IPSCs Induction And Directional Differentiation Of Sheep Umbilical Cord Mesenchymal Stem Cells

Umbilical Cord Mesenchymal Stem Cells(UCMSCs)are stem cells having capacities of self-renewing and multilineage differentiation.UCMSCs are characterized by rich source,uninvolved ethics and morals,low immunogenicity,strong multiplication ability in vitro and differentiation into various somatic cells,somuch attention was paid to relative studies.At present,researches or reports related to the isolation,cultivation and induced differentiation of sheep sUCMSCs are very limited although UCMSCs from human,rats,rabbits,pigs and other animals have been obtained.Sheep is important large animal model in basic biology and animal husbandry.This research,based on the improved isolation and cultivation systems,investigated the specialties of inducing sUCMSCs to Induced Pluripotent Stem Cells,iPSCs(iPSCs),the directed differentiation of sUCMSCs,and the mechanisms in vivo of sUCMSCs repairing damaged tissues,the experimental results showed as follows:1.UCMSCs were isolated with tissue block secondary adherence method and sUCMSCs were amplified in vitro with serum free culture system.Results showed that immunophenotyping of sUCMSCs obtained,showed a higher expression of CD90,CD 105 and CD44(expression quantity>95%)and a lower expression of CD45,CD34 and CD 14(expression quantity<2%).sUCMSCs obtained by tissue block secondary adherence showed a continuous passage after P12,and had ability to osteoblast,lipoblast and chondroblast.2.iPSCs were induced from sUCMSCs by using OSKM-four factors,Yamanaka.-Embryonic Stem Cells(ESCs)like colonies were obtained and identified after alkaline phosphatase staining.The results showed a positive colony with brownish red.Pluripotent factors expression of the cloning cells above was tested with results showing the clones expressed pluripotent factor OCT-4,S SEA-4 and Nanog.To detect the differentiation ability in vivo of clones of sUCMSCs,the clones were prepared into single-cell suspension,which were then injected into the immunodeficient mice under the arm.Teratoma tissues were collected and made into paraffin section for HE staining on week 4 after injection.Under inverted microscope,it was observed that tissues,such as muscles,hair follicles and glands,three germ layers have formed from the teratoma.3.The method of 5-azacitidine combined with horse serum was used to induce the sUCMSCs into myoblast.The expression status of myoblast labelled proteins(Desmin,MyHC and MyoD)and labelled molecules(Desmin,Myf5 and MyoG)in different differentiating phases were detected.In the injured tibialis anterior muscles,the muscle damaged model mice were injected with sUCMSCs,myoblast on the 14th day of differentiation and mitomycin C treated sUCMSCs.The mice tibialis anterior muscles at different time point after transplantion were collected and made into paraffin section for HE staining.The repair of damaged muscles in mice was examined.Results showed as followed:(1)During the induction differentiation,sUCMSCs,on the 3th day,Myf5 was first expressed;on the 7th day,some cells got their cellular morphology changed in rod,polygonal shape,fused and expressed myocyte labelled protein Desmin,MyHC and MyoD;on the 14th day,Desmin and MyoG gene expression increased,cell proliferation continued to slow down,fusion cells increased;on the 21th day,myotube-like cells appeared and Desmin gene expression increased sharply;(2)Repair phase started on the 2nd day after the transplant of sUCMSCs into damaged muscles in the mice;on the 10th day,the muscle fibers were arranged densely and blended with each other,and the damaged muscles basically returned to normal.After transplantation into the damaged muscles of mice,the differentiated myoblast entered into its repair phase on the 3rd day.The muscle fibers,on the 14th day,are arranged compact and blended with each other,and damaged muscles basically returned to normal.Mitomycin C treated sUCMSCs and control group started their restoration on day 2-3,and also no difference was found in the repairation of damaged muscles on day 10 and 14.In conclusion,sUCMSCs with normal form and in vitro proliferation ability,potential for multi-directional differentiation and capability of expressing MSC surface labelled molecule could be produced by using secondary adherence and serum free culture system.The obtained SUCMSCs could not only be induced into iPSCs,but also differentiate into myoblast that may repair the damaged muscles in mice.This study provided scientific basis for establishing perfect sheep iPS technology and exploring the mechanism of directional differentiation and repairing of MSCs.