Study On The Biological Properties Of Multipotent Mesenchymal Stem Cells Cultured With Low Serum And Their Immunoregulatory Effect On Human Th Lymphocytes

Background:Multipotent mesenchymal stem cells(MSC),isolated from many tissues,are multipotent stem cells with the capacity for self-renewal and multilineage differentiation.Their ease of isolation and expansion,their multipotency,their low immunogenicity and immune-modulatory properties present them as an ideal stem cell candidate for tissue engineering and regenerative medicine.An important issue in cellular therapeutics is the availability of alternative MSC sources,while key to the success of cell therapy is the biological properties of MSC.Understanding the biological properties of MSC will be beneficial for fully utilizing the therapeutic potential of MSC,therefore,there has been an increased interest in understanding the biology of MSC due to their potential use in cell-based therapy.To date,most therapeutic applications are based on adult bone marrow(ABM) MSC,but ABM MSC have many drawbacks,including the limited volume,the invasive procedures of their harvest,and the age-dependent decline in the absolute number and differential capacity.This has prompted researchers to look for alternative sources of MSC and suitable culture medium for MSC so as to meet the requirement of clinics and research.Objective:This study aims at identificating the biological properties of MSC cultured with low serum by evaluating their morphous,karyotype, growth kinetics,immunophenotype and differentiation potential during in vitro expansion so as to supply the rationale for their therapeutic potential for tissue engineering.Methods:①Cells were isolated from fetal bone marrow and lung as well as umbilical cord by adherent culture,cultured in the culture medium containing DMEM/DF12 as well as 2%fetal bovine serum,and purified by passaging in vitro.②The karyotype and growth curve of MSC were detected.③Cell cycle was assayed by flow cytometry.④The immunophenotype and the pluripotent marker expression were obtained by flow cytometry analysis.⑤MSC were induced for adipocytes,osteoblasts and chondrocytes in the induction media.Oil red 0,alizarin red and alcian blue staining was performed to examine lipid droplet,mineralization and acid mucopolysaccharide,respectively.The expression of lineage-specific markers such as PPAR-γ,osteocalcin and collagenⅡwas evaluated by flow cytometry analysis.⑥MSC from fetal bone marrow and lung could differentiate into neural cells by two-step introduction and the expression of neural markers including MAP2,Nestin and GFAP in differentiated cells were confirmed by confocal microscopy.⑦The two-step procedure was performed for hepatocyte introduction.The mRNA expression of hepatic specific markers such asαFP,albumin and cytokeratin 18(CK18) was examined by RT-PCR.Hepatocyte function was assessed by detecting glycogen by periodic acid schiff(PAS) staining and dosing albumin in culture supernatants.Results:①MSC cultured with low serum had adherent and spindle-shaped characterizations,grow rapidly,and reached confluence with a whirl pool-like appearance.②They maintained a normal karyotype and displayed the growth curve of 'S' type.③Most cells were in the G0/G1 phases and only a small proportion of cells actively proliferated as revealed by cell cycles analysis.④They highly expressed mesenchymal markers including CD13, CD29,CD44,CD49e,CD90,CD105,CD166,HLA classⅠ,and did not express hematopoietic and endothelial markers such as CD31,CD34,CD11b,CD45,CD117, CD133 and HLA classⅡ.Importantly,MSC could even express pluripotent markers of embryonic stem cells such as Oct4,Nanog,Sox2 and SSEA-4.The expression levels of all markers in MSC from fetal lung were remained stable through 25 passages.⑤The induced MSC showed positive staining of oil red 0,alizarin red and alcian blue and high expression of lineage-specific markers.The data confirmed their potentials to differentiate directedly into adipocytes,osteoblasts and chondrocytes;moreover,MSC from fetal lung (P25) possessed the similar differentiation potentials.⑥The expression levels of neural markers in the differentiated cells were significantly higher than those in control cells.⑦Compared with control cells,the expression of hepatocyte markers was upregulated in the induced cells that also demonstrated the capability of storing glycogen and secreting albumin.Conclusion:①Culture medium with low serum is good culture reagent for MSC.②MSC cultured in the medium show great proliferation potential and suffice the demand of seed cells for tissue engineering.③They are able to differentiate into adipocytes,osteoblasts and chondrocytes and MSC from fetal lung maintain the ability up to 25 passages,which make it possible to supply enough cells for tissue engineering of bone or cartilage.④MSC express pluripotent markers of embryonic stem cells and differentiat directedly towards extramensenchymal lineages such as neuroectoderm cells and hepatocytes of endoderm.⑤The biological properties of MSC are favourable,and MSC have emerged as excellent seed cells for tissue engineering and regenerative medicine. Background:Multipotent mesenchymal stem cells(MSC) are multipotent stem cells that can differentiate into tissues of mesodermal origin and can be isolated from various tissues.MSC have also been noted to have profound immunomodulatory effects on immune cells,leading to the modulation of several effector functions.Recently,a new CD4+ T-cell subset has been designated as Th17 cells that preferentially produce IL-17.Th17 cells have been widely accepted as important effector cells in the development of autoimmune and allergic diseases and host defenses against a group of pathogens.Whereas the exact mechanisms underlying the immunomodulatory functions of MSC remain largely unknown,these cells have been exploited in a variety of clinical trials aimed at reducing the burden of immune-mediated disease.Objective:This article aimed to investigate the immunoregulatory effects of fetal bone marrow-derived MSC on human T lymphocytes,especially Th17 cells,so as to broaden our understanding of the immunomodulatory properties of MSC and provide insights as to their potential for clinical use as a cell-based therapy for immune-mediated disorders.Methods:We cultured PBMC and CD4+ T cells with or without FBM-MSC,and we named the coculture of PBMC or CD4+ T cells with FBM-MSC as coculture cells.The culture media included PHA and rIL-2,if not specifically mentioned.We used qRT-PCR,ELISA and flow cytometry analyses to compare①the expression levels of IL-17 bewteen PBMC as well as CD4+ T cells and their coculture cells in the absence or presence of PHA and rIL-2;②the change of IL-17 expression when supplemented with additional IL-6,IL-6 neutralizing antibody and TGF-β1;③the expression levels of IL-1 and IL-23 between PBMC as well as CD4+ T cells and their coculture cells;④the expression of IFN-γbetween PBMC as well as CD4+ T cells and their coculture cells;⑤the mRNA expression of Foxp3 between PBMC and its coculture cell.Results:qRT-PCR,ELISA and flow cytometry data showed①that the molecular and cell expression levels of IL-17 in PBMC and CD4+ T cells were lower than those in their coculture cells with or without PHA and rIL-2,respectively(p<0.05);②that the addition of IL-6 increased the expression of IL-17(p<0.05),while the addition of IL-6 neutralizing antibody and TGF-β1 decreased the expression of IL-17(p<0.05);③that the increased concentration of IL-1 in the supernant of CD4+ T cells than that in the supernant of their coculture cells(p<0.05),and that no significant difference of IL-23 expression was found between PBMC as well as CD4+ T cells and their coculture cells(p>0.05);④that the expression levels of IFN-γproduced by Th1 cells in PBMC and CD4+ T cells were lower than those in their coculture cells(p<0.05);⑤that the mRNA expression of Foxp3 in PBMC was lower than that in their coculture cells(p<0.05).Conclusion:Our data demonstrated for the first time that FBM-MSC could positively regulated human Th17 cells.The mechanisms underlying the regulation might include IL-6 as well as IL-1,whereas IL-23 seemed not to involve in the investigated regulation.In addition,FBM-MSC might exert a negative role in IFN-γproducing Th1 cells,but maybe promote Treg.