Experimental Study Of Treatment Of Sarcopenia With Human Umbilical Cord Wharton's Jelly Mesenchymal Stem Cells

Part one:Experimental study about biological characteristics of human umbilical cord Wharton's Jelly mesenchymal stem cells and their differentiation into myogenic cellsObjectiveTo investigate the biological characteristics of mesenchymal stem cells(MSCs)from human umibilical cord Whalton's Jelly,and explore their possibility to differentiate into myogenic cells.And hUCMSCs were transfected with lentivirus carrying green fluorescent protein(GFP)gene to obtain hUCMSCs stably expressing the GFP reporter gene.MethodsFirst of all,we collected healthy and full-term fetal umbilical cord tissue,which was repeatedly washed with phosphate buffered saline(PBS),and cleared the blood vessels within the umbilical cord to leave the Wharton's Jelly tissues.The Wharton's Jelly tissues were cut into tissue sections(1 mm3),hUCMSCs were isolated from human umbilical cord by being digested with type II collagenase.And primary cultured cells were harvested at about 80%-90%confluency after about 10 days and whose cell morphology was observed.The 3rd passage cells were collected,then the immunophenotype of hUCMSCs was detected by fluorescence activated cell sorting(FACS).The expression of Sox2,Oct4 and Nanog in pluripotent stem cells was detected by immunofluorescence.5-azacytidine(5-Aza)was used to induce myogenic differentiation into myogenic cells in vitro.The expression of skeletal muscle stem cell markers Pax-7 and MyoD was detected by immunofluorescence.The lentivirus carrying GFP gene was transfected into hUCMSCs,and FACS was utilized to analyze its transfection rate.ResultsIt could be cultured and proliferated in vitro that a large number of adherent hUCMSCs isolated from human umbilical cord by being digested with collagenase.The primary cultured hUCMSCs showed epithelial-like or short-fibroblast-like morphology,and the passaged hUCMSCs showed typical long spindle-like or fibroblast-like cells.Flow cytometry analysis revealed that hUCMSCs were positive for CD29,CD44,CD73,CD90 and CD105,but had very low expression of CD14,CD34,CD45 and HLA-II.Immunofluorescence staining showed that hUCMSCs could express stem cell markers Oct4 and Sox2,but no expression of Nanog was found.And hUCMSCs express skeletal muscle stem cell markers Pax-7 and MyoD under 5-Aza induced conditions in vitro.After transfection with lentivirus carrying GFP gene,hUCMSCs can stably express GFP.The FACS analysis showed that the transfection rate can be increased to 50-70%,and a large number of hUCMSCs stably expressing the GFP reporter gene can be obtained.ConclusionOur study confirmed that the existence of MSCs in human umbilical cord Wharton's Jelly and they had the capacity of differentiating into myogenic cells.The hUCMSCs stably expressing GFP can be obtained by lentiviral transfection,which lays a solid foundation for the follow-up of hUCMSCs transplantation.Part two:Experimental study of treatment of sarcopenia with human umbilical cord Wharton's Jelly mesenchymal stem cellsObjectiveWe studied the effects of transplanted human umbilical cord Wharton's Jelly mesenchymal stem cells(hUCMSCs)in aged male C57BL/6J mice with sarcopenia induced by hindlimb suspension,and explored the potential mechanism.Methods1.Hindlimb suspension(HLS)was used to induce sarcopenia in 32 C57BL/6J mice(24-month-old).After two weeks of HLS,the posterior necks of mice were subcutaneously embedded with 25 mg of 5-bromo-2'-deoxyuridine(BrdU)sustained release tablets and mice were given freedom of movement.2.Then mice were randomized into tail vein transplant(TVT)group(n=8),muscular transplant(MT)group(n=8),TVT-control group(n=8)and MT-control group(n=8)according to whether stem cells were transplanted or not.hUCMSCs transplant groups were treated directly in both gastrocnemius muscles(0.5 × 106 hUCMSCs/limb in 50 ?l PBS)or via the tail vein(1 × 106 hUCMSCs in 100 ?1 PBS)weekly for 8 weeks.hUCMSCs are passaged for 5 to 6 generations which labeled GFP.Control groups were given an equivalent volume of PBS weekly for 8 weeks.3.Efficacy evaluation:Animals were weighed and sacrificed for studies after 8 weeks of hUCMSCs transplant.Then both sides of the gastrocnemius were immediately separated and weighed.The absolute and relative gastrocnemius muscle mass and cross-sectional areas of muscle fibers were compared between hUCMSCs groups and PBS groups.4.Mechanism investigation:(1)to observe the changes of GFP-labeled hUCMSCs in the gastrocnemius muscle by using fluorescence microscopy;(2)to count the number of nuclei expressing BrdU and Pax-7 by immunofluorescent staining and calculate their expression index;(3)to count the apoptotic nuclei by TUNEL and calculate the apoptotic index;(4)The expression of inflammatory cytokines TNF-a and IL-6,anti-inflammatory cytokines IL-4 was detected by Western blot and ELISA.5.Statistical analyses were performed using SPSS 13.0 software.Data are reported as means ± SD.Differences among means for transplant factors(TVT vs.MT)and treatment(hUCMSCs vs.PBS)were confirmed with two-way ANOVA.A repeated-measures ANOVA was used for longitudinal data from animals before and after HLS,and after hUCMSCs treatment.Statistical significance was set at P<0.05.Results1.The gastrocnemius muscle wet weight and muscle fiber cross-sectional area(CSA)were significantly increased in hUCMSCs-transplanted mice than in controls(P<0.05).Effect of hUCMSCs in sarcopenic mouse model;HLS reduced about 13%of the body weight of aged mice(P>0.05),but this was not significantly different among four groups.After 8 weeks of hUCMSCs transplant,weight continued to decrease,mice treated with hUCMSCs had less weight loss than controls,but this was not significant(P>0.05).hUCMSCs transplant enhanced absolute/relative gastrocnemius muscle mass and CSA of myofibers in hUCMSCs-transplanted mice compared to controls(P<0.05).Increased gastrocnemius muscle mass was more prominent in muscular transplant(MT)vs.tail vein transplant(TVT)groups(P<0.05).2.(1)GFP-labeled hUCMSCs were not found under fluorescence microscope;(2)Expression index of BrdU and Pax-7:hUCMSCs significantly increased BrdU and Pax-7 expression in the gastrocnemius muscle(MT:P<0.001,TVT:P<0.01;compared to relative control).Increased Pax-7 was more prominent in MT group compared to TVT group(P<0.05),but expression of BrdU was not different between the two groups.3.Nuclear apoptotic indices:Apoptotic indices indicated by TUNEL-positive myonuclei were significantly reduced after hUCMSC transplant(MT:P<0.001,TVT:P<0.01;compared to relative control).This reduction was more obvious in MT than TVT group,but there was no significant difference between the two groups(P>0.05).4.Cytokine expression:hUCMSC transplant significantly reduced expression of TNF-? and IL-6 and increased expression of IL-4 in the gastrocnemius muscle of TVT and MT groups(P<0.05).Conclusion1.hUCMSCs may ameliorate sarcopenia in aged male C57BL/6J mice induced by hindlimb suspension which significantly improved the weight of the gastrocnemius and cross-sectional area of the muscle fibers.2.hUCMSCs may be via activation of resident skeletal muscle satellite cells,reduction of apoptosis,and less chronic inflammation to promote muscle fiber proliferation in elderly.