现以识别自体肿瘤反应性的剧目，包括突变的特定新抗原-CD8 +淋巴细胞13。在组合离体扩增的自体肿瘤浸润淋巴细胞的过继转移用制备lymphodepleting方案和白细胞介素-2（IL-2）的全身给药会引起患者14亚转移性黑素瘤的实质性退化。尽管取得了令人鼓舞的临床前模型和患者中的结果，注入的T细胞的存活差和免疫抑制途径的存在似乎妥协过继性T细胞疗法的潜能。目前临床协议需要自体T细胞的广泛离体操作，以便获得大的数字。这导致具有不良存活终末分化的T细胞，降低海峡的产生iferative容量，以及高含量的PD-1 15。 继T细胞疗法的这种限制可以通过使用iPS细胞，可以提供自体T细胞免疫治疗无限源理论上克服。我们最近已报道黑素瘤的TIL的表达由仙台病毒（SeV载体）的四个转录因子介导的转导，OCT3 / 4，SOX2，KLF4和c-Myc的16个高层次的PD-1的重编程。虽然逆转录病毒载体需要整合到宿主染色体以表达重编程基因，SeV载体是非整合并最终被从细胞质淘汰。重新编程的效率是与SeV的系统高得多慢病毒或逆转录病毒载体6-8相比。此外，SeV载体能特异性重新编程在外周血单核细胞（PBMC）的T细胞，而通过慢病毒或逆转录病毒载体产生的一些的iPSC克隆可从非淋巴谱系6-8。在这里，我们详细介绍实施了人体黑素瘤的TIL的分离和活化和用于使用的SeV重新编程系统的TIL衍生的iPSC的生成的程序。

<table><tbody><tr><td>gentle MACS C Tubes</td><td>Miltenyi Biotec</td><td>130-093-237</td><td></td></tr><tr><td>gentle MACS Dissociator</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Tumor Dissociation Kit, human</td><td>Miltenyi Biotec</td><td>130-095-929</td><td></td></tr><tr><td>RPMI 1640</td><td>Life technologies</td><td>11875-093</td><td></td></tr><tr><td>Falcon 70 um Cell Strainer</td><td>BD</td><td>352350</td><td></td></tr><tr><td>BD Falcon 50ml Conical Cntrifuge tubes</td><td>BD</td><td>352070</td><td></td></tr><tr><td>IMDM</td><td>Life technologies</td><td>12440053</td><td></td></tr><tr><td>human AB serum</td><td>Life technologies</td><td>34005100</td><td></td></tr><tr><td>L-glutamine (200mM)</td><td>Life technologies</td><td>25030-081</td><td></td></tr><tr><td>2-mercaptoethanol (1000x, 55mM)</td><td>Life technologies</td><td>21985-023</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Life technologies</td><td>15140-122</td><td></td></tr><tr><td>gentamicin</td><td>Life technologies</td><td>15750-060</td><td></td></tr><tr><td>Ficoll-Paque PLUS</td><td>GE</td><td>17-1440-02</td><td></td></tr><tr><td>D-PBS (-)</td><td>Life technologies</td><td>14040-133</td><td></td></tr><tr><td>recombinant human (rh) IL-2</td><td>Aldesleukin, Prometheus Laboratories Inc.</td><td></td><td></td></tr><tr><td>Purified NA/LE Mouse Anti-Human CD3</td><td>BD</td><td>555329</td><td></td></tr><tr><td>Purified NA/LE Mouse Anti-Human CD28</td><td>BD</td><td>555725</td><td></td></tr><tr><td>X-VIVO 15</td><td>Lonza</td><td>04-418Q</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>HEPES</td><td>Life technologies</td><td>15630-080</td><td></td></tr><tr><td>N-Acetylcysteine</td><td>Cumberland Pharmaceuticals Inc.</td><td>NDC 66220-207-30</td><td></td></tr><tr><td>Falcon Tissue Culture Plates (6-well)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Falcon Tissue Culture Plates (24-well)</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Sendai virus vector</td><td>DNAVEC</td><td></td><td></td></tr><tr><td>SNL feeder cells</td><td>Cell Biolabs, Inc</td><td>CBA-316</td><td></td></tr><tr><td>mitomycin C</td><td>SIGMA</td><td>M4287</td><td>soluble in water (0.5 mg/ml)</td></tr><tr><td>gelatin</td><td>SIGMA</td><td>G1890</td><td></td></tr><tr><td>basic fibroblast growth factor (bFGF)</td><td>Life technologies</td><td>PHG0264</td><td></td></tr></tbody></table>

generation of induced pluripotent stem cells from human melanoma tumor-infiltrating lymphocytes

体外过继转移扩大了自体肿瘤浸润淋巴细胞（肿瘤浸润淋巴细胞）可以调节的转移性黑色素瘤患者显著的子集耐用和完整的答复。这种方法的主要障碍是转移的T细胞，引起端粒缩短的可行性减少的，并且从患者获得的肿瘤浸润淋巴细胞的数量有限。分化少T细胞端粒长就过继性T细胞治疗的理想T细胞亚群;然而，产生大量的这些分化较少的T细胞是有问题的。过继性T细胞疗法的这种限制可以通过使用诱导多能干细胞（iPS细胞）在理论上克服自我更新，维持多能性，具有细长的端粒，并提供自体T细胞的免疫治疗的一个无限源。在这里，我们提出了一个协议，以产生使用仙台病毒载体的重编程因子转导入肿瘤浸润淋巴细胞iPS细胞。该协议产生S完全重新编程，免费矢量克隆。这些TIL衍生的iPSC可能能够产生用于过继性T细胞疗法分化少患者 - 和肿瘤特异性T细胞。

图1显示了涉及黑素瘤的TIL与重组人IL-2，这是随后用抗CD3 / CD28激活和OCT3 / 4，KLF4，Sox2和c-Myc的基因转移到的TIL的初始膨胀过程的概要为iPS细胞的产生。通常情况下，对文化的TIL用的rhIL-2开始形成球体培养开始后21-28天。在这一点上，肿瘤浸润淋巴细胞准备用抗CD3激活/ CD28。 图2A示出的TIL，在培养用的rhIL-2在第21天，这是准备与被活化的抗CD3 / CD28。...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes at JoVE.com

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

generation-induced-pluripotent-stem-cells-from-human-melanoma-tumor

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.8K 次观看.  University of Michigan. The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

视频: Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

科研

发育生物学

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

从人多能干细胞的黑素细胞的无饲养推导

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

免疫与感染

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

医学

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

将小鼠成纤维细胞直接重编程为黑素细胞

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

<meta charset="utf8"></head><body>患者来源的黑色素瘤类器官：癌症免疫治疗的前沿

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

<meta charset="utf8"></head><body>作者聚焦：使用患者来源的类器官重建黑色素瘤复杂性以进行免疫治疗评估

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

揭示黑色素瘤患者来源的类器官模型的治疗机会

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

摘要

探索更多视频

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

从人多能干细胞的黑素细胞的无饲养推导

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

定向分化诱导性多潜能干细胞向T淋巴细胞

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

来自患者的细胞培养和分离的CD133

将小鼠成纤维细胞直接重编程为黑素细胞

揭示黑色素瘤患者来源的类器官模型的治疗机会

揭示黑色素瘤患者来源的类器官模型的治疗机会

揭示黑色素瘤患者来源的类器官模型的治疗机会