This work was supported by an NHLBI grant to K.M. and I.R.L. (1RO1HL119404). Carlos-Filipe Pereira was the recipient of a Revson Senior Biomedical Fellowship. We gratefully acknowledge the Mount Sinai hESC/iPSC Shared Resource Facility and S. D'Souza for assistance with materials and protocols. We also thank the Mount Sinai Flow Cytometry, Genomics, and Mouse facilities.

伦理学声明:小鼠细胞系衍生下列医学学院伊坎在西奈山的动物护理指导原则，并应遵守任何主办机构来完成。 C57BL / 6小鼠1.鼠标胚胎成纤维细胞（MEF）隔离<ol><li>设置定时交配13。一旦阴道塞的可视化，可以考虑0.5本节。 </li><li>分离插头上的日期插入女性和检查他们在10-11天，以确认妊娠。 </li><li>在13.5-14.5日通过CO安乐死怀孕的雌性小鼠吸入2颈椎脱位。 </li><li>浸泡怀孕雌性腹部用70％乙醇，解剖用无菌剪刀和镊子向下中线腹腔，并手术移除含有胚胎14子宫角。轻轻取出胚胎的同时切断剩余的腹部组织。 注:每边约恢复8胚胎4。 </li><li>放置子宫^ h含在10cm皿用5-10毫升无菌冷冻磷酸盐缓冲盐水（PBS）的胚胎orns。 </li><li>用无菌剪刀和镊子，使沿子宫角切口携带的胚胎囊隔离。转移囊到一个新的10cm皿5-10毫升无菌冷冻PBS并放置冰上。 </li><li>将几个囊到一个新的10cm皿5-10毫升无菌冷冻的PBS。用无菌剪刀和镊子轻轻划开囊（无切削太深），以免刺入胚胎。 </li><li>通过切割脐带分离从胎盘的胚胎。 </li><li>胚胎转移到含有5-10毫升冷冻PBS的新10cm皿和冰离开，而完成剩余解剖。 </li><li>斩首使用无菌镊子胚胎。小心砍掉胚胎用无菌剪刀和镊子，从断头地区开始开拓腹部正中线。钳位置下方ŧ他内脏器官（包括心脏，肝脏和肺）和刮出来15。 </li><li>切割用剪刀和镊子，并转移到含有10ml胰蛋白酶的50ml锥形管中剩余的组织切成小块。 </li><li>吸管上下10ml的移液管20-30次混合和分离组织。 </li><li>孵育在37℃的水浴的组织和胰蛋白酶混合物45分钟，偶尔漩。 </li><li>吸取上下10ml的移液管约5分钟以混合的内容。 </li><li>添加胰蛋白酶的细胞至3x标准的Dulbecco改良的Eagle培养基（DMEM）和10％胎牛血清（FBS），体积10微克/毫升青霉素/链霉素（P / S）和1mM L-谷氨酰胺（L-GLUT）中10厘米菜（〜10毫升）中，并在37℃下培养直至汇合（约2-4天）。文化约每10 cm培养皿1-2胚胎。 注:冻结的细胞，一旦板块都汇合。冷冻细胞可解冻并传代2次以上。细胞可以或者是解冻后冷冻前分裂2次，然后一次。 </li></ol> 2.病毒生产<ol><li>扩大293T细胞中，用10ml含10％FBS，1％P / S，和1％L- GLUT用于转染10的9cm培养皿中。 <ol><li> Trypsinize 293T细胞用2ml胰蛋白酶，孵育在37℃下5分钟，收集细胞到15ml试管，旋在300 XG和板适当（10毫升，每10cm皿）。 </li></ol></li><li> 24小时在转染前，分割293T细胞1的汇合10cm平板:6和种子于10cm明胶包衣（在PBS中的0.1％明胶）板。 注:每个病毒4板是必需的。 <ol><li>在明胶涂层板，至少添加将2ml 0.1％明胶溶液涂覆10厘米板的底部。孵育在37℃下至少20分钟。吸掉明胶之前，加入细胞板。 </li></ol></li><li>在15ml管中，加入84微克质粒（例如GATA2，Gfi1b，cFos的，或ETV6（子克隆到pFUW-TETO VECT或16）或FUW-M2rtTA 17），84微克psPAX2和42微克pMD2.G.加水（H 2 O），以弥补容积〜2毫升。