We would like to thank Patrick C. Lee, Cyril Ramathal, and Saravanan Karumbayaram (SK) for their assistance in performing the iPSC derivation and characterization experiments; Aaron Cooper for performing the iPSC analysis experiments; Vittorio Sebastiano and Renee A. Reijo Pera for directing the initial reprogramming efforts; SK, William E. Lowry, Jerome A. Zack, and Donald B. Kohn for directing the establishment of the UCLA GMP facilities permitting the conversion and characterization of clinical-grade iPSCs; Gustavo Mostoslavsky for providing us with the STEMCCA polycistronic reprogramming vector. This work is based on a research collaboration with Fibrocell Science and the Clinical Investigations for Dermal Mesenchymally Obtained Derivatives (CIDMOD) Initiative to generate safe personalized cellular therapeutics. This work was supported by funding from the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, The Phelps Family Foundation, Fibrocell Science, Inc., and the UCLA CTSI Scholar&#8217;s Award to JAB.

注:此方法是在报道敬畏等[10]研究使用。 1.重新编程人类成人皮肤成纤维细胞与STEMCCA <ol><li>解冻成人皮肤人成纤维细胞（HUFs），通道4或更低，在37℃水浴中2分钟，小心不要淹没与水的小瓶的顶部。 </li><li>放置1毫升浆液人成纤维细胞的成一个15毫升的锥形瓶中，并慢慢地放置4毫升标准福林媒体，预热到37℃的，在一个逐滴的方式，以稀释了二甲亚砜（DMSO）和重新悬浮细胞。 </li><li>离心小瓶，在200×g离心，在室温下10分钟。吸出上清液并重新悬浮在媒体的适当体积的细胞创造100000个细胞/ ml的悬浮液，然后加2ml成6孔板的一个孔中，预涂布20分钟用0.2％明胶。 注:妹妹以及与同等数量的每行条件的单元格都需要接种FOr中细胞计数/感染（MOI）计算的多重性。 </li><li>岩石板侧到另一边，并来回均匀地分布在细胞。在湿润的培养箱中孵育过夜，在37℃，5％的CO 2。 </li><li>对转导的当天，获得来自姐姐细胞计数（多个）以及（多个）初始福林电镀后约24小时。 </li><li>解冻2×10 8 TU，或等效，慢病毒浓缩物（STEMCCA）并稀释至1×10 8 TU / ml，在福林介质。 </li><li>转导的细胞在福林媒体10惯性矩比值补充有8微克/毫升的转染试剂。均匀混合导孔孵育过夜，在37℃，5％CO 2的加湿培养箱。 </li><li>慢病毒转导后约24小时，切换HUF媒体标准的人多能干细胞（PSC）中。 </li><li>天2到6，与人类PSC中每天都在变化的媒体。 </li><li>在第6天，板2 0.2％明胶包被的10厘米的板1.5〜1.75×10 6的MEF中HUF媒体12源自CF1小鼠。 </li><li>第7天，小心不要离心超过100 XG，利用室温胰蛋白酶升空的第7天的成纤维细胞重新编程，从6孔板和通道之一，同时在1:16的比例，由电镀所有的细胞均匀之间两个10 CM平板，用新鲜人PSC媒体说，前一天被镀的MEF。分发细胞聚集在顺时针螺旋移动并将其放置在37℃，5％CO 2的加湿培养箱中过夜。 </li><li>此后，大约播种重新编程的细胞上的MEF，每天48小时后，改变了人类花了PS​​C媒体与新鲜培养基。 </li><li> 3至4周后，挑单个菌落与典型ESC样形态用21号针头和亚克隆出到24孔板放置约8至10个单独的块特定菌落到各个孔中的0.2％明胶包被的24孔板预种子ED与CF1小鼠在人类PSC中派生的MEF。 注:扩张拾取菌落后，将菌落必须被表征为多能干通过胚状体的形成和多能性标记物表达的分析在蛋白质水平。 </li></ol> 2.矢量整合位点分析和STEMCCA切除<ol><li>如所述10分析STEMCCA整合位点由非限制性的线性扩增的PCR。 </li><li>后测定，以确保1整合到基因组中，附加STEMCCA的期望区域由抽吸掉旧人类ESC培养基中。然后，结合45微升浓腺Cre的PuroR病毒8微克/毫升的转染剂在3ml标准PSC介质24小时。 </li><li>在24小时的温育期后，吸脱混合的病毒上清和与人类的PSC介质洗细胞两次。放置连同2微克/毫升嘌呤霉素的新鲜人类PSC媒体，为期5天，改变媒体每日机智ħ新鲜的媒体和抗生素。 </li><li> 5天之后，剩余的亚克隆用21号针头的菌落到0.2％明胶包被的孔，在一个12孔板预先涂覆有从CF1mice衍生的MEF和扩大如上详述。 注:在足够的膨胀，以获得冷冻的储存，转化成无饲养条件是必需的。期望至少95％的干细胞集落的死亡，因为大多数的细胞将不会成功地转导，并会屈从于抗生素以上孵化5天。尽管腺Cre的PuroR集成在0.001-1％的效率进被感染细胞的宿主染色体，reexposing的因子的无菌落的样品，以嘌呤霉素保证100％的细胞死亡会确认没有整合发生13。 </li><li>解冻预先等分基底膜基质的一种小瓶在冰上，历时约2小时（制造商的建议），直至基质是液体和稀释出1:30在冷基础培养基的最终浓度。有限公司在孔中的六孔板中，每孔加入1ml期望数目。让坐在在室温下1小时。 </li><li>交叉影线（使用18号针，或玻璃或塑料"尖"），至少20至30的菌落从先前井涂上的MEF。要注意减少MEF振奋和板块转移。 <ol><li>通过一些不同的方法产生的交叉影线装置中，从更复杂的加热，拉，和精加工的玻璃巴斯德吸管的一个开放的火焰，在无菌塑料移液管尖的简单的使用或如在我们的情况下， 21-和/或18-号针头。 </li></ol></li><li>从涂好后孵化吸矩阵。 </li><li>通过小心刮擦从旧板卸下菌落件用200微升移液器并小心放置于3ml新鲜组合介质1在基质被覆的板构成。在37℃，5％CO 2的湿润培养箱孵育过夜。改变媒体48小时后传代。 </li><li>提取基因组DNA，从后切出，并无饲养转换人iPS细胞，在超过80％汇合，使用商用DNA提取试剂盒。 </li><li>适当切除与特异于STEMCCA慢病毒的外源整合的引物进行分析:基因组DNA-hendo-MycS进，5&#39;- acgagcacaagctcacctct-3&#39;;的gDNA-hWPRE反向，5&#39;-tcagcaaacacagtgcacacc-3&#39;。重构冻干的引物用PCR级水，以10μM的原液。 </li><li>运行的PCR与下列五个步骤的协议:初始变性95℃，3分钟; 35个循环以下每个的:变性在98℃持续20秒，引物退火在62℃持续15秒，并在72℃，15秒延伸;接着在72℃的单一周期最终延伸3分钟。 </li><li>通过称出1.5克琼脂糖并放置到50ml 1×Tris-醋酸-EDTA缓冲液使用3％的琼脂糖凝胶。微波直到琼脂糖溶解和发生在DNA凝胶染色为p罗珀稀释，混合，分配适当数量成卡式胶固化1小时，在室温下。 </li><li>负载12微升的PCR产物混合，用3微升样染料的成3％的琼脂糖凝胶。运行凝胶30分钟，在80 V. </li><li>可视化用紫外线光对缺少的频带的指示合理切除。 </li></ol>由前及后切除人iPS细胞使用定量PCR定量3.对基因表达的差异<ol><li>通过使用商业RNA提取试剂盒提取后切除和无饲养人iPS细胞转化的总RNA。 </li><li>反向转录高达1微克的总RNA通过使用商品化试剂盒按照制造商的说明进行操作。使用锚-寡（dT）18和随机六聚体引物。 注:确保PCR协议是专为超过或少于4 KB基因转录。将需要的任何基因STEMCCA原本整合我要作出特异性引物n要。 </li><li>重构冻干引物利用PCR级水至20μM的储备溶液。 </li><li>使用5纳克样品每20微升反应的，它包括10微米UPL探针，2倍的LightCycler 480探针万事达，和20μM的正向和反向引物。 </li></ol> 4.