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. J., Weissman, S. M., Urban, A. E., Snyder, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+genetic+variation+in+somatic+human+tissues.">Extensive genetic variation in somatic human tissues.</a> Proc Natl Acad Sci U S A. 109, 18018-18023 (2012).</li><li>Abyzov, 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=Somatic+copy+number+mosaicism+in+human+skin+revealed+by+induced+pluripotent+stem+cells.">Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.</a> Nature. 492, 438-442 (2012).</li></ol>

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?...

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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic Mix:&#160;</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.&#160;</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:&#160;</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&#215; MEM non-essential amino acid</td>
			<td>Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.5&#215; Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;0.1 mM &#946;-mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;bFGF (10 ng/ml, added fresh)</td>
			<td>Millipore</td>
			<td>GF003</td>
			<td></td>
		</tr>
		<tr>
			<td>Keratinocyte medium:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;EpiLife with 60&#160;&#181;M Calcium</td>
			<td>Invitrogen</td>
			<td>M-EPI-500-CA</td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Human keratinocyte growth supplement (HKGS)</td>
			<td>Invitrogen</td>
			<td>S-001-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS) medium:&#160;</td>
			<td></td>
			<td></td>
			<td>FBS medium is filtered using Stericup (Millipore, #SCGPU05RE)&#160; before use.</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum (FBS)</td>
			<td>Invitrogen</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Invitrogen</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5&#215; 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&#160;</td>
			<td>354234</td>
			<td>Aliquot Matrigel stock and store in -80&#176;C following manufacturer&#8217;s instructions. Stock concentration of Matrigel varies slightly from batch to batch (~9mg/ml). We recommend to use 200&#181;l matrigel for coating a 12-well plate (~150&#181;g/well).&#160;</td>
		</tr>
		<tr>
			<td>Coating Matrix Kit&#160;</td>
			<td>Invitrogen</td>
			<td>R-011-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids:&#160;</td>
			<td></td>
			<td></td>
			<td rowspan="5">Note that pCXLE-eGFP is only used for monitoring transfection efficiency and is not required for reprogramming.</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-eGFP</td>
			<td>Addgene</td>
			<td>27082</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hOct3/4-shP53F</td>
			<td>Addgene</td>
			<td>27077</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hSK</td>
			<td>Addgene</td>
			<td>27078</td>
		</tr>
		<tr>
			<td>-&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; pCXLE-hUL</td>
			<td>Addgene</td>
			<td>27080</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.&#160;</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 10mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Invitrogen</td>
			<td>17104-019</td>
			<td>Use at 1mg/ml</td>
		</tr>
		<tr>
			<td>TRA-160 antibody</td>
			<td>Millipore</td>
			<td>MAB4360</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>OCT4 antibody</td>
			<td>Santa Cruz</td>
			<td>SC-5279</td>
			<td>Use at 5&#181;g/ml</td>
		</tr>
		<tr>
			<td>NANOG antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF1997</td>
			<td>Use at 10&#181;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。 <p cl...

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

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 Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

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 ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

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) ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人胚胎或诱导多能干细胞对视网膜色素上皮细胞的快速定向分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

人诱导多潜能干细胞衍生肝细胞的药物发现方法

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...