准备一个管各因素（ 如 GATA2，Gfi1b，首席财务官，ETV6和FUW-M2rtTA）。 </li><li>加入250微升2M氯化钙2到包含所选基因的84微克组成2 mL萃取混合物中各管（GATA2，Gfi1b，首席财务官，ETV6或FUW-M2rtTA）+ 84微克psPAX2 + 42微克pMD2.G + H 2 O 。 </li><li>使用插入吸管援助巴斯德吸管，释放气泡进入2.25毫升DNA + H 2 O + 2M的氯化钙2混合。由于正在产生的气泡，将贴在玻璃吸管P1000的的前端，慢慢地喷射2毫升100毫N，N-双（2-羟乙基）-2-氨基乙烷磺酸（BES）盐水走在巴斯德吸管1毫升一次。 </li><li>孵育在培养罩所有管在室温下至少15分钟。 注:4.25毫升混合物（现在DNA + H 2 O + 2M 氯化钙 </子&gt; + BES生理盐水）将看上去有点浑浊。 </li><li>作为混合物孵化，吸媒体关闭293T板和加入10 mL的DMEM + 10％FBS + 1 2mM L-谷氨酰胺+ 25nM的氯喹。 注:该介质不包含P / S。 </li><li>温育至少15分钟后（或直到上293T细胞变化的媒体），添加4.25毫升DNA + H 2 O +的2M氯化钙2 + BES盐水混合物逐滴至293T细胞，跨越每个管4细胞板均匀分布（即略超过1毫升，每板中的混合物的）。 </li><li>在37℃的板过夜（O / N）。 </li><li>后24小时孵化，用4ml DMEM标准（见步骤1.15）替换所有板块的媒体。 </li><li>保存在32℃培养箱细胞从现在开始。 </li><li> 24小时步后2.10，媒体收集到单独的50ml试管。用4ml标准的DMEM替换从每块板收集的媒体。 注:每个初始管混合物，结果4板在16毫升收集媒体。 <ol><li>一个压脚提升培养12小时，重新收集媒体到同一个50ml试管和另外4 mL标准的DMEM取代。 </li><li>培养12小时后，收集媒体和添加到相同50ml试管。 注:各管应包含约48毫升含有病毒的媒介。 </li></ol></li><li>要通过0.45微米的低蛋白结合过滤器集中收集病毒，第一个过滤器收集到的介质。 </li><li>倾含病毒进入的病毒浓度离心管（约16毫升一时间）过滤介质。 </li><li>旋在4000 XG在4℃下25分钟，取出流通。 注意:浓缩介质和病毒的一种小体积将在过滤器是可见的。 </li><li>另外16毫升的过滤含病毒介质的添加到残留在过滤器中的浓缩培养基+病毒量和通过反转所述管混合。 </li><li>旋转管再次在4℃下4000×g离心25分钟，并丢弃流过。 </li><li>添加过滤剩余收集浓缩的培养基+病毒量留在过滤器中并通过反相管混匀。 </li><li>在4℃下10-20分钟旋转一次在4000 xg离心（取决于在过滤器的卷上），并确保浓缩培养基+病毒残留在过滤器的剩余体积为约1ml。如果大于1毫升浓培养基+病毒的保留在过滤器，以4,000 xg离心再次旋转1分钟，并重复直到1毫升在过滤器中留下。 </li><li>等份加入200μl浓缩病毒的进入1.5毫升管，并在-80℃冷冻或立即使用。 </li></ol> 3. MEF重编程<ol><li>预涂层6孔板用0.5ml 0.1％明胶的PBS中，每孔，确保明胶覆盖的阱的整个区域。在37℃培养箱放置并孵育至少20分钟。孵化后，取出板和吸出明胶。 </li><li>与以每孔25,000个细胞的密度标准DMEM板的MEF（150,000个细胞每6孔板）上从步骤3.1凝胶化6孔板（多个），并允许沉降O / N在37℃培养箱。 </li><li>通过混合50微升M2rtTA其余50微升GATA2，Gfi1b，cFos的，和ETV6的混合（每12.5微升）制备100μl的病毒鸡尾酒。 </li><li>由MEF吸媒体和加入2毫升的标准DMEM 8微克/毫升聚凝胺。 </li><li>转导的MEF平板的每个孔以15微升病毒鸡尾酒的，并在37°C孵育O / N。 注意:这是重编程过程的第0天。 </li><li>在第1天，在培养16-20小时后，用补充有1μg/ ml的阿霉素每阱2毫升的新鲜标准的DMEM替换培养基。 </li><li>制备添加有氢化可的松（10 -6 M），100ng / ml的干细胞因子（SCF），100纳克/毫升FMS相关酪氨酸激酶3配体（FLT3L），20纳克/毫升白细胞介素-3（IL-造血培养基3），20纳克/毫升白细胞介素-6（IL-6），和1μg/ ml的阿霉素。 </li><li>第4天aspiratE从每个孔中的介质和洗涤细胞用每孔1ml的PBS。 </li><li>抽吸PBS中，离解，用1毫升，每孔0.05％胰蛋白酶的细胞，并收集细胞。 </li><li>计数细胞用血细胞计数器，旋在300×g离心5分钟，重新悬浮于12ml在步骤3.7中所述的造血介质和板每6孔板60,000个细胞上0.1％明胶包被的平板（2毫升，每孔10,000细胞，每孔）。 </li><li>用新鲜的造血补充的文化传媒，每6天为文化的持续时间（35〜36天）更换介质。 </li><li>分析或者中间细胞或新出现的造血细胞样细胞通过免疫染色12，FACS分析8,12，定量RT-PCR检测12，和mRNA测序12确认采集hemogenic内皮或HSC状的标记和基因表达。 </li></ol> 4.胎盘聚集和菌落形成单位（CFU）的甲基纤维素测定<ol><li>从怀孕的C5解剖胎盘7BL / 6小鼠如先前所述18在E12.5和保持组织在冰上。 </li><li>启动胎盘解离19，洗净胎盘组织在2-5毫升0.2％I型胶原酶的PBS，用20％FBS和与由通过针头传代胎盘和PBS装入5ml的注射器的18G针机械解离至少3次。 </li><li>机械解离后，保持在2-5毫升胶原酶溶液的胎盘细胞在37℃下1.5小时。 </li><li>传代细胞通过20-25g的针头装配到5毫升注射器数次以进一步机械解离的胎盘细胞。 </li><li>通过70微米的细胞过滤器过滤单细胞悬液。 </li><li>计数与血球细胞中并在2000 cGy的照射有丝分裂失活20。 </li><li> 10毫升补充有100纳克/毫升的SCF，100纳克/毫升FLT3L，20纳克/ ml的IL-3，20纳克/ ml的IL-6和10ng / ml的血小板生成素（TPO）造血培养基添加到10cm的碟。 注:DOX未在该阶段需要。 </li><li>直接放置一个0.65微米的过滤器对造血培养基在盘中，允许其浮动以创建一个液 - 气界面，并设置菜一边。 </li><li>离解在步骤3.8-3.10描述每日25转导的MEF（来自步骤3.11衍生），并与来自步骤4.6胎盘集合体167,000细胞混合转导的MEFs 33000（1:6比）。 </li><li>要生成汇总18,21，先降速的MEF和胎盘细胞混合物从步骤4.9在300×克5分钟。吸媒体，并使用P200吸管悬浮沉淀的细胞在30-50微升标准的DMEM，绘制单细胞悬液到200微升nonbevelled的枪头。 </li><li>闭塞用石蜡膜枪头。放置阻塞尖入15ml离心管，并在尖端的覆盖端降速5分钟，在300 xg离心所以粒料形式。 </li><li>精心的15ml试管和地点卸下吸头尖端到P200移液管的端部。丢弃石蜡膜，然后从步骤4.8挤出细胞沉淀到浮置滤波器。 注:每板式过滤器最多3聚集。 </li><li>培养在浮动0.65微米的过滤器为4-5天的聚集体在37℃，5％的CO 2。 </li><li>收集用细胞刮刀的聚集物（至多3％滤波器），将它们结合起来，并用0.2％胶原酶消化，以下作为与整个胎盘做以解离细胞的相同的协议（步骤4.2-4.5）。 </li><li>解离后，洗聚集体与2-5毫升的PBS补充有5％FBS和300 XG降速细胞5分钟。 </li><li>悬浮于500μl的DMEM的聚集体无FBS，P / S，或L-GLUT。借此将500μl再悬浮，并将其添加到补充有10ng / ml的TPO，精心避免气泡的形成3毫升1％甲基纤维素的介质。在3 35×10mm的非处理培养皿该混合物的板等体积CFU测定12。 </li><li>作为对照，将培养照射胎盘聚集体没有如在步骤4.7-4.16如上所述重新编程的MEF 4-5天，并装在CFU实验中混合。 注:这些聚集本身并不能形成菌落。 </li><li>算手动后培养10-14天的显微镜，在37℃，5％CO 2条件下的造血集落。 </li><li>细胞离心涂片22拾取菌落来显示单个细胞的形态表型。 </li></ol>

