We thank Mrs. Matsumoto for the technical assistance.

1.细胞培养<ol><li>获得的细胞以诱导四倍体。迄今为止，已经证实，该技术可以应用到人类的成纤维细胞系的TIG-1，BJ，IMR-90和端粒酶永生TIG-1（TIG-HT）。 </li><li>生长在基本培养基细胞用α修改或适合于细胞类型进行研究补充有10％（V / V）热灭活的胎牛血清（FBS）的通过，在5％孵育（体积/任何其他细胞培养基ⅴ） 的 CO 2气氛中于37℃。传代细胞每3天或4天不超过汇合密度。 注:群体倍增水平（PDL）应在传代每次评估细胞生长和细胞年龄计算。的PDL可以从接种到培养皿（N1），并在使用下述式（X是初始PDL）的下传代（N 2）收获细胞数量的细胞数来计算。 PDL = X +（日志N1 - 日志N2）/ 2日志</li></ol> 2.诱导和埃斯塔从二倍体成纤维细胞四倍体细胞blishment <ol><li>诱导四倍体没有动摇过。 注意:某些细胞株（ 如 TIG-1）可与demecolcine（DC）的如下成为连续处理几乎完全四倍体。 <ol><li>传代细胞插入每天一或两个100毫米培养皿治疗前以诱导四倍体。一般，准备每盘5×10 5到1×10 6个细胞用于四倍体细胞的诱导。 </li><li>治疗指数生长的细胞含有0.1微克/毫升DC 4天网上平台。 </li><li>替换包含直流介质与不含药物的培养基，并在37℃孵育，在5％（体积/体积） 的 CO 2气氛中的细胞。细胞通常在1周内恢复增殖。 </li></ol></li><li>诱导四倍体与摇断。 注意:大多数细胞成为上述（2.1）中描述的，与直流的简单处理的结果是二倍体和四倍体群体的混合物。因此，p的一些修改rocedure消除二倍体人口是必要的如下（ 图1）。 <ol><li>传代细胞分为几个T75烧瓶一天治疗前诱导四倍体。通常，至少准备5×10 6个细胞（每烧瓶1×10 6个细胞）为四倍体细胞的诱导。 </li><li>治疗指数生长的细胞含有0.1微克/毫升DC 16中 - 18小时逮捕细胞的有丝分裂。逮捕在有丝分裂细胞中所需要的时间可能取决于靶细胞，并应通过初步实验来确定，以获得尽可能多的有丝分裂细胞成为可能。例如，速度较慢的生长的细胞应与直流待处理的速度比生长细胞较长的时间。 注:如果细胞可用于本步骤太长治疗，他们可能会经历有丝分裂滑脱并附着在餐具表面，使得不可能以收集通过摇断丝分裂细胞中的下一个步骤。 </li><li>收集DC-逮捕有丝分裂细胞通过摇动关方法，WHICH松散地粘附的有丝分裂的细胞由烧瓶中并用培养基洗涤后离心的轻轻摇动（300 XG，5分钟）收集。通过在这个步骤中的适当的方法计数细胞数目。 </li><li>补种收集有丝分裂细胞成60个100mm培养菜肴和治疗与0.1微克/毫升的DC细胞额外3天。种子每盘大于1×10 6个有丝分裂的细胞，因为细胞的小于10％的可存活这种治疗。 </li><li>含直流用不含药物的培养基介质更换和等待细胞恢复增殖，通常在1周内出现。 </li></ol></li><li>传代细胞原二倍体细胞相类似。 </li></ol> 3. DNA倍体的检查注意:这是一个行之有效的方法和技术手册应提交详细的程序和技术技巧。 <ol><li>收获的细胞（5×10 5 - 1 10 6）通过用溶液CON培养细胞泰宁0.05％胰蛋白酶和0.02％EDTA的10分钟，在37℃，并用含10％FBS的培养基中和胰蛋白酶。 </li><li>离心细胞介质和通过在5ml磷酸盐缓冲盐水的（PBS）然后离心（300 XG，5分钟）再悬浮清洗。 </li><li>通过以下两种方法之一制备用于DNA分析的细胞<ol><li>一种分析非固定细胞10方法 注意:可以使用用于细胞周期分析市售的试剂盒来进行此方法。