Name: generation of integration-free human induced pluripotent stem cells using hair-derived keratinocytes
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Description: Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

The authors wish to thank Harene Ranjithakumaran and Stacey Jackson for technical support. This work was supported in part by grants from the National Health and Medical Research Council (R.C.B. Wong, A. P&#233;bay), the University of Melbourne (R.C.B. Wong), Retina Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay) and the Ophthalmic Research Institute of Australia (R.C.B. Wong, S.S.C. Hung, A. P&#233;bay); Australian Research Council Future Fellowship (A. P&#233;bay, FT140100047), Cranbourne Foundation Fellowship (R.C.B. Wong); intramural funding from the National Institutes for Health (R.C.B. Wong, S.S.C. Hung) and operational infrastructure support from the Victorian Government.

从个人人类头发样品的集合需要通过人的研究伦理委员会在宿主机构伦理委员会批准，并应符合进行与机构准则。 从弹拨头发1.隔离的角质形成细胞<ol><li>解冻细胞外基质（ECM）解决方案（ 即基质胶）在冰O / N。 </li><li>用预冷的枪头，加入200微升的ECM溶液到12毫升冷冻的DMEM / F12培养基。外套一个12孔板用1ml稀释的ECM溶液到每孔中。孵育涂板O / N在37℃。在使用前预热DMEM / F12培养基洗涤包被的平板。 </li><li>通过将手指靠近发根和拔除毛发在一个快速和平滑的运动收集来自个人的头的原代人的头发。检查以确认每个收集的头发中含有一个完整的毛囊并确定联合国的头发的生长阶段明镜解剖显微镜（ 图1）。 <ol><li>收集5-10生长期的毛发从各个提取角质。过程弹拨毛发尽快确保角化细胞的成功的推导。 注意: 图1示出在生长周期的不同阶段的毛发的形态。毛发生长初期是在生长阶段，上皮周围的头发的长度，称为ORS的可见外层。另一方面，休止期头发代表头发在休止期和不具有可见的ORS，但有一个明确的细胞覆盖的毛发的根，被称为休止凸起的球。 </li></ol></li><li>将头发样品到含有10ml抗生素混合料在室温洗5分钟的培养皿。 </li><li>用消毒剪刀和镊子，剪除过多的毛干，留下了头发的片段用一个完整的毛囊和周围0.5 - 毛干为1厘米。 </li><li>仔细TRAnsfer 1发片段插入使用镊子将ECM涂覆的板的各孔中。 </li><li>使用1毫升吸管，加入约3滴敲除血清替代品（KSR）培养基（〜100 - 200微升）到头发样品。 注意:保持头发的片段密切接触板面，以允许附件和随后的角质细胞生长。加入过量的介质，在该步骤中可以使头发片段浮起并减小成功头发附着的机会。 </li><li>孵育头发样品在37℃的培养箱中5％的CO 2，并允许毛囊附着O / N。第二天，轻轻再添3滴KSR介质（100〜 - 200微升），以保持头发湿润的样品。 </li><li>观察该头发样本每日以确认毛囊已经成功附着。通常附着毛囊内1-3天明显。厚厚的毛发通常更容易附着相比细毛，因为细毛有较高倾向浮起阻碍连接过程。 <ol><li>一旦头发已经附着到涂板，加入500μl的KSR培养基到孔中。添加媒体时头发样品可能没有牢固地附着要格外小心。从这一点来说，改变媒体每2天。 注:后3-7天（ 图2A-B）副产物角质应该可以看到从发片段的ORS区域。让角质形成细胞在下一节介绍的传代细胞前长到14天。 </li></ol></li></ol> 2.维护和角质形成细胞传代<ol><li>预涂层用涂料矩阵包一个6孔板。添加680微升稀释介质的从所述试剂盒向每个孔，接着6.8微升涂布基质溶液。摇板，以确保在平板上涂料基液的均匀分布。 </li><li>允许涂覆的板孵育30分钟，在室温。拆除之前的角质形成细胞的电镀镀层Matrix解决方案。 </li><li>离解的角质形成细胞为单细胞悬浮液，用500μL每孔0.25％胰蛋白酶的为2-5分钟，在37℃。灭活用500μl含培养基的血清（ 例如，胎牛血清（FBS）的培养基）的胰蛋白酶，并收集到无菌的15毫升管。删除使用消毒镊子头发的片段。 </li><li>确定使用血球收获的细胞的数目。 </li><li>在200离心细胞3分钟×g下。洗涤细胞，用PBS和重复离心。吸出PBS，重悬细胞沉淀在新鲜的角质形成细胞培养基。 </li><li>板向下4×10 4的角质形成细胞进入ECM涂覆后的板在角质形成细胞培养基的存在下的一个阱。保持角化细胞在37℃培养箱中，用5％的CO 2（ 图2C）。改变媒体每天都和传代细胞时，文化是〜80％汇合，WHI通道可能需要约2-7天，取决于角化细胞的传代次数和细胞增殖率。派生角质应该扩大使用标准慢速冷冻方法冷冻保存16产生足够的冷冻库存。 注意:所述的培养条件下支持角化细胞具有典型的鹅卵石形态（ 图2C）的同源性的群体的膨胀。偶尔，一些差异化的角质形成细胞，具有大而扁平的形态也失去复制能力，可以在培养中观察到。角化细胞的表征可以通过免疫染色采用抗细胞角蛋白14来执行，如前所述17。支原体后使用市售的试剂盒1-3通道建立角化细胞系也应进行测试。 </li></ol> 3.从一代人iPS细胞角质形成细胞利用游离型载体<ol><li>预涂层的6孔板0.1％明胶（2毫升/孔）。允许明胶涂覆至少20分钟，在室温。 </li><li>对于重编程的实验中，使用角质形成不迟于通道6离解角质成单细胞悬浮液与1ml每孔0.25％胰蛋白酶为2-5分钟，在37℃。灭活胰蛋白酶用1毫升含有培养基的血清（ 例如，胎牛血清介质）的，并收集在一个15毫升管。 </li><li>离心细胞3分钟，在200×g离心和重悬细胞的角质形成细胞培养基中。