Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs.
Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs.
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DOI：
10.1371/journal.pone.0011834
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发表时间：
2010-07-28
期刊：
影响因子：
3.7
通讯作者：
Chen IS
中科院分区：
文献类型：
--
作者：
Kamata M;Liang M;Liu S;Nagaoka Y;Chen IS
人类诱导多能干细胞（hiPSC）为再生疗法提供了新的可能性。为了实现这一潜力，有效监测这些hiPSC分化为特定谱系至关重要。在这里，我们描述了对特定microRNA（miRNA）敏感的慢病毒报告载体，以显示携带与不同报告分子缀合的多个miRNA靶序列的单个载体可用于监测hiPSC形成和随后从人胎儿成纤维细胞（HFF）分化。报告载体编码与人胚胎干细胞（hESC）特异性miRNA（miR-302 a和miR-302 d）的靶标缀合的EGFP和与分化细胞特异性miRNA（miR-142- 3 p、miR-155和miR-223）的靶标缀合的mCherry。该载体用于追踪HFF到iPSC的重编程。用该报告载体和编码4种重编程因子（OCT 4、SOX 2、KLF 4和cMYC）的载体共转导的HFF在hiPSC形成的早期阶段大多数对EGFP呈阳性（67%）。EGFP表达逐渐消失，mCherry表达增加，表明分化细胞特异性miRNA减少，hESC特异性miRNA表达减少。在hiPSC分化成胚状体后，这些hiPSC中的大部分重新获得EGFP表达，并且这些细胞中的一些变得对EGFP呈单阳性。进一步分化成神经谱系显示出由EGFP或mCherry表达划分的不同结构。这些发现表明，miRNA依赖性报告载体可以是在hiPSC重编程和随后分化为谱系特异性细胞期间监测活细胞的有用工具。
Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved, it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here, we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p, miR-155, and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4, SOX2, KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies, a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells.
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