背景:染色体稳定性对于多能干细胞(PSC)的稳态和早期胚胎发育至关重要。当使用PSC作为原始材料时,染色体缺陷可能会增加再生医学中的致癌风险。然而,关于PSC染色体稳定性维持的详细机制尚不完全清楚。
方法:在标准条件下培养小鼠胚胎干细胞(细胞系D3)和人胚胎干细胞(细胞系H9)。为了证实RetSat蛋白在PSC有丝分裂染色体上的负载,在小鼠胚胎成纤维细胞(MEFs)的PSC自发分化试验和iPSC重编程试验中进行免疫染色,分别。此外,qPCR,免疫沉淀,LC-MS/MS和免疫印迹法研究RetSat的表达,以及RetSat与共粘素/凝缩素组分的相互作用。进行RNA测序和畸胎瘤形成测定以评估具有RetSat缺失的小鼠胚胎干细胞的致癌风险。
结果:我们报道了一个PSC高表达基因,RetSat,在染色体稳定中起着关键作用。我们鉴定了位于有丝分裂染色体上的RetSat蛋白,特别是在干细胞阳性细胞中,例如胚胎干细胞(ESC)和诱导多能干细胞(iPSC)。我们发现染色体不稳定,例如染色体桥接,当下调RetSat时,小鼠和人ESCs中的滞后和相间微核。RetSat敲除小鼠ESCs上调癌症相关基因通路,并在畸胎瘤形成试验中显示出较高的致瘤能力。机械上,我们证实RetSat与粘附素/凝缩素成分Smc1a和Nudcd2相互作用。RetSat缺失损害了Smc1a的染色体负荷剂量,Smc3和Nudcd2。
结论:总之,我们报道RetSat是多能干细胞染色体凝聚的关键稳定剂.这突出了RetSat在早期胚胎发育中的关键作用,以及RetSat作为评估多能干细胞质量的有效生物标志物的潜在价值。
BACKGROUND: Chromosome stability is crucial for homeostasis of pluripotent stem cells (PSCs) and early-stage embryonic development. Chromosomal defects may raise carcinogenic risks in regenerative medicine when using PSCs as original materials. However, the detailed mechanism regarding PSCs chromosome stability maintenance is not fully understood.
METHODS: Mouse embryonic stem cells (line D3) and human embryonic stem cells (line H9) were cultured under standard conditions. To confirm the loading of RetSat protein on mitotic chromosomes of PSCs, immunostaining was performed in PSCs spontaneous differentiation assay and iPSC reprogramming assay from mouse embryonic fibroblasts (MEFs), respectively. In addition, qPCR, immunoprecipitation, LC-MS/MS and immunoblotting were used to study the expression of RetSat, and interactions of RetSat with cohesin/condensin components. RNA sequencing and teratoma formation assay was conducted to evaluate the carcinogenic risk of mouse embryonic stem cells with RetSat deletion.
RESULTS: We reported a PSC high-expressing gene, RetSat, plays key roles in chromosome stabilization. We identified RetSat protein localizing onto mitotic chromosomes specifically in stemness positive cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found dramatic chromosome instability, e.g. chromosome bridging, lagging and interphase micronuclei in mouse and human ESCs when down regulating RetSat. RetSat knock-out mouse ESCs upregulated cancer associated gene pathways, and displayed higher tumorigenic capacities in teratoma formation assay. Mechanistically, we confirmed that RetSat interacts with cohesin/condensin components Smc1a and Nudcd2. RetSat deletion impaired the chromosome loading dosage of Smc1a, Smc3 and Nudcd2.
CONCLUSIONS: In summary, we reported RetSat to be a key stabilizer of chromosome condensation in pluripotent stem cells. This highlights the crucial roles of RetSat in early-stage embryonic development, and potential value of RetSat as an effective biomarker for assessing the quality of pluripotent stem cells.