每孔板1×10 5 -1.5×10 5角朊细胞的糊化板O / N。 </li><li>于次日（第0天），检查板，以保证细胞50-60％汇合。 注意:如果角质小于〜50％汇合，他们转染后也可能不会增长。 </li><li>在第0天，执行对角化细胞的游离型重编程载体的转染，使用8微升转染试剂的比率为2微克总质粒DNA。<ol><li>以0.5微克的每个质粒4（pCXLE-EGFP，pCXLE-hOct3 / 4-shP53F，pCXLE-HSK，pCXLE-讫）和稀释的100微升减少血清培养基（ 如，OPTI-MEM）的。 注意:质粒pCXLE-eGFP的用于监视在角化细胞的转染效率和不需要成功重新编程。 </li><li>添加8微升转染试剂来稀释的DNA。轻弹管混合。允许该DNA反应混合物休息原状在室温15分钟。 </li><li>更换角化细胞培养基用新鲜培养基。 </li><li>轻轻的DNA反应混合物用5％的CO 2添加到角质细胞培养和孵化在37℃培养箱4小时。 注意:不建议在执行角质形成细胞转染延长O / N，因为他们表现出低生存染后。 </li><li> 4小时后转染后，更换角化细胞培养基用新鲜培养基。 </li></ol></li><li>在第1天（第1天后反染），改变角化细胞培养基中并通过估计在荧光显微镜下的eGFP阳性细胞百分比检查转染效率。通常&gt; 60％-70％的转染效率观察使用该协议的主要角质形成细胞。 </li><li>第2天，按步骤3.5节重复转染。连续两次转染通常会达到&gt; 90％的转染效率。角质形成细胞培养通常是由第二次转染的端部到达汇合。 <ol><li>制备小鼠胚胎成纤维细胞（MEF）的送料器的10cm皿的每个重编程反应。准备有丝分裂失活的MEF饲养如前所述18。 </li><li>预涂层10cm皿用5ml 0.1％明胶的至少20分钟。板4×10 6％的FBS媒体10 cm培养皿有丝分裂灭活的MEF。使细胞沉降并附着O / N在37℃培养箱，用5％的CO 2。 </li></ol></li><li>在第3天，哈尔如在步骤3.2和3.3中所述归属用0.25％胰蛋白酶的角质形成细胞。重悬在KSR培养基中的角质形成细胞和板90％，从1以及一个6孔板的角质形成细胞的成10cm的MEF饲养皿KSR培养基。 </li><li>从第4天起，每天换了KSR媒介。 <ol><li>或者18天之后，更换培养基每隔一天。 注:非重编程角化细胞将停止增殖，同时具有限定边界，类似于人类胚胎干细胞（胚胎干细胞）hiPSC菌落会从第14天 - 21。 </li></ol></li><li>从21日起隔离hiPSC殖民地人工清扫。避免hiPSC殖民地，它们紧密地聚集在一起，只能挑hiPSC殖民地有明确从别人分开。 注:hiPSC殖民地可以采摘较早，但与定义的边界殖民地可能不是很明显，在较早的时间点给小菌落大小。重编程效率差异可能会有所不同erent病人的角质形成细胞，并且还通过其他因素如通道数或角质形成细胞的增殖率的影响。 <ol><li>对于hiPSC殖民地人工清扫，使用26G针头切解剖显微镜下hiPSC殖民地。切hiPSC殖民地到约300小块 - 600微米长。转让从一个殖民地hiPSC件到一个新的MEF饲养板（2.5×10 4 MEF /厘米2）建立iPS细胞的克隆系。最初，文化的12孔板或器官培养的菜肴之一以及每个hiPSC克隆行，然后扩展到6孔板格式。 </li><li>维持iPS细胞在KSR介质上MEF饲养建立无性系。一旦培养达到70％ - 80％的汇合与hiPSC菌落〜1.5mm的直径，使用根据制造商的说明标准的酶传代方法（的Accutase，分散酶或胶原酶IV）的通道人iPS细胞。 </li></ol></li><li>建立特征线hiPSCs到确认多能性及其分化成细胞代表在体外和/或体内 13,19三种胚层的能力。此外，使用市售的试剂盒定期测试建立hiPSC线支原体，以确保它们是无病原体。定期监测的hiPSC线通过核型分析和基于阵列的拷贝数变异（CNV）分析基因组稳定性。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+point+of+large-scale+collections+of+human+induced+pluripotent+stem+cells.">What is the point of large-scale collections of human induced pluripotent stem cells.</a> Nat Biotechnol. 31, 875-877 (2013).</li><li>Utikal, 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=Immortalization+eliminates+a+roadblock+during+cellular+reprogramming+into+iPS+cells.">Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.</a> Nature. 460, U1145-U1112 (2009).</li><li>Xu, 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=Proliferation+rate+of+somatic+cells+affects+reprogramming+efficiency.">Proliferation rate of somatic cells affects reprogramming efficiency.</a> J Biol Chem. 288, 9767-9778 (2013).</li><li>Liu, 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=Late+passage+human+fibroblasts+induced+to+pluripotency+are+capable+of+directed+neuronal+differentiation.">Late passage human fibroblasts induced to pluripotency are capable of directed neuronal differentiation.</a> Cell Transplant. 20, 193-203 (2011).</li><li>Huallachain, M., Karczewski, K. 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The authors have no conflict of interest.

