WNT and VEGF/PDGF signaling regulate self-renewal in primitive mesenchymal stem cells

Therapeutic use of mesenchymal stem cells (MSCs) is hampered due to poor growth and self-renewal potential. They are also affected by passaging. This study found that xeno-free medium (XM) maintained morphology, self-renewal, and differentiation potential in primitive (p) MSCs. In contrast, growth in fetal bovine serum medium (FM) resulted in gradual changes in morphology, differentiation capability and reduced colony-forming efficiency. Transcriptomic analysis showed upregulation of genes involved in self-renewal, cell cycle, and DNA replication in XM-grown pMSCs. However, differentiation and senescence genes were upregulated in FM-grown cells. The expression of selected genes was corroborated by qRT-PCR analyses. We also examined changes in chromatin structure by MNase-seq. We proposed WNT and VEGF/PDGF signaling pathways are involved in self-renewal and TGFB, and PI3K signaling pathways are responsible for the induction of senescence in pMSCs. Our findings may help expand pMSCs for large-scale preclinical and clinical studies, which have been limited thus far.

间充质干细胞（mesenchymal stem cells, MSCs）的治疗应用受到增殖能力弱、自我更新潜能不足的制约，且其生物学特性易受传代培养的影响。本研究发现，无血清培养基（xeno-free medium, XM）可维持原始间充质干细胞（primitive (p) MSCs）的细胞形态、自我更新能力与分化潜能。与之相对，在胎牛血清培养基（fetal bovine serum medium, FM）中培养的细胞会逐渐出现形态改变，分化能力下降，且集落形成效率降低。转录组分析结果显示，经XM培养的pMSCs中，参与自我更新、细胞周期调控及DNA复制的基因表达显著上调；而FM培养的细胞中，与细胞分化及衰老相关的基因则呈现表达上调趋势。我们通过实时荧光定量逆转录聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）验证了部分筛选基因的表达水平。此外，本研究还通过微球菌核酸酶测序（micrococcal nuclease sequencing, MNase-seq）检测了染色质结构的动态变化。本研究提出，WNT信号通路与VEGF/PDGF信号通路参与调控pMSCs的自我更新过程，而转化生长因子β（Transforming Growth Factor beta, TGFB）及磷脂酰肌醇3-激酶（Phosphoinositide 3-kinase, PI3K）信号通路则介导了pMSCs的衰老诱导。本研究结果可为实现pMSCs的大规模体外扩增提供理论支撑，以解决此前限制大规模临床前及临床研究的细胞来源瓶颈问题。