Monodelphis domestica induced pluripotent stem cells reveal metatherian pluripotency architecture

Marsupials have been a powerful comparative model to understand mammalian biology. However, because of the unique characteristics of their embryology, marsupial pluripotency architecture remains to be fully understood, and nobody has succeeded in developing embryonic stem cells (ESCs) from any marsupial species. We have developed an integration-free induced pluripotent stem cell (iPSC) reprogramming method and established validated iPSC lines from two fully inbred strains of the gray short-tailed opossum (Monodelphis domestica). A comprehensive characterization of the M. domestica skin fibroblasts and their reprogrammed iPSCs was performed by genome-wide mRNA sequencing. The established monoiPSCs showed a significant (6,181 DE genes) but highly uniform (between clone r2 at 95% CI = 0.973 ± 0.007) resetting of the cellular transcriptome during reprogramming and were highly similar to eutherian ESCs and iPSCs in their overall transcriptomic and functional profiles. However, monoiPSCs showed unique regulatory architecture of the core pluripotency transcription factors and were more like epiblasts. Our results suggest POU5F1 and the splice variant specific expression of POU5F3 synergistically regulate the opossum pluripotency gene network. It is plausible that POU5F1, POU5F3 splice variant XM_016427856.1, and SOX2 form a self-regulatory network. NANOG expression, however, was specific to monoiPSCs and epiblasts, and displayed a distinct expression profile in embryonic cells. Furthermore, POU5F1 was highly expressed in trophectoderm cells, whereas all other pluripotency transcription factors were significantly downregulated, suggesting that the regulatory architecture of core pluripotency genes of marsupials may be distinct from that of eutherians. Overall design: To better understand marsupial pluripotency architecture, a genome-wide comparative gene expression analysis of the M. domestica skin fibroblasts and their reprogrammed iPSCs was performed.

有袋类（marsupials）长期以来都是解析哺乳动物生物学的经典比较模型。然而，由于其胚胎发育的独特特征，有袋类多能性调控架构仍有待全面解析，且迄今尚无任何有袋类物种的胚胎干细胞（embryonic stem cells, ESCs）成功建系的报道。本研究建立了一种无整合型诱导多能干细胞（induced pluripotent stem cell, iPSC）重编程方法，并从两个完全近交系的灰短尾负鼠（Monodelphis domestica）中构建了经过验证的iPSC细胞系。我们对灰短尾负鼠皮肤成纤维细胞及其重编程得到的iPSC开展了全基因组mRNA测序（genome-wide mRNA sequencing）的全面表征。所获得的负鼠iPSC（monoiPSCs）在重编程过程中实现了细胞转录组的显著重编程（共鉴定出6181个差异表达基因（differentially expressed genes, DE genes）），且克隆间一致性极高（95%置信区间内克隆间决定系数r²为0.973 ± 0.007），其整体转录组与功能特征与真哺乳类ESCs及iPSC高度相似。然而，monoiPSCs的核心多能性转录因子调控架构具有独特性，更接近于上胚层细胞。研究结果表明，POU5F1与POU5F3的剪接变体协同调控负鼠多能性基因网络。推测POU5F1、POU5F3剪接变体XM_016427856.1与SOX2可形成自我调控网络。不过，NANOG的表达仅特异性存在于monoiPSCs与上胚层细胞中，且在胚胎细胞中呈现独特的表达谱。此外，POU5F1在滋养层细胞中高表达，而其余多能性转录因子的表达均显著下调，这提示有袋类核心多能性基因的调控架构可能与真哺乳类存在显著差异。总体实验设计：为深入解析有袋类多能性调控架构，本研究对灰短尾负鼠皮肤成纤维细胞及其重编程得到的iPSC开展了全基因组比较基因表达分析。