Pathologic Bladder Microenvironment Attenuates Smooth Muscle Differentiation of Skin Derived Precursor Cells: Implications for Tissue Regeneration

Smooth muscle cell containing organs (bladder, heart, blood vessels) are damaged by a variety of pathological conditions necessitating surgery or organ replacement. Currently, regeneration of contractile tissues is hampered by lack of functional smooth muscle cells. Multipotent skin derived progenitor cells (SKPs) can easily be isolated from adult skin and can be differentiated in vitro into contractile smooth muscle cells by exposure to FBS. Here we demonstrate an inhibitory effect of a pathologic contractile organ microenvironment on smooth muscle cell differentiation of SKPs. In vivo, urinary bladder strain induces microenvironmental changes leading to de-differentiation of fully differentiated bladder smooth muscle cells. Co-culture of SKPs with organoids isolated from ex vivo stretched bladders or exposure of SKPs to diffusible factors released by stretched bladders (e.g. bFGF) suppresses expression of smooth muscle markers (alpha SMactin, calponin, myocardin, myosin heavy chain) as demonstrated by qPCR and immunofluorescent staining. Rapamycin, an inhibitor of mTOR signalling, previously observed to prevent bladder strain induced de-differentiation of fully differentiated smooth muscle cells in vitro, inhibits FBS-induced smooth muscle cell differentiation of undifferentiated SKPs. These results suggest that intended precursor cell differentiation may be paradoxically suppressed by the disease context for which regeneration may be required. Organ-specific microenvironment contexts, particularly prevailing disease, may play a significant role in modulating or attenuating an intended stem cell phenotypic fate, possibly explaining the variable and inefficient differentiation of stem cell constructs in in vivo settings. These observations must be considered in drafting any regeneration strategies.含平滑肌细胞的器官（膀胱、心脏、血管）可因多种病理状态受损，进而需接受手术治疗或器官置换。当前，功能性平滑肌细胞的匮乏严重阻碍了收缩性组织的再生修复进程。多能皮肤来源祖细胞（Skin Derived Progenitor Cells, SKPs）可从成年皮肤中便捷分离，且可在体外通过胎牛血清（Fetal Bovine Serum, FBS）诱导分化为收缩性平滑肌细胞。本研究证实，病理状态下的收缩器官微环境会对SKPs的平滑肌细胞分化产生抑制作用。在体内环境中，膀胱机械应力可诱导微环境发生改变，进而导致完全分化的膀胱平滑肌细胞出现去分化现象。通过将SKPs与离体拉伸膀胱分离得到的类器官共培养，或将SKPs暴露于拉伸膀胱释放的可扩散因子（如碱性成纤维细胞生长因子（basic Fibroblast Growth Factor, bFGF））中，均可抑制平滑肌标志物（α平滑肌肌动蛋白（alpha Smooth Muscle Actin, α-SMA）、钙调蛋白、心肌素、肌球蛋白重链）的表达，该结论已通过实时定量PCR（quantitative Real-Time Polymerase Chain Reaction, qPCR）与免疫荧光染色得以验证。雷帕霉素作为哺乳动物雷帕霉素靶蛋白（mammalian Target of Rapamycin, mTOR）信号通路的抑制剂，此前被证实可在体外阻断膀胱机械应力诱导的完全分化平滑肌细胞去分化；本研究发现其同样可抑制FBS诱导的未分化SKPs平滑肌细胞分化。上述结果表明，本应用于组织再生的前体细胞分化，反而可能因再生所需的疾病微环境受到抑制，这一现象看似矛盾。器官特异性微环境，尤其是当前存在的疾病状态，可能在调控或削弱预定的干细胞表型命运方面发挥关键作用，这或许可以解释为何体内环境中干细胞构建体的分化效率低下且结果不稳定。在制定任何再生修复策略时，均需充分考量上述研究发现。