Thummarati Parichut, Kino-Oka Masahiro
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.
Front Bioeng Biotechnol. 2020 Sep 23;8:578140. doi: 10.3389/fbioe.2020.578140. eCollection 2020.
Skeletal muscle comprises a heterogeneous population of myoblasts and fibroblasts. Autologous skeletal muscle myoblasts are transplanted to patients with ischemia to promote cardiac regeneration. In damaged hearts, various cytokines secreted from the skeletal muscle myoblasts promote angiogenesis and consequently the recovery of cardiac functions. However, the effect of skeletal muscle fibroblasts co-cultured with skeletal muscle myoblasts on angiogenic cytokine production and angiogenesis has not been fully understood. To investigate these effects, production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) was measured using the culture medium of monolayers prepared from various cell densities (mono-culture) and proportions (co-culture) of human skeletal muscle myoblasts (HSMMs) and human skeletal muscle fibroblasts (HSMFs). HSMM and HSMF mono-cultures produced VEGF, whereas HSMF mono-culture produced HGF. The VEGF productivity observed in a monolayer comprising low proportion of HSMFs was two-fold greater than that of HSMM and HSMF mono-cultures. The production of VEGF in HSMMs but not in HSMFs was directly proportional to the cell density. VEGF productivity in non-confluent cells with low cell-to-cell contact was higher than that in confluent cells with high cell-to-cell contact. The dynamic migration of cells in a monolayer was examined to analyze the effect of HSMFs on myoblast-to-myoblast contact. The random and rapid migration of HSMFs affected the directional migration of surrounding HSMMs, which disrupted the myoblast alignment. The effect of heterogeneous populations of skeletal muscle cells on angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) incubated with fabricated multilayer HSMM sheets comprising various proportions of HSMFs. Co-culturing HSMFs in HSMM sheet at suitable ratio (30 or 40%) enhances endothelial network formation. These findings indicate the role of HSMFs in maintaining cytokine balance and consequently promoting angiogenesis in the skeletal muscle cell sheets. This approach can be used to improve transplantation efficiency of engineered tissues.
骨骼肌由成肌细胞和平滑肌细胞组成的异质群体构成。自体骨骼肌成肌细胞被移植到缺血患者体内以促进心脏再生。在受损心脏中，骨骼肌成肌细胞分泌的多种细胞因子促进血管生成，从而促进心脏功能的恢复。然而，与骨骼肌成肌细胞共培养的骨骼肌成纤维细胞对血管生成细胞因子产生和血管生成的影响尚未完全了解。为了研究这些影响，使用由不同细胞密度（单培养）和人骨骼肌成肌细胞（HSMMs）与人骨骼肌成纤维细胞（HSMFs）比例（共培养）制备的单层培养基来测量血管内皮生长因子（VEGF）和肝细胞生长因子（HGF）的产生。HSMM和HSMF单培养产生VEGF，而HSMF单培养产生HGF。在包含低比例HSMFs的单层中观察到的VEGF生产力比HSMM和HSMF单培养高两倍。HSMMs中而非HSMFs中VEGF的产生与细胞密度成正比。细胞间接触少的非汇合细胞中的VEGF生产力高于细胞间接触多的汇合细胞。检查单层中细胞的动态迁移以分析HSMFs对成肌细胞与成肌细胞接触的影响。HSMFs的随机快速迁移影响周围HSMMs的定向迁移，从而破坏成肌细胞排列。使用与包含不同比例HSMFs的人造多层HSMM片一起孵育的人脐静脉内皮细胞（HUVECs）评估骨骼肌细胞异质群体对血管生成的影响。以合适比例（30%或40%）在HSMM片中共同培养HSMFs可增强内皮网络形成。这些发现表明HSMFs在维持细胞因子平衡并因此促进骨骼肌细胞片中血管生成方面的作用。这种方法可用于提高工程组织的移植效率。
