目标:迄今为止,已经做出了许多努力来建立猪胚胎干(pES)细胞,但没有成功。胚胎外内胚层(XEN)细胞可以自我更新并分化为内脏内胚层和壁内胚层。XEN细胞来源于胚泡内细胞团的原始内胚层,并且可以是细胞重编程中的中间状态。
方法:从猪多能干细胞(pPSC)产生猪XEN细胞(pXENCs),并通过RNA测序和免疫荧光分析表征。在嵌合小鼠胚胎中研究了pXENCs的发育潜力。
结果:将源自猪pPSC的猪XEN细胞在补充有bFGF的N2B27培养基中成功扩增至少30代。RNA测序和免疫荧光分析表明,pXENC表达了鼠和犬XEN标记Gata6,Gata4,Sox17和Pdgfra,但不表达多能标记Oct4,Sox2和TE标记Cdx2。此外,当注射到四细胞期小鼠胚胎中时,这些细胞有助于XEN。补充Chir99021和SB431542促进了pXENCs的多能性。
结论:我们成功地衍生了pXENC,并表明补充Chir99021和SB431542赋予它们多能性。我们的研究结果为研究猪诱导多能干细胞的重编程机制提供了新的资源。
OBJECTIVE: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming.
METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos.
RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs.
CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.