Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.
Improved methods for reprogramming human dermal fibroblasts using fluorescence activated cell sorting.
复制标题
改进的方法，使用荧光激活的细胞分选来重编程人皮肤成纤维细胞。
DOI：
10.1371/journal.pone.0059867
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Noggle SA
中科院分区：
文献类型：
--
作者：
Kahler DJ;Ahmad FS;Ritz A;Hua H;Moroziewicz DN;Sproul AA;Dusenberry CR;Shang L;Paull D;Zimmer M;Weiss KA;Egli D;Noggle SA
目前通过病毒感染从人皮肤成纤维细胞获得诱导多能干细胞（iPSC）系的方法依赖于昂贵且冗长的方案。导致获得细胞系所需时间的一个主要因素是研究人员在感染后早期时间点从含有转化或部分重编程细胞和成纤维细胞的混合细胞群中鉴定完全重编程的独特候选克隆的能力。未能在衍生过程的早期选择高质量的集落导致细胞系需要增加的维护和不可靠的实验结果。在此，我们描述了一种改进的iPSC系衍生方法，其使用荧光激活细胞分选（FACS）在感染后第7-10天分离表达细胞表面标志物签名CD 13 NEGSSEA 4 POSTra-1- 60 POS的单细胞。该技术前瞻性地分离完全重编程的iPSC，并耗尽亲本和“污染的”部分重编程的成纤维细胞，从而在不使用限定的小分子混合物的情况下显著减少产生iPSC系所需的时间和试剂。FACS衍生的iPSC系表达多能性的常见标志物，并且在体外和体内具有自发分化潜力。为了证明FACS对于高通量iPSC产生的适用性，我们使用整合（逆转录病毒）或非整合（仙台病毒）重编程载体衍生了228个单独的iPSC系，并对这些系的子集进行了广泛的表征。本研究中使用的iPSC系来源于来自各种组织来源的76个独特样品，包括从健康或疾病患者收获的活检产生的新鲜或冷冻成纤维细胞。
Current methods to derive induced pluripotent stem cell (iPSC) lines from human dermal fibroblasts by viral infection rely on expensive and lengthy protocols. One major factor contributing to the time required to derive lines is the ability of researchers to identify fully reprogrammed unique candidate clones from a mixed cell population containing transformed or partially reprogrammed cells and fibroblasts at an early time point post infection. Failure to select high quality colonies early in the derivation process results in cell lines that require increased maintenance and unreliable experimental outcomes. Here, we describe an improved method for the derivation of iPSC lines using fluorescence activated cell sorting (FACS) to isolate single cells expressing the cell surface marker signature CD13NEGSSEA4POSTra-1-60POS on day 7–10 after infection. This technique prospectively isolates fully reprogrammed iPSCs, and depletes both parental and “contaminating” partially reprogrammed fibroblasts, thereby substantially reducing the time and reagents required to generate iPSC lines without the use of defined small molecule cocktails. FACS derived iPSC lines express common markers of pluripotency, and possess spontaneous differentiation potential in vitro and in vivo. To demonstrate the suitability of FACS for high-throughput iPSC generation, we derived 228 individual iPSC lines using either integrating (retroviral) or non- integrating (Sendai virus) reprogramming vectors and performed extensive characterization on a subset of those lines. The iPSC lines used in this study were derived from 76 unique samples from a variety of tissue sources, including fresh or frozen fibroblasts generated from biopsies harvested from healthy or disease patients.
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影响因子：
48
作者：
Lin, Tongxiang;Ambasudhan, Rajesh;Yuan, Xu;Li, Wenlin;Hilcove, Simon;Abujarour, Ramzey;Lin, Xiangyi;Hahm, Heung Sik;Hao, Ergeng;Hayek, Alberto;Ding, Sheng
通讯作者：
Ding, Sheng
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
5.2
作者：
Henderson, JK;Draper, JS;Andrews, PW
通讯作者：
Andrews, PW
影响因子：
1.2
作者：
Sundberg, Maria;Jansson, Linda;Narkilahti, Susanna
通讯作者：
Narkilahti, Susanna
影响因子：
64.5
作者：
Takahashi, Kazutoshi;Yamanaka, Shinya
通讯作者：
Yamanaka, Shinya