Degeneration of smooth muscles in sphincters can cause debilitating diseases such as fecal incontinence. Skeletal muscle-derived cells have been effectively used in clinics for the regeneration of the skeletal muscle sphincters, such as the external anal or urinary sphincter. However, little is known about the in vitro smooth muscle differentiation potential and in vivo regenerative potential of skeletal muscle-derived cells. Myogenic progenitor cells (MPC) were isolated from the skeletal muscle and analyzed by flow cytometry and in vitro differentiation assays. The differentiation of MPC to smooth muscle cells (MPC-SMC) was evaluated by immunofluorescence, flow cytometry, patch-clamp, collagen contraction, and microarray gene expression analysis. In vivo engraftment of MPC-SMC was monitored by transplanting reporter protein-expressing cells into the pyloric sphincter of immunodeficient mice. MPC derived from human skeletal muscle expressed mesenchymal surface markers and exhibit skeletal myogenic differentiation potential in vitro. In contrast, they lack hematopoietic surface marker, as well as adipogenic, osteogenic, and chondrogenic differentiation potential in vitro. Cultivation of MPC in smooth muscle differentiation medium significantly increases the fraction of alpha smooth muscle actin (aSMA) and smoothelin-positive cells, while leaving the number of desmin-positive cells unchanged. Smooth muscle-differentiated MPC (MPC-SMC) exhibit increased expression of smooth muscle-related genes, significantly enhanced numbers of CD146- and CD49a-positive cells, and in vitro contractility and express functional Cav and Kv channels. MPC to MPC-SMC differentiation was also accompanied by a reduction in their skeletal muscle differentiation potential. Upon removal of the smooth muscle differentiation medium, a major fraction of MPC-SMC remained positive for aSMA, suggesting the definitive acquisition of their phenotype. Transplantation of murine MPC-SMC into the mouse pyloric sphincter revealed engraftment of MPC-SMC based on aSMA protein expression within the host smooth muscle tissue. Our work confirms the ability of MPC to give rise to smooth muscle cells (MPC-SMC) with a well-defined and stable phenotype. Moreover, the engraftment of in vitro-differentiated murine MPC-SMC into the pyloric sphincter in vivo underscores the potential of this cell population as a novel cell therapeutic treatment for smooth muscle regeneration of sphincters.

中文翻译：

---

产生肌源性祖细胞来源的平滑肌细胞，用于括约肌再生。

括约肌平滑肌的退化会导致大便失禁等使人衰弱的疾病。骨骼肌来源的细胞已在临床中有效地用于骨骼肌括约肌的再生，例如肛门外括约肌或尿道括约肌。然而，人们对骨骼肌来源细胞的体外平滑肌分化潜力和体内再生潜力知之甚少。从骨骼肌中分离出肌源性祖细胞（MPC），并通过流式细胞术和体外分化测定进行分析。通过免疫荧光、流式细胞术、膜片钳、胶原收缩和微阵列基因表达分析评估MPC向平滑肌细胞（MPC-SMC）的分化。通过将表达报告蛋白的细胞移植到免疫缺陷小鼠的幽门括约肌中来监测 MPC-SMC 的体内植入。源自人骨骼肌的 MPC 表达间充质表面标记，并在体外表现出骨骼肌原性分化潜力。相反，它们缺乏造血表面标志物，以及体外成脂、成骨和软骨形成分化潜力。在平滑肌分化培养基中培养 MPC 显着增加 α 平滑肌肌动蛋白 (aSMA) 和平滑蛋白阳性细胞的比例，同时保持结蛋白阳性细胞的数量不变。平滑肌分化的 MPC (MPC-SMC) 表现出平滑肌相关基因表达增加、CD146 和 CD49a 阳性细胞数量显着增加、体外收缩性和表达功能性 Cav 和 Kv 通道。MPC 向 MPC-SMC 的分化还伴随着其骨骼肌分化潜力的降低。去除平滑肌分化培养基后，大部分 MPC-SMC 仍呈 aSMA 阳性，表明其表型已明确获得。将小鼠 MPC-SMC 移植到小鼠幽门括约肌中，显示基于宿主平滑肌组织内 aSMA 蛋白表达的 MPC-SMC 植入。我们的工作证实了 MPC 产生具有明确且稳定表型的平滑肌细胞 (MPC-SMC) 的能力。此外，体外分化的小鼠 MPC-SMC 在体内植入幽门括约肌，强调了该细胞群作为括约肌平滑肌再生的新型细胞治疗方法的潜力。