用这种方法制备的样品应当立即进行分析，因为细胞不固定在过程和染色的核将随着时间的推移损坏。 <ol><li>洗用含有250mM的蔗糖的40mM柠檬酸盐缓冲液的细胞。随后处理细胞用0.1％（体积/体积）的Nonidet P40和30微克/毫升胰蛋白酶250微升在含有250mM的蔗糖在室温下10分钟200 40微升mM柠檬酸盐缓冲液中。 </li><li>加入0.5微克/毫升胰蛋白酶INH 200微升ibitor和0.1微克/毫升RNA酶A在含有250mM的蔗糖的40mM柠檬酸盐缓冲液，并温育细胞，在室温下10分钟。 </li><li>加入200微升的0.4毫克/在含有250mM的蔗糖的40mM柠檬酸盐缓冲溶液的碘化丙啶（PI），并在室温下孵育10分钟的细胞进行染色细胞核。 </li></ol></li><li>一种分析固定的细胞的方法<ol><li>修复用80％乙醇的细胞通过在1毫升的PBS悬浮细胞，加入4毫升100％乙醇的溶液，同时混合该细胞悬浮液，并离开它在室温下30分钟。在此固定剂的细胞可以储存在-20℃下几个星期。 </li><li>通过在5mL PBS中重悬它们洗涤细胞，然后离心（300 XG，5分钟）。治疗细胞用0.5毫克/毫升RNA酶A并用50微克/毫升的碘化丙啶（PI）（0.5毫克/毫升RNA酶A 475微升和1毫克25微升/毫升，PI），在室温下30分钟染色它们。 </li></ol></li></ol></li><li>分析的T DNA含量他用流式细胞仪用488 nm激光和红色通道排放过滤器网格（长传&gt; 610纳米）。分析在同一时间从真正二倍体细胞制备来评估靶细胞的倍性的参比样品。 <ol><li>备选地，添加荧光标准珠以评估二倍体细胞对珠的荧光强度比。使用"脉冲高度与脉冲宽度"流选项仪单四倍体细胞和二倍体细胞11团块区别对待。 </li></ol></li></ol> 4.检查染色体计数及核型分析注意:这些都是行之有效的程序和技术手册或制造商的协议应提交详细的程序和技术技巧。 <ol><li>治疗指数生长的细胞用0.1微克/毫升DC 4小时，逮捕细胞分裂中期。 </li><li>准备染色体核型。 <ol><lI&gt;收获细胞（至少5×10 5个细胞）通过胰蛋白酶消化，在含有FBS的培养基中止。 </li><li>离心细胞在培养基（300 XG，5分钟），并用在37℃的低渗溶液（5mL，0.075M的KCl）10分钟治疗。 </li><li>修复和暂停在卡诺固定液细胞（甲醇:乙酸= 3:1）。 </li><li>细胞悬液 - （35微升25）通过删除一小体积散布细胞到载玻片。如暴露于热蒸汽附加步骤可能是必要的，以改善染色体蔓延。 </li></ol></li><li>染色细胞原位杂交染色体计数或核型分析或多色荧光（渔业部）。 <ol><li>染色体计数。 <ol><li>染色细胞用5％吉姆萨溶液（或含有荧光显微镜0.5毫克/毫升RNA酶A 5微克/毫升，PI）15 - 20分钟。 </li><li>数字拍摄至少50中期细胞。 </li><li>使用图像分析softwa计数每个细胞的染色体数目再接触并使用照片编辑软件重叠染色体手动分离后。虽然染色体数目可以手动进球，推荐电脑计分。 </li></ol></li><li>核型分析 注:本分析应该由经过培训的技术人员或专业的公司来进行。 <ol><li>根据一个标准的G显带技术12染色的细胞。 </li><li>分析染色体，使标准karyograms 12。 </li></ol></li><li>渔业部 注:该步骤是可选的，并且如果必要应进行。 <ol><li>使用用于根据制造商的方案13的人染色体的多色FISH探针染色的细胞。 </li><li>分析使用带有适当的滤波器荧光显微镜和渔业部分析13软件的染色体。 </li></ol></li></ol></li></ol>