转换到GMP级条件<ol><li>超过到GMP级的条件下转换后切除人iPS细胞的第一天，做一个融合了媒体人组成的联合PSC媒体1（联合媒体1）和临床级良好生产规范（GMP）标准的媒体（联合媒体2 ）以80:20的比例。 </li><li>抽吸掉旧合并介质1和放置3毫升新合并的介质1和2中，预热的，在80:20比率。每日与此特定比率改变媒体3天。位置细胞放回37℃，5％CO 2的培养箱中培养。 </li><li>第4天，使合并的介质1和2，在50:50的比率。吸过旧媒体和replace为预热介质，与组合介质1和2，在50:50的比率。与此特定比率改变媒体3天。位置细胞放回37℃，5％CO 2的培养箱中培养。 </li><li>第7天，使该组合介质1和2，在20:80比率。抽吸掉旧的介质，并与预热的介质替换，与组合介质1和2的20:80的比例。与此特定比率改变媒体3天。位置细胞放回37℃，5％CO 2的培养箱中培养。 </li><li>在第10天，使合并的介质1和2在0:100的比例。抽吸掉旧的介质，并用预温热培养基替换，与组合介质1和2在0:100的比例。每天都在这个特定的比例发生变化的媒体。电池现在处于完全确定无异物媒体。位置细胞放回37℃，5％CO 2的培养箱中培养。 注:在这整个转换过程中，常规传代与18号针头是需要保持适当的群体大小和密度。在P计划菌落大小，汇合和分化的基础上assaging细胞每4至5天。 </li><li>外套1井在一6孔板具有限定无异物衬底作为制造商推荐的。 </li><li>机械通过一个融合的很好，已经转化为无异物介质条件下，从6孔板与18号针头。重要的是，把新的媒体（组合介质2）上的细胞用1×ROCK抑制剂1小时，传代到预调节介质之前。 </li><li>吸出合成底物溶液脱新印版，该细胞被转移到。删除所有包含从旧板的干细胞团块媒体和转移到新的板块。 </li><li>位置细胞放回37℃，5％CO 2培养箱中培养2天最小物理干扰（理想的是板，一旦进入培养箱，不直接触及以任何方式），以允许最大附件。 注:电池需要每天更换介质。持续passagin克每4天将是非常重要的。使用21号针头，具有最佳ESC样形态（高核质比）的菌落通道仅部分。这将确保选择正确hiPSC殖民地，将生长良好，并最终产生均匀的殖民地。总时间ESC推导像殖民地是15-20天之间的初始综合媒体更改后。 </li><li>由第一交叉线冻结向下GMP级别后切除人iPS细胞的所有菌落通过使用18号针头。升空所有部分用200μl的移液管，并在4℃下5分钟，含有该细胞的所有媒体转移到15毫升锥形瓶中并在离心机200×g离心。 </li><li>而将细胞离心，使冷冻媒体包括冷组合介质2等体积的，并与最终DMSO的7.5％浓度的冷冻介质。 </li><li>细胞沉淀后，吸出旧媒体，并逐滴重新暂停颗粒与冷冻介质。立即TRansfer到冷冻小瓶并置于-80℃冷冻机中。 </li></ol> 5.表征GMP级后切除人iPS细胞<ol><li>以确保多潜能标志物的转换后的适当的表达，可使用定量PCR来量化的关键的多能性标记物（SOX2，OCT4和Nanog）通过使用表1中的相同的步骤中列出的引物表达如上述在第3步QPCR方法遵循如在步骤3中列出的相同步骤。 </li><li>使用流式细胞仪来检测按照试剂盒中列为以前发表10标准条件非人唾液酸Neu5Gc的（N- -glycolylneuraminic酸）。 </li><li>轻轻用1×磷酸盐缓冲盐水（PBS）洗涤细胞培养板用的细胞。 </li><li>通过使用1倍的解离试剂5分钟，在室温下解离的菌落。 </li><li>在步骤5.4的5分钟的温育时间，准备封闭溶液（在所提供盒）通过使0.5％冰冷的PBS阻断剂。 </li><li>标签管流式细胞分析的以下一组:未染色; 4&#39;，6-二脒基-2-苯基吲哚（DAPI）的DAPI;对照抗体1:200;初级抗体1:200;二次抗体1:200驴抗鸡IgG（H + L）;样品的MEF;采样后切除细胞矩阵;和样品后切除细胞合成基质。 </li><li>轻轻加入2ml阻断溶液并转让内容到15毫升锥形管中去除细胞聚集来自步骤5.4。离心机在80×g离心5分钟，在4℃。 </li><li>用封闭溶液和离心机如在步骤5.7所述洗细胞两次。 </li><li>轻轻在100μl阻断与每种抗体的对应稀释体积溶液重新悬浮大约1×10 6个细胞。孵育轻轻摇动，在4℃下进行1小时。 </li><li>重复洗涤三次如在步骤5.8。 </li><li>再暂停400微升稀释剂卜的细胞沉淀FFER（从盒），用1:对管2，6，7 100的DAPI，及8列出在步骤5.6。 </li><li>通过一个40微米的过滤细胞株，并通过流量运行仪。 </li></ol>