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The authors have no conflict of interest to disclose.

从容易实现的体细胞产生骨髓干细胞从头提供了一个独特的方法来研究体外造血，并有机会可能这个技术应用到人的制度。这个翻译将产生一个新的工具来研究人类血液病在培养皿中，以及提供药物测试平台和基因定位机会以治疗与新疗法或HSC移植众多障碍。在该领域，最近的研究上的能力扩展到产生骨髓干细胞从头 ，展示的重编程过程的几个特征的重要性。这些措施包括起始细胞群和TF鸡尾酒的选择，以及如何培养条件（共培养小生）和补充（细胞因子，小分子等）显著影响24-26重新编程的效率。几个研究正在不通过行驶施加重编程技术对人体系统一个多能干中间，27,28不希望的步骤在这些方法中。 下面是是第经由发育过程，以产生由体细胞的HSC细胞样细胞，同时避免了使用多能性因子和多能性的中间体的形成的协议。生成的HSC样细胞出现通过一个发展过程的发展，首先需要通过细胞向旅行hemogenic内皮中间类似于EHT。在含有内皮细胞和造血基因表达谱重新编程内皮样中间体的形式20天。 HSC细胞样细胞从第35天含有细胞表面免疫表型和基因表达概况高度相似的HSCs，并可以产生CFU测定红细胞和粒细胞集落这些中间细胞出现。这表明，与其他大多数研究中，这种重新编程协议概括体外复杂的发育过程，可以烫发它涉及胚胎造血造血和血液疾病过程的进一步研究。这些研究允许在如何对待和治疗存在血液病，以及如何操纵造血为此在众人进一步研究。这可以通过在特定患者的成纤维细胞诱导hemogenic程序体外疾病模型建立完成。用这些模型中，正常和病变造血可以精细地解剖和研究，以及进行药物筛选和基因编辑治疗与感兴趣疾病相关的/正确的造血缺陷。 用小鼠成纤维细胞支持这种重编程过程的各种有益的品质。成纤维细胞本身是很容易从供体小鼠获得，使细胞采集简单。翻译该技术对人类将因此仅仅必要病人皮肤样本，使得采集的患者特异性血液制品和随后的细胞德刺痛血液病的治疗一个可行的选择。此外，成纤维细胞是从造血干细胞非常表观遗传学上不同的，表明所选择的转录因子的能力，在此重新编程协议既表观遗传学再加压成纤维细胞的身份，并且还通过改变起始细胞29,30的后生存储器煽一个hemogenic程序。虽然移植研究还不证明这些来源的细胞充分多向植入的功能，看到如果这些因素诱导，在人的系统产生完全功能的造血细胞将是极大的兴趣的一个hemogenic程序。 该协议内的几个步骤可以在困难的情况下，重新编程期间进行修改，如转导之前接种的细胞的数量和病毒的用于转导的量。使用每6孔板（步骤3.2）150,000个细胞用vi的前述体积最佳结果看出RUS（步骤3.3和3.5），但更多的细胞可以在暴露于该病毒的细胞死亡的情况下使用。同样地，病毒的较小体积可用于应细胞死亡是一个问题，只要重新编程进如（可通过FACS分析，CFU测定法，以及基因分析选中）。确保病毒2.13-2.19产量适量所需的协议的其余部分的成功，步骤。步骤3.5-3.7也是至关重要的，细胞作为成功转导是诱发本hemogenic计划至关重要。每转导的细胞的孔的DOX的适当量必须保持一致，新鲜，以确保转基因（因此需要频繁更换培养基）的表达。尽管不是绝对必要，使用DOX当在黑暗中的细胞培养物可有助于防止DOX功能的丧失。转导后，细胞应密切通过形态学在显微镜下监测和通过各种分析方法（如FACS和CFU测定）测定以确保小号uccessful病毒转导和重新编程。如预期重编程的细胞可以不采取许多形态变化，要求前面提到的分析方法，作为适当重新编程的最佳指标。 最近的研究已在TF鸡尾酒或成纤维细胞作为起始细胞群体31-33利用这种重新编程协议的不同的功能，如GATA2。这些方法也出现在重编程过程中诱发发展计划。重新编程9,10,25,26中其他策略已示出的造血龛的重要性。确定所有的辅助重编程的因素将是发展产生轻易达到的特定患者细胞善意的造血干细胞的协议至关重要。总之，在这里我们描述了一个战略，以便产生通过诱导GATA2，Gfi1b，首席财务官，并从诱发发展过程HSC样细胞小鼠成纤维细胞ETV6表达。该技术将被转换为人体系统，其中，在与其他已发表的研究结合，将允许生成患者特异性血制品适用于各种临床应用。