患者特异性人iPS细胞的产生提供了用于在体外研究发病在患病细胞类型的独特方法，并且还提供了用于药物筛选平台，以确定新的分子，可以拯救疾病表型。使用人iPS细胞这种疾病建模方法已取得了令人鼓舞的结果，适用于各种疾病，包括长QT综合征，亨廷顿氏病，帕金森氏病和肌萎缩性侧索硬化22。若干举措已经展开，建立以病人特异性iPS细胞，其中包括在美国，欧洲，澳大利亚，中国，韩国和日本财团23,24大型图书馆。 在这里，一个协议从头发角质形成细胞衍生的iPS细胞建立描述，它具有方便和快速轨道建立疾病特异性iPS细胞的大规模库的潜力。该协议提供了两个好处:第一，游离系统在本协议中使用的生成使用六重编程因子鸡尾酒集成无iPS细胞（OCT4，SOX2，KLF4，L-MYC，LIN28，为的shRNA P53）在相对 ​​高效率的4。相比于逆转录病毒或慢病毒介导的重编程方法，利用附加型载体的重新编程避免在国外转基因的基因组不可取的整合，因此，许多举措有利于利用集成的无重编程的方法建立大型hiPSC库，如附加型载体，仙台病毒或mRNA，综合以上方法重新编程23。 其次，头发是方便，可以由患者无需使用侵入性程序或医务人员的出勤收获自己。这为来自不同地区的患者自己的头发样本中一个独特的机会，收集和邮件实验室进行角质推导和随后的重新编程。为支持这一战略的可行性，我们未公布的数据表明，角质形成细胞可以成功地从保存在4℃至10天的弹拨头发隔离。 这里所描述的方法将允许角质细胞从弹拨头发推导。虽然角质形成细胞可以从只是一个单一的头发得到，我们的建议是收集至少5生长期的毛发的角质细胞的推导。此协议的一个限制是某些毛发可能不附加井和角化细胞可能无法生长。较厚的头发往往附着比较好，而汗毛需要电镀，以避免浮动，增强附着在培养液量减少。一旦角化细胞衍生，最好扩大并使用标准的慢速冷冻冷冻保存方法16冻结向下股票。随后的角质形成细胞重编程，可以在执行这个协议描述的以下步骤。重要的是要注意，起始细胞的增殖速率可能影响重编程效率，在晚期传代以降低重编程效率观察细胞达到细胞衰老25-27。在这方面，使用角质形成不迟于通道6用于重编程的实验。另外，重新编程的效率可能不同个体的角质细胞之间变化。 最近的研究表明，在多种类型的体组织28的广泛的遗传嵌合体，以〜报道具有躯体的CNV在基因组中29皮肤成纤维细胞的30％。这些拷贝数变异可以通过在DNA复制，DNA修复或转座子动员错误引起的。它也有可能是长时间的紫外线照射到皮肤可能会导致在表皮细胞，包括成纤维细胞和角化细胞的其他拷贝数变异，但这种程度还不是很清楚。如角质形成细胞的CNV将在重编程过程中进行过，它是即时portant来执行例行核型或CNV分析，以确保所建立的人iPS细胞维持基因组稳定性。总之，我们在这里所描述的方法用于产生人iPS细胞的自发衍生的角质形成细胞，其可用于疾病建模和再生医学。