<ol>
	<li>Rabinovitch, P., 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=Predictors+of+progression+in+Barrett's+esophagus+III:+baseline+flow+cytometric+variables.">Predictors of progression in Barrett's esophagus III: baseline flow cytometric variables.</a> Am. J. Gastroenterol. 96 (11), 3071-3083 (2001).</li><li>Galipeau, P., 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=NSAIDs+modulate+CDKN2A,+TP53,+and+DNA+content+risk+for+progression+to+esophageal+adenocarcinoma.">NSAIDs modulate CDKN2A, TP53, and DNA content risk for progression to esophageal adenocarcinoma.</a> PLoS Med. 4 (2), e67 (2007).</li><li>Olaharski, 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=Tetraploidy+and+chromosomal+instability+are+early+events+during+cervical+carcinogenesis.">Tetraploidy and chromosomal instability are early events during cervical carcinogenesis.</a> Carcinogenesis. 27, 337-343 (2006).</li><li>Liu, 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=p53-independent+abrogation+of+a+postmitotic+checkpoint+contributes+to+human+papillomavirus+E6-induced+polyploidy.">p53-independent abrogation of a postmitotic checkpoint contributes to human papillomavirus E6-induced polyploidy.</a> Cancer Res. 67, 2603-2610 (2007).</li><li>Davoli, T., de Lange, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+causes+and+consequences+of+polyploidy+in+normal+development+and+cancer.">The causes and consequences of polyploidy in normal development and cancer.</a> Annu Rev Cell Dev Biol. 27, 585-610 (2011).</li><li>Fox, D., Duronio, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoreplication+and+polyploidy:+insights+into+development+and+disease.">Endoreplication and polyploidy: insights into development and disease.</a> Development. 140, 3-12 (2013).</li><li>Fujiwara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis+failure+generating+tetraploids+promotes+tumorigenesis+in+p53-null+cells.">Cytokinesis failure generating tetraploids promotes tumorigenesis in p53-null cells.</a> Nature. 437, 1043-1047 (2005).</li><li>Ganem, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokinesis+failure+triggers+hippo+tumor+suppressor+pathway+activation.">Cytokinesis failure triggers hippo tumor suppressor pathway activation.</a> Cell. 158 (4), 833-848 (2014).</li><li>Kuznetsova, 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=Chromosomal+instability,+tolerance+of+mitotic+errors+and+multidrug+resistance+are+promoted+by+tetraploidization+in+human+cells.">Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells.</a> Cell cycle. 14 (17), 2810-2820 (2015).</li><li>Vindel&#248;v, L., Christensen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent+and+proteolytic+enzyme-based+techniques+for+nuclear+isolation+and+DNA+content+analysis.">Detergent and proteolytic enzyme-based techniques for nuclear isolation and DNA content analysis.</a> Methods Cell Biol. 41, 219-229 (1994).</li><li>Darzynkiewicz, Z., Juan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content+measurement+for+DNA+ploidy+and+cell+cycle+analysis.">DNA content measurement for DNA ploidy and cell cycle analysis.</a> Curr Protoc Cytom. , Chapter 7: Unit 7.5 (2001).</li><li>Knutsen, T., Bixenman, H., Lawce, H., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+analysis+guidelines+preliminary+report.">Chromosome analysis guidelines preliminary report.</a> Cancer Genet Cytogenet. 52 (1), 11-17 (1991).</li><li>Liehr, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+FISH+probe+sets+and+their+applications.">Multicolor FISH probe sets and their applications.</a> Histol. Histopathol. 19 (1), 229-237 (2004).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+bipolar+spindles+with+two+centrosomes+in+tetraploid+cells+established+from+normal+human+fibroblasts.">Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.</a> Hum. Cell. 25 (3), 78-85 (2012).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from normal human fibroblasts.</a> Front. Oncol. 3, 198 (2013).</li><li>Ohshima, S., Seyama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+proliferative+tetraploid+cells+from+telomerase-immortalized+normal+human+fibroblasts.">Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.</a> Genes, Chromosome Cancer. 55 (6), 522-530 (2016).</li><li>Di Leonardo, 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=DNA+rereplication+in+the+presence+of+mitotic+spindle+inhibitors+in+human+and+mouse+fibroblasts+lacking+either+p53+or+pRb+function.">DNA rereplication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function.</a> Cancer Res. 57, 1013-1019 (1997).</li><li>Andreassen, P., Lohez, O., Lacroix, F., Margolis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetraploid+state+induces+p53-dependent+arrest+of+nontransformed+mammalian+cells+in+G1.">Tetraploid state induces p53-dependent arrest of nontransformed mammalian cells in G1.</a> Mol. Biol. Cell. 12, 1315-1328 (2001).</li><li>Vogel, C., 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=Crosstalk+of+the+mitotic+spindle+assembly+checkpoint+with+p53+to+prevent+polyploidy.">Crosstalk of the mitotic spindle assembly checkpoint with p53 to prevent polyploidy.</a> Oncogene. 23, 6845-6853 (2004).</li><li>Aylon, Y., Oren, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53:+Guardian+of+ploidy.">p53: Guardian of ploidy.</a> Mol. Oncol. 5 (4), 315-323 (2011).</li><li>Uetake, Y., Sluder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+progression+after+cleavage+failure+:+mammalian+somatic+cells+do+not+possess+a+&amp;#34;tetraploidy+checkpoint&amp;#34.">Cell cycle progression after cleavage failure : mammalian somatic cells do not possess a &amp;#34;tetraploidy checkpoint&amp;#34.</a> J. Cell Biol. 165, 609-615 (2004).</li><li>Ganem, N., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+the+proliferation+of+polyploid+cells.">Limiting the proliferation of polyploid cells.</a> Cell. 131, 437-440 (2007).</li><li>Ho, C., Hau, P., Marxer, M., Poon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+of+p53+for+maintaining+chromosomal+stability+during+tetraploidization.">The requirement of p53 for maintaining chromosomal stability during tetraploidization.</a> Oncotarget. 1 (7), 583-595 (2010).</li></ol>

