Analysis Of The Composition Of Cell Subsets In Skeletal Muscle Tissue And Their Role In Skeletal Muscle Regeneration

Skeletal muscle accounts for 40%to 50%of the body weight of the adult animals.It is a vital motion generation organ of human body.It is also an indispensable component of the musculoskeletal system.The skeletal muscle with a strong regeneration ability.Skeletal muscle stem cells that is satellite cells(SCs)play important role in skeletal muscle regeneration.Muscle stem cells,also known as satellite cells,are located between the muscle fiber membrane and the basement membrane in adult skeletal muscle.SCs are in a resting state under normal conditions.However,when muscles are damaged,SCs are activated into the cell cycle,and rapid division produces a large number of myogenic progenitor cells.MPCs differentiate to form new myotubes to complete the repair of damaged muscles.Dealing with minor injuries(such as muscle strain,sharp cuts,etc.),skeletal can be repaired by SCs under the control of microenvironment(necrotic muscle fibers,extracellular matrix and growth factors),however with severe muscle defects(such as surgical resection),which was also called muscle Volumetric muscle loss(VML),it could not be repaired but form a large number of scar tissue in the damage area.At present,the clinical treatment of VML is limited.For existing treatments,it is impossible to restore damaged muscle to pre-injury function levels.Therefore,it is urgent to find a new VML treatment method and to reveal the mechanisms under the skeletal muscle regeneration.The goal of tissue engineering is to repair or replace the damaged tissues by combining seed cell with biocompatible scaffolds and culturing them in a specific condition.It is with great potential to repair VML.However,lack of knowledge about skeletal muscle regeneration and repair mechanisms limits the development of muscle tissue engineering.Extracellular matrix(ECM),growth factors and skeletal muscle interstitial cells constitute the SCs microenvironment?Skeletal muscle ECM not only provides a binding site for SCs and myotubes,but also glycoproteins in ECM facilitates the attachment of growth factor precursors,providing an environmentally friendly environment for SCs to sense external mechanical stimulation and growth factor regulation.Recently,a variety of interstitial cells have been found in skeletal muscle,such as side population cells,PW1+cells,fibro/adipogenetic progenitors(FAPs),etc.Some of them promote myogenic effects during muscle development and regeneration,such as fibro/adipogenetic progenitors(FAPs),Tcf4+cells.However,is the decellularized skeletal muscle ECM still able to play a role in regulating SCs?What is the mechanisms that some interstitial cells play a positive role in myogenic processes?These question still not clear nowTherefore,the purpose of this study is:(1)To explore the interaction between MPCs and skeletal muscle ECM;(2)To study the role of rapidly adhering cells in skeletal muscle in the process of myogenesis;(3)To analyze skeletal muscle cell atlas by single cell transcriptome sequencing and mechanisms of rapidly adhering cells in myogenesis.Based on this,my study is divided into the following three partsPart I.To explore the interaction between mice MPCs and skeletal muscle ECMIn order to investigate the effects of skeletal muscle ECM on the growth and differentiation of MPCs,we combined acellular matrix slices as tissue engineering muscle scaffold muscle with muscle progenitor cells(MPCs),then induce them to differentiate into tissue engineered muscle bundles.We found that MPCs could attached,grow,and differentiated on skeletal muscle acellular scaffolds.However,the resulting tissue engineered muscle bundles with low myogenic efficiency and without the basic physiological functions of natural skeletal muscle which suggested that only MPCs and extracellular matrix and basic myogenic differentiation culturing environment are not enough,though MPCs play a key role in skeletal muscle regeneration.Part ?.The function of mice RACs in the process of myogenesisSkeletal muscle regeneration is a process coordinated by various cell types.Previous studies have found that skeletal muscle interstitial cells,such as fibro/adipogenic progenitors and Tcf4+,play an important role in the regulation of muscle stem cell division,self-renewal,and differentiation.However,the role of rapidly adhering cells(RACs)in muscle during muscle regeneration remains unclear.In this chapter,we first constructed a skeletal muscle organ culture system in which RACs were co-cultured with myogenic progenitor cells(MPCs)to induce myogenic differentiation.The immunofluorescence result of myosin heavy chain(MHC)staining showed that RACs could promote the expression of MHC in myogenic differentiation of MPCs.The transmission electron microscopy results of skeletal muscles organoid showed that RACs could promote the formation of sarcomeres.Moveheat results,physiological quantification of skeletal muscle organs,showed that the RACs and MPCs co-culture group skeletal muscle organoids were significantly stronger than mono-culture group.The above results show that RACs can significantly improve the myogenic efficiency of MPCs in skeletal muscle organ culture systems.At the same time,Skeletal muscle organoid technology provided a new model for studying muscle regeneration.Part?.Dissecting cell diversity and connectivity in skeletal muscle for myogenesisThe mechanisms under the phenomenon that RACs play positive role in promoting myogenesis capability of MPCs in the skeletal muscle organ formation process is still unknown.To explore this,firstly,we analyzed the cell types in RACs by single-cell transcriptome sequencing technique.The results showed that there are 7 subpopulations constructed RACs,one of which are new cell types:teno-myogenic cells(TMCs)Based on the single cell transcriptome sequencing data,the relationship between cell MPCs and RACs was calculated by the in silico receptor-ligand pairing number method.The calculation results showed that extracellular matrix proteins and growth factors(VEGFA and HBEGF)secreted by RACs are involved in the myogenic process of skeletal muscle organs.Finally,we verified the prediction of the heat map of receptor-ligand pairing relationships between RACs and MPCs by trans-well experiments.In this chapter,we first reported the 7 cell subpopulations of RACs.More importantly,the mechanism of RACs promote the myogenesis capability of MPCs provides clues for strategy of skeletal muscle tissue engineering.