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James A. Byrne (JAB) and Agustin Vega-Crespo receive research funding from Fibrocell Science, Inc. JAB is a scientific consultant for Fibrocell Science, Inc. No other authors have any competing interests to disclose.

我们描述推导因子人iPS细胞无，使他们通过临床未来人类疗法将这些细胞转化为GMP级的条件，下游细胞分化相关的方法论。虽然该协议是广泛地适用于多种细胞类型的，我们选择了重新编程的人皮肤成纤维细胞，因为很容易提取从患者和他们的适用性，以个性化的人类治疗。一旦限制是尽可能补救作为充分分化为临床相关细胞衍生关注图14中，提出了转化为GMP级别的条件将变得更加相关的...

Adult human cells have been shown to be capable of undergoing epigenetic remodeling and reprogramming, as a result of lentiviral-based expression of four key transcription factors4,5. An important advancement in the reprogramming field was the use of a single excisable lentiviral stem cell cassette (STEMCCA), which housed all four reprogramming transcription factors that allowed a precise stoichiometric ratio of protein expression6. Additionally, when transduced in specific multiplicity of infection ranges, STEMCCA can lead to predominantly single genomic integration events during the reprogramming process7. The introduction of an excisable version of STEMCCA, which utilizes Cre/loxP technology followed by excision of the reprogramming vector after derivation of the stem cell line, enabled factor-free human induced pluripotent stem cell (hiPSC) lines to be derived8. Additionally, in order to enhance therapeutic applications of hiPSCs, a novel, quick, and readily applicable methodology for good manufacturing practice (GMP)-grade cell line conversion, from xeno-containing to xeno-free conditions, needed to be implemented. Here, we discuss a relevant methodology that more precisely assesses integrated gene expression differences, specifically when integrated into an intron, and clinical-grade cell conversion into putative GMP conditions. 
Previous research has used only relatively insensitive microarray transcriptional analysis to analyze gene expression differences in integrated genes after STEMCCA transduction3,9. Here, we introduce the methodology of sensitive quantitative polymerase chain reaction (PCR) analysis, to further examine integrated gene expression differences. Importantly, current safe-harbor criteria discard hiPSCs that have genes with viral integrations, thus limiting the applicability of these cells for downstream human cellular therapeutics9. We propose that the status quo may change with the use of fully characterized and transgene-free intronically reprogrammed hiPSCs. Additionally, we introduce a robust GMP-grade cell conversion protocol that can be readily applied to a variety of different cell types, which were originally derived under xeno-containing conditions10. This provides significant opportunities for the development of future cell reprogramming experiments, which require clinical-grade conditions to maintain human therapeutic relevance. 
These methodologies provide a foundation upon which current safe-harbor criteria may be expanded to include characterized STEMCCA reprogrammed hiPSC lines that maintain a normal gene expression profile after STEMCCA excision from the integrated intron. Also, full conversion into clinical-grade conditions, free from non-human animal antigens, will help to incorporate many more cell types, which have previously been reprogrammed and characterized only in xeno-containing conditions. These methodologies combined, are persuasive grounds for the US Food and Drug Administration (FDA) to consider expanding their limited approval from human embryonic stem cell (ESC)-based therapeutics to hiPSC-based therapeutics11. 
We recently detailed the derivation of a factor-free hiPSC line that was fully characterized and converted into putative clinical-grade conditions10. Here, we detail the protocol for hiPSC derivation by utilizing the STEMCCA lentivirus. These stem cells then undergo an excision process followed by gene expression characterization. Finally, the hiPSCs are converted over into GMP-grade conditions by a slow conversion methodology.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Media and reagents&lt;/strong&gt;</td></tr><tr><td>DMEM/F12 (basal media)</td><td>Invitrogen (Carlsbad, CA, USA)</td><td>11330057</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen</td><td>16000044</td><td></td></tr><tr><td>Minimum essential medium (MEM) non-essential amino acids (NEAA), 100x</td><td>Invitrogen</td><td>11140050</td><td></td></tr><tr><td>Glutamax, 100x</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>PenStrep, penicillin-streptomycin, 100x</td><td>Invitrogen</td><td>15140-122</td><td>Thaw at 4 &amp;deg;C, aliquot and store at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Knockout serum replacement</td><td>Invitrogen</td><td>10828028</td><td>Thaw at 4 &amp;deg;C, aliquot and store at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Trypsin/EDTA, 