尽管经过多年的研究,猪诱导的多能干细胞(piPSCs)与种系嵌合能力尚未建立。此外,在PiPSC中定义幼稚状态的关键转录因子(TFs)也仍然难以捉摸,即使内细胞团(ICM)中的TF被认为是幼稚多能性的关键分子决定因素。在这项研究中,干扰素调节因子1(IRF-1)在ICM中的表达高于滋养外胚层(TE)。但IRF-1对piPSC多能性维持的影响尚未确定。
分析早期ICM的转录组概况以确定高度互连的TF。携带这些TFs报告分子的细胞用作体细胞核转移的供体细胞,以检测胚泡中的表达模式。接下来,IRF1-Flag在DOX-hLIF-2ipiPSCs和AP染色中过表达,qRT-PCR,和RNA-seq进行检查IRF-1对多能性的影响。然后,用GFP标记DOX-hLIF-2ipiPSCs中IRF-1的表达,并进行qRT-PCR以确定GFP阳性和GFP阴性细胞之间的差异。接下来,进行ChIP-Seq以鉴定IRF-1的靶基因。用野生型piPSCs中的IL7和IRF-1过表达piPSCs中的STAT3磷酸化抑制剂进行处理,以证实JAK-STAT3信号通路在IRF-1多能性调节中的作用。此外,在重新编程期间,IRF-1被过表达并敲低以确定重编程效率的变化。
筛选出IRF-1在猪ICM中的表达高于d6〜7SCNT胚泡的TE。首先,IRF-1在piPSCs中的过表达被观察到促进形态学,AP染色,和多能性基因的表达谱,如细胞接近原始状态时所预期的。基因,KEGG途径,与分化过程相关的GO术语也下调。接下来,在野生型piPSC中,IRF-1启动子激活的高水平荧光与piPSCs中初始相关基因的较高表达相关。ChIP-Seq分析表明与JAK-STAT通路相关的基因,IL7和STAT3的表达被IRF-1激活。观察到STAT3磷酸化的抑制剂可以逆转IRF-1过表达细胞中引发的基因的表达,但在培养基中添加IL7对细胞形态无明显变化,AP染色结果,或多能性相关基因的表达。此外,重编程过程中IRF-1的击倒似乎降低了重编程效率,而过表达发挥相反的作用。
在猪早期胚泡的ICM中表达的IRF-1增强了piPSCs的多能性,部分通过促进JAK-STAT途径。
Despite years of research, porcine-induced pluripotent stem cells (
piPSCs) with germline chimeric capacity have not been established. Furthermore, the key transcription factors (TFs) defining the naïve state in
piPSCs also remain elusive, even though TFs in the inner cell mass (ICM) are believed to be key molecular determinants of naïve pluripotency. In this study, interferon regulatory factor 1 (IRF-1) was screened to express higher in ICM than trophectoderm (TE). But the impact of IRF-1 on maintenance of pluripotency in
piPSCs was not determined.
Transcriptome profiles of the early ICM were analyzed to determine highly interconnected TFs. Cells carrying these TFs\' reporter were used to as donor cells for somatic cell nuclear transfer to detect expression patterns in blastocysts. Next, IRF1-Flag was overexpressed in DOX-hLIF-2i piPSCs and AP staining, qRT-PCR, and RNA-seq were conducted to examine the effect of IRF-1 on pluripotency. Then, the expression of IRF-1 in DOX-hLIF-2i piPSCs was labeled by GFP and qRT-PCR was conducted to determine the difference between GFP-positive and GFP-negative cells. Next, ChIP-Seq was conducted to identify genes target by IRF-1. Treatment with IL7 in wild-type
piPSCs and STAT3 phosphorylation inhibitor in IRF-1 overexpressing
piPSCs was conducted to confirm the roles of JAK-STAT3 signaling pathway in IRF-1\'s regulation of pluripotency. Moreover, during reprogramming, IRF-1 was overexpressed and knocked down to determine the change of reprogramming efficiency.
IRF-1 was screened to be expressed higher in porcine ICM than TE of d6~7 SCNT blastocysts. First, overexpression of IRF-1 in the piPSCs was observed to promote the morphology, AP staining, and expression profiles of pluripotency genes as would be expected when cells approach the naïve state. Genes, KEGG pathways, and GO terms related to the process of differentiation were also downregulated. Next, in the wild-type piPSCs, high-level fluorescence activated by the IRF-1 promoter was associated with higher expression of naïve related genes in
piPSCs. Analysis by ChIP-Seq indicated that genes related to the JAK-STAT pathway, and expression of IL7 and STAT3 were activated by IRF-1. The inhibitor of STAT3 phosphorylation was observed could revert the expression of primed genes in IRF-1 overexpressing cells, but the addition of IL7 in culture medium had no apparent change in the cell morphology, AP staining results, or expression of pluripotency related genes. In addition, knockdown of IRF-1 during reprogramming appeared to reduce reprogramming efficiency, whereas overexpression exerted the converse effect.
The IRF-1 expressed in the ICM of pigs\' early blastocyst enhances the pluripotency of piPSCs, in part through promoting the JAK-STAT pathway.