Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells.
Reversible Immortalization Enables Seamless Transdifferentiation of Primary Fibroblasts into Other Lineage Cells.
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DOI：
10.1089/scd.2016.0035
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发表时间：
2016-08
影响因子：
4
通讯作者：
Fei Xie;Kerui Gong;K. Li;Mingliang Zhang;Judy C. Chang;Shizhong Jiang;Lin Ye;Jiaming Wang;Yuting Tan;Y. Kan
中科院分区：
文献类型：
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作者：
Fei Xie;Kerui Gong;K. Li;Mingliang Zhang;Judy C. Chang;Shizhong Jiang;Lin Ye;Jiaming Wang;Yuting Tan;Y. Kan
成纤维细胞可以直接转分化为其他体细胞，如心肌细胞、造血细胞和神经元。体细胞分化而不首先产生诱导的多能干细胞（iPSC）的优点是它避免了分化的细胞被残留的iPSC污染，这可能导致畸胎瘤。然而，由于来自活检的原代成纤维细胞在重复培养期间经历衰老，因此可能难以生长足够数量的转分化细胞用于未来的治疗目的。为了避免这个问题，我们可逆永生化的原代成纤维细胞，通过使用piggyBac转座子提供人端粒酶逆转录酶（hTERT）基因hTERT加SV40大T。这两种方法都能使成纤维细胞连续生长而不衰老，并且都不会在免疫缺陷小鼠中引起畸胎瘤形成。然而，用hTERT加SV40大T抗原永生化的成纤维细胞积累染色体重排，而用hTERT永生化的成纤维细胞保留正常核型。为了将hTERT永生化的成纤维细胞转分化为其他体细胞谱系细胞，我们用附加型OCT 4瞬时转染它们，并在神经细胞生长条件下用转座酶培养它们以去除转座子。三能神经祖细胞无缝和有效地产生。因此，具有hTERT的原代成纤维细胞的可逆永生化将允许潜在的基于自体细胞的治疗剂绕过并模拟iPSC产生。
Fibroblasts can be transdifferentiated directly into other somatic cells such as cardiomyocytes, hematopoietic cells, and neurons. An advantage of somatic cell differentiation without first generating induced pluripotent stem cells (iPSCs) is that it avoids contamination of the differentiated cells with residual iPSCs, which may cause teratoma. However, since primary fibroblasts from biopsy undergo senescence during repeated culture, it may be difficult to grow transdifferentiated cells in sufficient numbers for future therapeutic purposes. To circumvent this problem, we reversibly immortalized primary fibroblasts by using the piggyBac transposon to deliver the human telomerase reverse transcriptase (hTERT) gene hTERT plus SV40 Large T. Both approaches enabled fibroblasts to grow continuously without senescence, and neither caused teratoma formation in immunodeficient mice. However, fibroblasts immortalized with hTERT plus SV40 large T antigen accumulated chromosomal rearrangements, whereas fibroblasts immortalized with hTERT retained the normal karyotype. To transdifferentiate hTERT-immortalized fibroblasts into other somatic lineage cells, we transiently transfected them with episomal OCT4 and cultured them under neural cell growth condition with transposase to remove the transposon. Tripotent neural progenitor cells were seamlessly and efficiently generated. Thus, reversible immortalization of primary fibroblasts with hTERT will allow potential autologous cell-based therapeutics that bypass and simulate iPSC generation.