0.5%</td><td>Invitrogen</td><td>15400-054</td><td>Dilute stock out to 0.05% in 1x PBS</td></tr><tr><td>Basic fibroblast growth factor</td><td>GlobalStem (Rockville, MD, USA)</td><td>GSR-2001</td><td>Reconstitute to 10 &amp;micro;g/ml stock in 0.1% bovine serum albumin dissolved in 1x PBS and store at &amp;minus;80 &amp;deg;C</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Millipore (Billerica, MA, USA)</td><td>ES-007-E</td><td></td></tr><tr><td>Matrigel (basement membrane matrix)</td><td>BD Biosciences (San Jose, CA, USA)</td><td>356231</td><td>Dilute stock Matrigel vial with 10 ml of DMEM/F12 while on ice for a 1:2 dilution. Aliquot and store at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>CELLstart (Synthetic Substrate)</td><td>Invitrogen</td><td>A1014201</td><td></td></tr><tr><td>Stemmolecule Y27632</td><td>Stemgent (Cambridge, MA, USA)</td><td>04-0012-02</td><td></td></tr><tr><td>Puromycin</td><td>Invitrogen</td><td>A1113802</td><td></td></tr><tr><td>LightCycler 480 Probes Master</td><td>Roche (Basel, Switzerland)</td><td>4707494001</td><td></td></tr><tr><td>ProFreeze-CDM Medium/freezing medium</td><td>Lonza (Basel, Switzerland)</td><td>12-769E</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich (St. Louis, MO, USA)</td><td>D8418</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>100 BP DNA Ladder</td><td>Invitrogen</td><td>15628019</td><td></td></tr><tr><td>SYBR Safe DNA Gel Stain 10,000x</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Agarose</td><td>Bio-Rad Laboratories, Inc. (Hercules, CA, USA)</td><td>161-3101</td><td></td></tr><tr><td>Gelatin, from porcine skin</td><td>Sigma-Aldrich</td><td>G1890-100G</td><td>Make stock at 0.2% in PBS, autoclave and store at room temperature.</td></tr><tr><td>mTeSR1</td><td>StemCell Technologies (Vancouver, BC, Canada)</td><td>5850</td><td>Combine Supplement 5x with the basic medium, aliquot and store at 4 &amp;deg;C for up to 2 weeks.</td></tr><tr><td>Stemedia NutriStem XF/FF Culture Medium</td><td>Stemgent</td><td>05-100-1A</td><td>Thaw at 4 &amp;deg;C O/N, aliquot and store at 4 &amp;deg;C for up to 2 weeks.</td></tr><tr><td>Primocin</td><td>InvivoGen (San Diego, CA, USA)</td><td>ant-pm-1</td><td></td></tr><tr><td>Accutase (Dissociation Reagent)</td><td>Invitrogen</td><td>A1110501</td><td></td></tr><tr><td>Donkey anti-Chicken IgG AlexaFluor 488</td><td>Jackson ImmunoResearch (West Grove, PA, USA)</td><td>703-546-155</td><td></td></tr><tr><td>Polybrene/transfection agent</td><td>Millipore</td><td>TR-1003-G</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Plasticware&lt;/strong&gt;</td></tr><tr><td>12-well plates</td><td>VWR (West Chester, PA, USA)</td><td>29442-038</td><td></td></tr><tr><td>6-well plates</td><td>VWR</td><td>29442-042</td><td></td></tr><tr><td>10-cm plates</td><td>Sigma-Aldrich</td><td>Z688819</td><td></td></tr><tr><td>18-gauge needle</td><td>Fisher Scientific (Pittsburgh, PA, USA)</td><td>148265D</td><td></td></tr><tr><td>21-gauge needle</td><td>Fisher Scientific</td><td>14-829-10D</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>BD LSRII Flow Cytometer</td><td>KSystem by Nikon (Tokyo, Japan)</td><td></td><td></td></tr><tr><td>BD FACSDiva Version 6.1.3 Software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Kits&lt;/strong&gt;</td></tr><tr><td>PureLink Genomic DNA Mini Kit</td><td>Invitrogen</td><td>K182000</td><td></td></tr><tr><td>KAPA HiFi Hotstart ReadyMix PCR Kit</td><td>KAPA Biosystems (Wilmington, MA, USA)</td><td>KK2601</td><td></td></tr><tr><td>High Pure RNA Isolation Kit</td><td>Roche</td><td>11828665001</td><td></td></tr><tr><td>Transcriptor First Strand cDNA Synthesis Kit</td><td>Roche</td><td>4379012001</td><td></td></tr><tr><td>Sialix anti-Neu5Gc Basic Pack Kit</td><td>Sialix (Newton, MA, USA)</td><td>Basic Pack</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Media&lt;/strong&gt;</td></tr><tr><td>Combined media 1</td><td>StemCell Technologies and Stemgent</td><td></td><td>Consists of equal parts mTeSR1 and Nutristem</td></tr><tr><td>Combined media 2</td><td>StemCell Technologies and Stemgent</td><td></td><td>Consists of equal parts TeSR2 and Nutristem</td></tr><tr><td>HUF Media</td><td></td><td></td><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/F12 [DMEM/F12] supplemented with 10% fetal bovine serum, 1x non-essential amino acids, 1x Glutamax, and 1x Primocin</td></tr><tr><td>Human Pluripotent Stem Cell Media</td><td></td><td></td><td>DMEM/F12 supplemented with 20% knockout serum replacement, 1x Glutamax, 1x non-essential amino acids, 1x Primocin, 1x &amp;beta;-mercaptoethanol, and 10 ng/ml basic fibroblast growth factor.</td></tr><tr><td colspan="4">DMEM/F12, Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium/F12; PBS, phosphate-buffered saline.</td></tr></tbody></table>