造血是一个复杂的发育过程，其中的造血干细胞（HSCs）出芽存在于各种胚胎造血网站，如主动脉-性腺-中肾和胎盘1,2 hemogenic内皮。无法培养造血干细胞在体外阻止此过程的深入分析，以及这些研究的临床应用。规避此限制，先前的研究试图获得造血干细胞从头或者通过使用重新编程媒体4,5-多能干细胞（的PSCs）3，或在体细胞诱导的可塑性和定向分化的分化。这些研究，但是，不产生临床安全engraftable细胞或允许最终造血发育研究"的菜。" 从体细胞的成纤维细胞p由山及其同事建立的新颖工作以产生诱导性多能干细胞（iPS细胞）rovides转录因子（TF）在重编程的细胞命运6,7-基于过表达策略的框架。这项工作已经促使调查人员在若干领域通过容易获得体细胞重编程TF产生细胞类型的选择。这里所描述的重编程策略的目标是诱导从使用具有转换这些发现对人体系统重新编程特定病人的成纤维细胞，以研究人类造血体外的目标基于TF重新编程的方法和小鼠体细胞hemogenic过程生成患者特异性血制品用于疾病建模，药物检验，和干细胞移植。 第一步，以确保在该小鼠系统适当重新编程是开发担任的读出为CD34的表达，在内皮祖细胞和造血干细胞的已知标记的报道一致。要做到这一点，huCD34-和tTA的TETO-H2BGFP转基因小鼠系被用于与Generate双转基因小鼠胚胎成纤维细胞（MEF），现在表示34 / H2BGFP，发荧光绿于CD34启动子8的激活。这允许各种已知被要求在不同的点造血说明书和发育过程中的TF的筛选。在PMX retrovial载体18开始的TF（通过文献挖掘和GFP标签保留从之前造血干细胞的分析确定描述34 / H2BGFP只），34 / H2BGFP MEF中与AFT024 HSC-支持基质细胞的所有因素和培养转。检测34 / H2BGFP激活后，转录因子随后从重新编程的鸡尾酒删除，直到转录因子的活化记者一组最佳被确定。此初始屏幕之后，将因素转移到DOX诱导pFUW载体系统，以允许转录因子的控制的表达。由于这两个DOX可控系统是不兼容的（34 / H2BGFP细胞和pFUW诱导矢量），由MEF野生型C57BL / 6小鼠被要求。此外，还必须提供适当的微环境，使hemogenesis继续创造多向祖细胞克隆。 试图体细胞重编程到造血干细胞和祖细胞（骨髓干细胞）目前的研究已达到成功的9-11不同层次。迄今为止，小鼠和具有长期和自我更新的重新填充能力的人类移植骨髓干细胞的产生尚未使用同一组转录因子实现。在这个协议中，我们提供先前建立的策略的详细描述可重复地诱导hemogenesis在MEF中。我们证明，引进了全套转录因子（GATA2，Gfi1b，首席财务官和ETV6）的最小的能够煽动体外复杂的发展计划，规定了可发展的造血和造血重新编程的临床应用，可进一步研究12的平台。

