背景:成人骨骼肌含有具有高成肌和植入潜力的常驻肌肉干细胞(MuSC),使它们适用于细胞治疗和再生医学方法。然而,MuSC的纯化过程仍然是其在临床中使用的主要障碍。的确,肌肉组织酶解离引发应激信号通路的大量激活,其中P38和JNKMAPK,与MuSC静止的过早丧失有关。虽然这些途径在MuSC的肌源性进展中的作用已经确立,它们的解离诱导的激活对这些细胞功能的影响程度仍有待研究。
方法:我们通过药理学方法评估了P38和JNKMAPK诱导对干细胞标记表达和MuSC激活状态的影响。通过体外测定和体内移植实验评价MuSC功能性。我们对P38和JNKMAPK(分别为SB202190和SP600125)的药理学抑制剂纯化的人MuSC的转录组与可用的RNAseq资源进行了比较分析。
结果:我们监测了肌肉解离过程中鼠MuSC中的PAX7蛋白水平,并显示出两步下降,部分取决于P38和JNKMAPK活性。我们表明,在整个MuSC分离过程中同时抑制这些途径可以保留干性标记的表达并限制其过早激活,导致体外存活和扩增的改善以及体内植入的增加。通过对新鲜分离的人MuSC的比较RNAseq分析,我们提供的证据表明,我们在鼠MuSC中的发现可能与人类MuSC相关.基于这些发现,我们实施了净化策略,显着提高人MuSC的回收率。
结论:我们的研究强调了P38和JNKMAPK活性的药理学限制,作为定性和定量改善人类MuSC纯化过程的合适策略,这可能对基于细胞的疗法非常感兴趣。
BACKGROUND: Adult skeletal muscle contains resident muscle stem cells (MuSC) with high myogenic and engraftment potentials, making them suitable for cell therapy and regenerative medicine approaches. However, purification process of MuSC remains a major hurdle to their use in the clinic. Indeed, muscle tissue enzymatic dissociation triggers a massive activation of stress signaling pathways, among which P38 and JNK MAPK, associated with a premature loss of MuSC quiescence. While the role of these pathways in the myogenic progression of MuSC is well established, the extent to which their dissociation-induced activation affects the functionality of these cells remains unexplored.
METHODS: We assessed the effect of P38 and JNK MAPK induction on stemness marker expression and MuSC activation state during isolation by pharmacological approaches. MuSC functionality was evaluated by in vitro assays and in vivo transplantation experiments. We performed a comparative analysis of the transcriptome of human MuSC purified with pharmacological inhibitors of P38 and JNK MAPK (SB202190 and SP600125, respectively) versus available RNAseq resources.
RESULTS: We monitored
PAX7 protein levels in murine MuSC during muscle dissociation and demonstrated a two-step decline partly dependent on P38 and JNK MAPK activities. We showed that simultaneous inhibition of these pathways throughout the MuSC isolation process preserves the expression of stemness markers and limits their premature activation, leading to improved survival and amplification in vitro as well as increased engraftment in vivo. Through a comparative RNAseq analysis of freshly isolated human MuSC, we provide evidence that our findings in murine MuSC could be relevant to human MuSC. Based on these findings, we implemented a purification strategy, significantly improving the recovery yields of human MuSC.
CONCLUSIONS: Our study highlights the pharmacological limitation of P38 and JNK MAPK activities as a suitable strategy to qualitatively and quantitatively ameliorate human MuSC purification process, which could be of great interest for cell-based therapies.