Study On The Therapy Of Lung Injury By Type ? Alveolar Epithelial Cells Induced From Mesenchymal Stem Cells

By conducting biological characteristics and inducing differentiation of stem cells not only enrich research contents of stem cells saving animal genetic resources,but also provide new application in these stem cells.Pulmonary-derived mesenchymal stem cells(PMSCs)with active proliferative capacity to proliferate in vitro and differentiate into numerous functional type ? alveolar epithelial cells that has become one of the feasible ways to solve the respiratory diseases.In recent years,the commonly used method of enforced over expression of exogenous transcription factor which can regulate cell differentiation and cell fate determination is time consuming.Moreover,the expression levels of the exogenous genes in cells is out of control which present a formidable obstacle to the therapeutic use of induced cells in clinic applications for lack of security.CRISPR/Cas9 system has arised a new idea for the differentiation of cells.This study mainly established PMSCs line and CSPCs line of livestock and avian embryos,studied the biological characteristics and multi-differentiation potential.And,treated PMSCs derived from pig embryo as the object of this research,we try to use the CRISPR/Cas9-mediated gene activation system to induce their differentiation toward AT ? and evaluate AT ? functionality of lung wound healing,the main results were as follows:1.The fetal pig PMSCs,fetal sheep PMSCs,goose embryo PMSCs and fetal sheep CSPCs were isolated using collagenase digestion and could be substantially passaged in vitro.The morphology of the cells displayed typical fibroblast-like patterns.And,RT-PCR,immunofluorescence and flow cytometry analysis demonstrated that the PMSCs could express mesenchymal marker genes CD29,CD44,CD71,CD73,CD90 and CD105.The CSPCs of the fetal sheep were positive for specific marker genes CD29,CD166,ITGB1,ALCAM,FGFR3,COL1A2 and SOX9.Colony-forming cell assay and cell population dynamics assay of homogenous population have proved that PMSCs possessed the self-renewal and proliferation capability.In addition,the isolated cells can be induced to differentiate not only into mesoblastic cells such as adipocytes,osteoblasts and chondrocytes in vitro,but also cells of non-mesenchymal origin.2.In comparison with SAGM induction group and PneumaCult?-ALI medium induction group independently,conversion of PMSCs into AT ? by CRISPR/Cas9-mediated activators could achieve more effective inductive effects in three differentiation experiments.Here we present a predictive system Mogrify to predict the transcription factors necessary to induce cell conversion.And,the key transcription factors were currently identified by q-PCR experimental testing.Western blot experiments and q-PCR detection of pro-SPC protein and SPC,SPB mRNA expression levels were obviously increased in the differentiated cells by the combined effect of the fused VPR with dCas9 and a synthetic single-guide RNA in activating transcription factors GATA6,HOXA5 and NR2F1 expression.Flow cytometry analyses of PMSCs for their percentage or extents of differentiated cells demonstrated that the results were significantly elevated by CRISPR/Cas9-mediated activators.And,the differentiated PMSCs presented epithelia-like cobblestone cell morphology.Immunofluorescence micrographs demonstrated that the induced cells could express SFTPC,SPC and SPB marker genes of type ? alveolar epithelial cells.In addition,lamellar bodies of AT ? were identified through transmission electron micrographs.3.Bleomycin which was instilled into the tracheal lumen caused mice lung injury characterized by pulmonary inflammation or fibrosis.And,transplanted PMSCs and AT ? through caudal vein expressing phenotypic markers of type ? alveolar epithelial cells were observed in lung sections.Both transplantation of PMSCs and AT ? efficiently participated in lung tissue repair.Morever,transplantation of AT ? into bleomycin-injured mouse lungs was more effective to improve and ameliorate injured lung comparing with transplantation of PMSCs,demonstrated by decreased collagen deposition,obvious rapid repair of inflamed and fibrotic effects,increased survival and recovery of 95% body weight.Taken together,the multilineage differentiation potential of PMSCs and CSPCs derived from livestock and avian embryos were successfully confirmed and studied in our experiment,and we have successfully induced PMSCs from pig embryos to differentiate into AT ? by CRISPR/Cas9-mediated activators.The induced cells exhibited characters of functional cells by marker genes expression and animal model cell-transplantation assays.And it provides an experimental basis for the research of lung wound healing.This technique of manipulation cell fate by directly activating specific endogenous genes provides a simple,safe and effective tool to study cell reprogramming.