Yuan Ye, Park Jinkyu, Tian Yuchen, Choi Jungmin, Pasquariello Rolando, Alexenko Andrei P, Dai Aihua, Behura Susanta K, Roberts R Michael, Ezashi Toshihiko
1Bond Life Sciences Center, University of Missouri, Columbia, MO 65211 USA.
2Division of Animal Sciences, University of Missouri, Columbia, MO 65211 USA.
Cell Death Discov. 2019 Jun 17;5:104. doi: 10.1038/s41420-019-0184-4. eCollection 2019.
Understanding essential signaling network requirements and making appropriate adjustments in culture conditions are crucial if porcine pluripotent stem cells (PSC) are to achieve their full potential. Here, we first used two protein factors (LIF and FGF2) and kinase inhibitor combinations in attempts to convert primed type lentiviral-reprogrammed porcine induced PSC (Lv-piPSC) into naïve-like state and developed a medium called FL6i. In addition to FGF2 and LIF, this medium contained inhibitors of MAPK14, MAPK8, TGFB1, MAP2K1, GSK3A and BMP. Crucially, the usual TGFB1 and BMP4 protein components of many stem cell media were replaced in FL6i with inhibitors of TGFB1 and BMP. With this medium, Lv-piPSC were readily transformed from their original primed state into cells that formed colonies with typical features of naïve-state stem cells. The FL6i medium also assisted generation of naïve-type piPSC lines from porcine embryonic fibroblasts with non-integrating episomal plasmids (Epi-piPSC). These lines, despite retaining variable amounts of vector DNA, expressed higher endogenous and than Lv-piPSC. They have been cultured without obvious morphological change for >45 passages and retained pluripotent phenotypes in terms of upregulation of genes associated with pluripotency, low expression of genes linked to emergence of somatic cell lineages, and ability to generate well differentiated teratomas in immune-compromised mice. FL6i conditions, therefore, appear to support elevated pluripotent phenotypes. However, FL6i was less able to support the generation of embryonic stem cells from porcine blastocysts. Although colonies with dome-shaped morphologies were evident and the cells had some gene expression features linked to pluripotency, the phenotypes were ultimately not stable. Pathway analysis derived from RNAseq data performed on the various cell lines generated in this study suggest the benefits of employing the FL6i medium on porcine cells reside in its ability to minimize TGFB1 and BMP signaling, which would otherwise de-stabilize the stem cell state.
如果猪多能干细胞（PSC）要充分发挥其潜力，了解基本的信号网络要求并在培养条件上进行适当调整至关重要。在此，我们首先使用两种蛋白质因子（LIF和FGF2）以及激酶抑制剂组合，试图将引发型慢病毒重编程的猪诱导PSC（Lv-piPSC）转化为幼稚样状态，并开发了一种名为FL6i的培养基。除了FGF2和LIF外，这种培养基还含有MAPK14、MAPK8、TGFB1、MAP2K1、GSK3A和BMP的抑制剂。至关重要的是，FL6i中许多干细胞培养基中常见的TGFB1和BMP4蛋白质成分被TGFB1和BMP的抑制剂所取代。使用这种培养基，Lv-piPSC很容易从其原始的引发状态转化为形成具有幼稚状态干细胞典型特征的集落的细胞。FL6i培养基还辅助从猪胚胎成纤维细胞中使用非整合附加体质粒（Epi-piPSC）生成幼稚型piPSC系。这些细胞系尽管保留了可变数量的载体DNA，但比Lv-piPSC表达更高的内源性 和 。它们已经培养了超过45代而没有明显的形态变化，并且在与多能性相关的基因上调、与体细胞谱系出现相关的基因低表达以及在免疫缺陷小鼠中产生分化良好的畸胎瘤的能力方面保留了多能表型。因此，FL6i条件似乎支持增强的多能表型。然而，FL6i在支持从猪囊胚生成胚胎干细胞方面能力较弱。尽管具有圆顶形形态的集落很明显，并且细胞具有一些与多能性相关的基因表达特征，但最终表型并不稳定。对本研究中生成的各种细胞系进行的RNAseq数据通路分析表明，在猪细胞上使用FL6i培养基的好处在于其能够最小化TGFB1和BMP信号传导，否则这会破坏干细胞状态。