人类诱导多能干细胞（人iPS细胞）的发现已经彻底改变了再生医学的领域中，提供用于产生患者特异性干细胞1-3的一种可行的方法。人iPS细胞已经成功地从不同的体细胞类型，包括成纤维细胞4,5，造血细胞6,7，从尿8肾小管上皮细胞和角质形成细胞9,10产生的。迄今为止，皮肤成纤维细胞和造血细胞代表了最常用的细胞来源，用于产生病人的具体的iPSC。可以说，这是由于这样的事实，即皮肤活检和采血是常规的医疗程序，并已在许多国家建立大型生物库患者血液或皮肤的样品。 相较于血细胞和皮肤成纤维需要侵入性的提取方法，角质代表一个方便的细胞类型的hiPSC产生。 KeratinocytES是形成皮肤的外表皮屏障，并且还发现，在指甲和头发角蛋白11的富上皮细胞。特别是，角朊细胞可以在毛囊，即覆盖毛干连同内根鞘（IRS）的细胞（12， 图1）的外部的蜂窝层的外根鞘（ORS）中找到。由于毛发收集是一个简单的过程，不需要医务人员的协助下，它提供了一个机会，让患者收集并发送自己的头发样品的实验室，这将极大地方便了hiPSC代病人样本的采集。表皮角化细胞也有较高的重编程效率和更快的重编程动力学相比的成纤维细胞，加入到用角质形成细胞作为起始细胞hiPSC代9,13的优点。此外，人iPS细胞还可以使用毛囊内其他细胞群产生，包括位于毛囊14,15的底部的毛乳头细胞。 IPSC的代发使用来源的细胞以前的报告中经常使用逆转录病毒或慢病毒为基础的重编程方法9,14,15。然而，这些病毒方法的重新编程过程中引进国外转基因不良基因组整合。相比较而言，使用游离型载体的代表一个可行的，非病毒重新编程方法来生成积分-自由iPSCs的4。我们以前曾开发出一种简单，经济有效和非病毒的方法来有效地重新编程角化细胞到使用附加型载体13人iPS细胞。在这里，我们提供的角质细胞衍生的人iPS细胞，包括来自弹拨头发，扩展和维护的角质形成和随后的重新编程，以产生iPS细胞的角质形成细胞的推导的生成的详细协议。

