Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines
Preserving self-renewal of porcine pluripotent stem cells in serum-free 3i culture condition and independent of LIF and b-FGF cytokines
复制标题
在无血清 3i 培养条件下保持猪多能干细胞的自我更新且不依赖于 LIF 和 b-FGF 细胞因子
DOI：
10.1038/s41420-017-0015-4
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发表时间：
2018-02-14
影响因子：
7
通讯作者：
Wang, Huayan
中科院分区：
文献类型：
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作者：
Ma, Yangyang;Yu, Tong;Wang, Huayan
真正的猪多能干细胞的来源仍然是一个关键问题，因为目前还没有猪胚胎干细胞(ESCs)，而且大多数保持猪诱导多能干细胞(PiPSCs)的培养条件是基于小鼠和人的iPS细胞的条件。在本研究中，我们建立了多西环素诱导的猪iPS细胞系(DOX-IPSCs)，并用它来筛选维持PiPSCs自我更新的最佳培养条件。我们发现LIF和b-FGF是猪细胞重编程所必需的，但不是维持PiPSCs自我更新和多能性所必需的细胞因子。通过认真筛选，制备了含有CHIR99021、SB431542和PD0325901三种抑制剂，BMP4、SCF和IL-6三种细胞因子以及人血小板裂解物(PL)的无血清3I培养液。在3I条件下，多西环素诱导的IPSCs可以在不添加多西环素的情况下长期传代，随着内源性多能基因表达的增加，中间状态的piPSCs的扁平形态可以转变为幼稚的形态。此外，从5.5日龄囊胚期分离的PPSC细胞株在3I培养液中能持续存在，内源性多能基因OCT4、Esrrb和STELLA的表达显著增加。我们的发现指导了一种新的重新编程策略，即使用3i条件来维持并将启动的piPSCs转换为幼稚的多能性状态。传统的LIF/b-FGF条件和3I条件的结合可能有助于我们找到一种合适的重编程方法来产生猪IPSCs的幼稚状态。
Derivation of bona fide porcine pluripotent stem cells is still a critical issue because porcine embryonic stem cells (ESCs) are not available yet, and most of the culture conditions to maintain porcine induced pluripotent stem cells (piPSCs) are based on conditions for mouse and human iPS cells. In this study, we generated a doxycycline-inducible porcine iPS cell line (DOX-iPSCs) and used it to screen the optimal culture condition to sustain the self-renewal of piPSCs. We found that LIF and b-FGF were required for porcine cell reprogramming, but were not essential cytokines for maintaining the self-renewal and pluripotency of piPSCs. A serum-free 3i medium, which includes three inhibitors CHIR99021, SB431542, and PD0325901, three cytokines BMP4, SCF, and IL-6, and human platelet lysates (PL), was made through serious selections. In 3i condition, the doxycycline-inducible iPSCs could be passaged for a long term without the addition of doxycycline, and the flattened morphology of intermediate state piPSCs could convert to the naïve-like morphology with the increase in endogenous pluripotent gene expressions. Additionally, pPSC cell line isolated from 5.5 days blastocysts could be sustained in 3i medium and the expression of endogenous pluripotent genesOCT4,ESRRB, andSTELLAwas significantly increased. Our finding directed a new reprogramming strategy by using 3i condition to maintain and convert primed piPSCs into naïve-like pluripotent state. A combination of traditional LIF/b-FGF conditions and 3i condition may help us to find out an appropriate reprogramming approach to generate the naïve state of porcine iPSCs.