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>0.45 &amp;mu;M filters</td><td>Corning</td><td>430625</td><td></td></tr><tr><td>Amicon Ultra centrifugal filters</td><td>Millipore</td><td>UFC900324</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>25030-081</td><td></td></tr><tr><td>FBS</td><td>Gemini&#039;s Benchmark</td><td>100-106</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>18 G needles</td><td>BD</td><td>305195</td><td></td></tr><tr><td>20 G needles</td><td>BD</td><td>305175</td><td></td></tr><tr><td>25 G needles</td><td>BD</td><td>305125</td><td></td></tr><tr><td>Collagenase Type I</td><td>Sigma</td><td>C0130-100MG</td><td></td></tr><tr><td>TrypLE Express</td><td>Invitrogen</td><td>12605-010</td><td></td></tr><tr><td>Myelocult media</td><td>Stem Cell Technologies</td><td>M5300</td><td></td></tr><tr><td>SCF</td><td>R &amp;amp; D Systems</td><td>455-MC</td><td></td></tr><tr><td>Flt3L</td><td>R &amp;amp; D Systems</td><td>427-FL</td><td></td></tr><tr><td>IL-3</td><td>R &amp;amp; D Systems</td><td>403-ML</td><td></td></tr><tr><td>IL-6</td><td>R &amp;amp; D Systems</td><td>406-ML</td><td></td></tr><tr><td>TPO</td><td>R &amp;amp; D Systems</td><td>488-TO</td><td></td></tr><tr><td>Doxycycline</td><td>Sigma</td><td>D9891-1G</td><td></td></tr><tr><td>Polybrene (hexadimethrine bromide)</td><td>Sigma</td><td>AL-118</td><td></td></tr><tr><td>Durapore 0.65 &amp;mu;M membrane filters</td><td>Millipore</td><td>DVPP14250</td><td></td></tr><tr><td>Methylcellulose media</td><td>Stem Cell Technologies</td><td>Methocult M3434</td><td></td></tr><tr><td>Hydrocortisone</td><td>Stem Cell Technologies</td><td>07904</td><td></td></tr><tr><td>C57BL/6 mice</td><td>The Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G-1890 100g</td><td></td></tr><tr><td>pFUW-tetO</td><td>Addgene</td><td>Plasmid #20321</td><td></td></tr><tr><td>Gata2</td><td>Origene</td><td>MR226728</td><td></td></tr><tr><td>Gfi1b</td><td>Origene</td><td>MR204861</td><td></td></tr><tr><td>cFos&amp;nbsp;</td><td>Addgene</td><td>Plasmid #19259</td><td></td></tr><tr><td>Etv6</td><td>Origene</td><td>MR207053</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>Plasmid #12260</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>Plasmid #12259</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5670-100g</td><td></td></tr><tr><td>FUW-M2rtTA</td><td>Addgene</td><td>Plasmid #20342</td><td></td></tr><tr><td>35 mm x 10 mm culture dishes</td><td>Thermo Scientific</td><td>171099</td><td></td></tr></tbody></table>

reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

造血是一个复杂的发展过程，在不同的胚胎网站开始。 Hemogenic内皮细胞存在于这些网站，并给通过手机初露头角的23引起造血干细胞。这个过程目前还无法通过将造血干细胞或造血前体文化，因此需要一种方法在体外以某种方式获得这些细胞进行复制，或者通过HSPC 体外扩增或产生从头 。该协议表明了我们试图通过获得TF表达，这些细胞在MEF中新技术。 图1示出了总体重编程过程。 MEF的产生和扩建后，细胞转导与GATA2，Gfi1b，首席财务官，ETV6和FUW-M2rtTA。后扩张和接触DOX开始转基因活化3天，将细胞解离，并在第4天分裂到明胶包被的平板和在补充造血介质。 在长期培养后转导重新编程的媒体，采用细胞形态学清晰的变化。在20天的细胞采用内皮细胞形态由MEF不同。进一步培养至35天的结果在几个回合的造血细胞样细胞来自内皮样中间体（使用34 / H2BGFP的MEF时）和染色阳性造血标记SCA1和CD45（ 图2），它们的 GFP +涌现。这种染色表明，这些细胞获得提示hemogenic感应的表型标记。进一步培养结果在表达CD45同时保持huCD34记者表达细胞。在胎盘聚合培养细胞通过克隆电位和生成含有各种造血形貌和爆炸样细胞（ 图3）中的甲基纤维素的菌落。 EP-together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/54372/54372fig1.jpg" /> 图1: 策略中的MEF hemogenic感应。示出的重编程过程，并在其中的每个步骤。一般重编程过程首先包括MEF膨胀，随后分裂在适当的密度到明胶包被的6孔板上。细胞与GATA2，Gfi1b，首席财务官，ETV6和FUW-M2rtTA的鸡尾酒转。细胞用补充有DOX标准DMEM中进一步培养。在第4天的细胞胰蛋白酶化并分成6孔板中。每6天介质被改变，并在所选择的时间点的细胞可以通过FACS，CFU，或者基因表达的测定法进行分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54372/54372fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 负载/ 54372 / 54372fig2.jpg"/&gt; 图2: 造血干细胞前体，从新兴的诱导。阳性细胞34 / H2BGFP染色SCA1和CD45表现出hemogenic程序的诱导。 （A）中该图像显示染色CD45和SCA1同时与重编程的细胞的潜在明图像荧光GFP细胞的合并。只有扁的细胞群的一个子集表示SCA1，内皮/ hemogenic标记，仅圆形造血细胞样细胞从这些细胞对于CD45，泛造血标记物染色红色出现。 （B）此图像显示染色和GFP荧光的细胞没有明图像，清楚地显示出与SCA1 +细胞的 CD45 +细胞的密切关联。比例尺=100μM的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54372/54372fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图3" src="/files/ftp_upload/54372/54372fig3.jpg" /> 图3:CD45 + 造血细胞 的生成 。 （A）约12.7％，与GATA2，Gfi1b，首席财务官和ETV6转34 / H2BGFP的MEF是GFP + CD45 +重编程后的35天。清髓性形貌和爆炸样细胞可以看出以下的CD45的细胞离心涂片+镀在甲基纤维素10-14天分选的细胞后的H＆E染色（B）。这表明，采用造血功能与这个最小集转录因子转导后重新编程细胞的克隆潜能研究。比例尺=100μM的。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54372/54372fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program at JoVE.com

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

重新编程的小鼠胚胎成纤维细胞转录因子诱导Hemogenic计划

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

reprogramming-mouse-embryonic-fibroblasts-with-transcription-factors

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.1K 次观看.  Icahn School of Medicine at Mount Sinai. The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

视频: Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

科研

发育生物学

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

促纤维化信号增强因子介导的小鼠胚胎成纤维细胞再编程诱导心肌细胞的抑制

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

重新用四个转录因子的小鼠胚胎成纤维细胞诱导多能干细胞的产生，强力霉素诱导慢病毒转导系统

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

生物学

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...