<table><tbody><tr><td>Antibiotic Mix:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>250 ng/ml Antimycotic amphotericin B</td><td>Sigma</td><td>A2942-20ml</td><td>Antibiotic mix is made up in PBS.&amp;nbsp;</td></tr><tr><td>1X Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>PBS (-)</td><td>Invitrogen</td><td>14190-144</td><td></td></tr><tr><td>Knockout Serum Replacement (KSR) medium:&amp;nbsp;</td><td></td><td></td><td>KSR medium is filtered using Stericup (Millipore, #SCGPU05RE) before use. bFGF is added fresh to the media before use.</td></tr><tr><td>20% knockout serum replacement (KSR)</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>DMEM/F12 with glutamax</td><td>Invitrogen</td><td>10565-042</td><td></td></tr><tr><td>1&amp;times; MEM non-essential amino acid</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>&amp;nbsp;0.5&amp;times; Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;nbsp;0.1 mM &amp;beta;-mercaptoethanol</td><td>Invitrogen</td><td>21985</td><td></td></tr><tr><td>&amp;nbsp;bFGF (10 ng/ml, added fresh)</td><td>Millipore</td><td>GF003</td><td></td></tr><tr><td>Keratinocyte medium:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;EpiLife with 60&amp;nbsp;&amp;micro;M Calcium</td><td>Invitrogen</td><td>M-EPI-500-CA</td><td></td></tr><tr><td>1&amp;times; Human keratinocyte growth supplement (HKGS)</td><td>Invitrogen</td><td>S-001-5</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS) medium:&amp;nbsp;</td><td></td><td></td><td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE) before use.</td></tr><tr><td>10% fetal bovine serum (FBS)</td><td>Invitrogen</td><td>26140079</td><td></td></tr><tr><td>DMEM&amp;nbsp;</td><td>Invitrogen</td><td>11995-073</td><td></td></tr><tr><td>0.5x Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>2 mM L-glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>0.25% trypsin-EDTA</td><td>Invitrogen</td><td>25200-056</td><td></td></tr><tr><td>Extracellular Matrix (ECM):</td><td></td><td></td><td></td></tr><tr><td>Matrigel</td><td>Corning&amp;nbsp;</td><td>354234</td><td>Aliquot Matrigel stock and store in -80&amp;deg;C following manufacturer&amp;rsquo;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&amp;micro;l matrigel for coating a 12-well plate (~150&amp;micro;g/well).&amp;nbsp;</td></tr><tr><td>Coating Matrix Kit&amp;nbsp;</td><td>Invitrogen</td><td>R-011-K</td><td></td></tr><tr><td>Plasmids:&amp;nbsp;</td><td></td><td></td><td>Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-eGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hOct3/4-shP53F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>-&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>Transfection reagent Fugene HD</td><td>Promega</td><td>E231B</td><td></td></tr><tr><td>Gelatin (from porcine skin)</td><td>Sigma</td><td>G1890</td><td>Make up 0.1% gelatin in distilled water. Autoclave before use.&amp;nbsp;</td></tr><tr><td>Reduced Serum medium: OPTI-MEM</td><td>Invitrogen</td><td>31985062</td><td></td></tr><tr><td>Accutase</td><td>Sigma</td><td>A6964-100ml</td><td></td></tr><tr><td>Mouse embryonic fibroblast (MEF) feeder</td><td></td><td></td><td>MEF can be inactivated by mitomycin C treatment or irradiation as described previously 16.</td></tr><tr><td>26G needle</td><td>Terumo</td><td>NN2613R</td><td></td></tr><tr><td>6-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>12-well plate (tissue culture treated)</td><td>BD Biosciences</td><td>353043</td><td></td></tr><tr><td>10 cm dish (tissue culture treated)</td><td>BD Biosciences</td><td>353003</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td>Use at 10 mg/ml</td></tr><tr><td>Collagenase IV</td><td>Invitrogen</td><td>17104-019</td><td>Use at 1 mg/ml</td></tr><tr><td>TRA-160 antibody</td><td>Millipore</td><td>MAB4360</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>OCT4 antibody</td><td>Santa Cruz</td><td>SC-5279</td><td>Use at 5 &amp;micro;g/ml</td></tr><tr><td>NANOG antibody</td><td>R&amp;amp;D Systems</td><td>AF1997</td><td>Use at 10 &amp;micro;g/ml</td></tr><tr><td>MycoAlert Detection kit</td><td>Lonza</td><td>LT07-418</td><td></td></tr></tbody></table>

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.

