The Mechanism Of 3D Culture Decrease The Size Of Mesenchymal Stem Cells

Mesenchymal stem cell(MSC)is multipotent stem cell,which can be derived from the bone marrow,umbilical cord,adipose tissue and other tissue sources.They have special biological characteristics,such as long-term proliferation ability,ability to differentiate into different cell lines and secretion of a variety of cytokines.In addition,MSC have low immunogenicity thus devoid of immune rejection after allogeneic transplantation.Numerous previous studies have indicated that MSC promote the repair and regeneration of various tissues,and over 600 clinical trials world wide for different diseases are undergoing.However,cultured MSC have a serious problem that has affected their clinical use.MSC amplified in 2D condition cause severe pulmonary vascular obstruction after intravenous injection,leading to reduced levels of circulating MSC in the blood,low homing and engraftment of MSC to injured tissues,thus impairing their therapeutic efficacy.The lung obstruction of MSC is largely caused by enlargement of MSC after ex vivo expansion in 2D culture.Our earlier studies have shown that 3D culture can greatly reduce the volume of MSC which had been cultured for passages in 2D condition,thus reducing pulmonary obstruction and enhancing homing and engraftment of MSC to ischemic myocardium;however,the mechanisms underlying the cell size reduction are not clear.In the present study,we found that MSC cultured under 3D culture condition,treated with actin cytoskeleton depolymerized agent Cytochalasin D or functionally blocked with an antibody against integrin beta 1,showed depolymerized actin cytoskeleton,significantly reduced levels of extended parallel shuttle actin fiber bundles,and decreased amounts of F-actin.Meanwhile,the volume of MSC gradually but significantly reduced under these conditions.Our results further showed that MSC after the above treatments produced and released more extracellular vesicles(EVs),which contained higher levels of actin.Importantly,associated with these changes,MSC gradually reduced their volume,which was particularly evident in the first 36 hours of 3D culture.And consistently,MSC gradually increased the release of extracellular vesicles in the first 36 hours of 3D culture,suggested that the degree of MSC volume decrease is associated with the increase of the vesicle release which contained higher levels of actin.In conclusion,we have demonstrated for the first time that MSC under 3D culture condition reduce their cell size by increasing vesicle excretion,which is mediated by depolymerized actin cytoskeleton,and decreased F-actin expression.Our findings are important in understaning the mechnisms of MSC size reduction after 3D culture,and the benefit of 3D cultured MSC in trafficking and homing to tissue injuries.