作者宣称，他们没有竞争的经济利益。

从二倍体细胞通过化学试剂四倍体诱导的主要问题，无论是由胞质抑制剂或由主轴抑制剂，是细胞常成为二倍体和四倍体群体的混合物，和四倍体细胞必须从二倍体细胞中分离出来。免费二倍体细胞的四倍体种群隔离最常用的方法通过有限稀释使用FACS或克隆。然而，这些程序是费力和不容易执行。在这份报告中，我们提出了一个新的协议建立无正常人成纤维细胞二倍体人口增殖细胞四倍体。该协...

多倍体已经观察到，不仅在哺乳动物物种的专门的组织，而且在各种病理状况，如癌症和变性疾病。多倍体（主要是四倍体）细胞通常在人体组织中，诸如Barrett食管1,2或鳞状子宫颈3,4上皮内病变的癌前病变观察到的，并已经被认为是恶性非整倍体细胞的来源在那些组织5 6。虽然有人建议，四倍体转化为异倍体细胞可以是在肿瘤发生的早期阶段的关键事件，参与这一过程的机制尚未完全了解。这部分是因为没有体外模型已经可以在那里未转化多倍体人体细胞可以传播。 一些研究人员已经通过代双核细胞通过异烟肼诱导四倍体在非转化人类上皮细胞ibiting胞质7-9。在该方法中，然而，不必要的二倍体细胞，必须通过荧光激活细胞分选（FACS）消除7,8- 或通过有限稀释9克隆。因为这些程序是艰苦和不易执行，简单的方法来建立非转化四倍体细胞被期望在这一领域的研究。 在本报告中，我们描述了一个协议，通过相对简单的程序，以建立正常的人类成纤维细胞或端粒酶永生化人成纤维细胞增殖四倍体细胞。该过程使用的主轴毒药demecolcine（DC）逮捕由摆脱与DC进一步处理收集在有丝分裂二倍体细胞，并有丝分裂细胞。与DC治疗时间延长二倍体有丝分裂细胞转化为四倍体G1期细胞，而这些细胞增殖生长停滞了以下药物清除几天后的四倍体细胞。该协议提供用于创建一个有用的模型来研究染色体的不稳定性和多倍体的人类细胞的致瘤潜力之间的关系的有效方法。

<table><tbody><tr><td>MEM-&amp;alpha;</td><td>Sigma-Aldrich</td><td>M8042-500ML</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>172012-500ML</td><td></td></tr><tr><td>Demecolcine solution (10 &amp;mu;g/mL in HBSS)</td><td>Sigma-Aldrich</td><td>D1925-10ML</td><td></td></tr><tr><td>BD CycleTES Plus DNA Reagent Kits</td><td>BD Biosciences</td><td>#340242</td><td>For examination of DNA ploidy by flow cytometry</td></tr><tr><td>Human chromosome multicolor FISH probe 24XCyte</td><td>MetaSystems</td><td>#D-0125-060-DI</td><td>Specialized filter set and software for mFISH analysis are necessary</td></tr><tr><td>Isis imaging system with mFISH&amp;nbsp; software&amp;nbsp;</td><td>MetaSystems</td><td></td><td>Specialized probe kit is necessary</td></tr></tbody></table>

establishment of proliferative tetraploid cells from nontransformed human fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

根据我们的经验，TIG-1细胞可以几乎完全四倍体用0.1微克/毫升直流4天（ 图2A）制成由简单的连续治疗。与此相反，其他成纤维细胞株，如BJ或IMR-90，和TIG-HT细胞，成为由摇离方法二倍体和按照相同的治疗四倍体群体，和有丝分裂细胞的分离的混合物中的过程中是必要的的直流处理（通常为16 -治疗开始后18小时）（ 图2B，C）。与DC的额外治疗3天后?...

Watch this Scientific Journal Video about Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts at JoVE.com

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

从非转化人成纤维细胞增生四倍体细胞的建立

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

establishment-proliferative-tetraploid-cells-from-nontransformed

Research

JoVE Journal

Developmental Biology

7.2K 次观看.  Saitama Medical University. Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

视频: Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

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) ...

发育生物学

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

基于 rna 的人原代成纤维细胞重组成诱导多能干细胞

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

生物学

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

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

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

通过转录因子的强制表达，人类纤维细胞的造血再编程

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

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 ...

神经科学

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 ...

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 ...

生物工程

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

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

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-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 ...