毛发经过生长周期3个不同阶段:生长期（生长阶段），退行期（回归阶段）和休止期（休息阶段）20,21。生长期毛囊中含有上皮多层;这些层包括ORS，IRS和毛干（ 图1）。毛发生长初期最终经历过渡到退行期阶段，该阶段的特点是ORS的和终止的毛干分化的细胞凋亡。最后，退行期的头发过渡到休止期，在细胞凋亡停止，休止期毛囊变得与静态特征休止期隆起20,21。 图1示出了休止期头发和毛发生长初期的形态。我们通常利用生长期的头发角质细胞推导。按照此协议，角质细胞副产物可以观察到头发附着（ 图2A）以后，早在3天，将继续为proliferate（ 图2B）。根据我们的经验，一些生长期的毛发可能无法连接或不遵守角质细胞生长;因此，收集至少5 - 10从各个生长期的毛发，以确保成功角质细胞的隔离。随后，将角质细胞可传代到新的板，并保持多个通道（ 图2C）。要注意，如在第2描述的，相对于基质胶与KSR培养基上有一个涂层矩阵的角化细胞培养基角化细胞的更好的成长是很重要的。 扩建之后，角质形成细胞可以被重新编程为32天后重新编程的第3 图3A描述显示了一个典型的角质细胞衍生的hiPSC殖民地生成iPS细胞。这是常见的观察一些分化在hiPSC殖民地的中心。一旦人工采摘，派生的iPS细胞通常显示出高核细胞质比率和规定菌落绑定元（ 图3B）。人iPS细胞的建立的细胞系可以接着如先前13,19所述，如多能标记物的OCT4，NANOG和TRA-160（ 图3C-E）的表达进行表征为多能性。 <img alt="图1" src="/files/ftp_upload/53174/53174fig1.jpg" /> 图1.在不同的生长阶段弹拨的毛发代表图像。（A）表示在生长期或休止期的毛发。相位对比图像，显示在（B）的休止期和（C）的生长期一个拨弦头发。 HS =毛干; IRS =内根鞘; ORS =外毛根鞘。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53174/53174fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 的弹拨头发人物头发衍生的角质形成细胞2.代表性图像。相位对比图像镀下来（A）中3天，（B）的10天。白色箭头标记角质形成细胞从弹拨头发生长。第3天角质形成细胞与传代后，一个典型的鹅卵石形态（C）相衬图像。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53174/53174fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53174/53174fig3.jpg" /> 图的角质细胞衍生的iPS细胞代表性的图像。一个32天重编程的文化呈现出角质细胞衍生的hiPSC共同的（A）相衬图像罗尼。 （ 二）手动选择角质细胞衍生的iPS细胞具有形态类似于胚胎干细胞。 iPS细胞多能性与标志（C）NANOG和（D）OCT4和免疫（E）的角质细胞衍生的iPS细胞TRA-160。比例尺= 100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53174/53174fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes at JoVE.com

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

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 ...

generation-integration-free-human-induced-pluripotent-stem-cells

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.4K 次观看.  University of Melbourne. 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. 

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

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

科研

发育生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

从人类毛囊神经嵴干细胞的分离和培养的

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

神经科学

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

成人皮肤角质形成细胞的生成与培养

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

成人人成纤维细胞的推导和他们直接转换成可扩展的神经祖细胞

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

免费接驳适应，文化和传代使用完整的基因敲除血清替代馈线中的人类iPS细胞

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

生物学

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Cryopreserving和恢复人类iPS细胞血清替代馈线中使用完整的基因敲除

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

使用非整合方法分离成人人体皮肤纤维细胞与诱导多能干细胞的生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...