derivation and characterization of a transgene-free human induced pluripotent stem cell line and conversion into defined clinical-grade conditions

人类诱导多能干细胞（人iPS细胞）可与慢病毒为基础的重编程方法来生成。然而，残留在基因组中的活性转录区潜在的致癌基因的痕迹，限制了其潜在的用于人类治疗应用1使用。此外，来自于干细胞重编程或分化为治疗相关的衍生物的非人类抗原排除来自于一个人的临床背景下2正在使用这些人iPS细胞。在这段视频中，我们提出了一个程序，重新编程和分析因子人iPS细胞无无外源转基因。这些人iPS细胞然后可用于在含有慢病毒的特异性内含子的基因表达异常进行分析。该分析可以使用灵敏的定量聚合酶链反应（PCR），其具有以前用于检测基因表达的3分歧较不敏感的技术的优点进行。全部转化成临床级良好生产规范（GMP）的条件下，使人类的临床意义。我们的协议提供了另一种方法，提供的电流安全港标准将扩大和包括因子无其特征为人类治疗应用，用于导出GMP级人iPS细胞，这应消除任何免疫原性风险因非人抗原hiPSC基衍生物。这个协议是广泛地适用于任何类型的慢病毒重编程的细胞，并提供一个可重复的方法用于将重编程的细胞成GMP级别的条件。

我们提出了一个协议，用于通过使用所述STEMCCA慢病毒为基础的重编程方法推导临床级因子-自由人iPS细胞。 图1A示出了三个不同的预切hiPSC线代表图象，与上一层的MEF的STEMCCA方法重新编程之后。所述STEMCCA重新编程方法的主要优势在于一致重编程成功由多个科学家实现，在不同的研究组和位置。 图1B给出了后切除逆转录-PCR凝胶，显示出一个特定的亚克隆（2.3），这是完全?...

Watch this Scientific Journal Video about Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions at JoVE.com

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

推导与表征的转基因无人类诱导多能干细胞系，并转换成定义的临床级条件

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

derivation-characterization-transgene-free-human-induced-pluripotent

Research

JoVE Journal

Biology

7.8K 次观看.  University of California, Los Angeles (UCLA). We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions. 

视频: Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

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 ...

科研

发育生物学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

神经科学

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

在常规免疫细胞化学鉴定无血清条件下人类iPS细胞的小规模的传播

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

无异条件下多原代羊水和膜细胞重新编程

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

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) ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

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 ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

生物学

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...