Mesenchymal Stem Cells Promote Proliferation And Terminal Differentiation Of B Cells

Background:Mesenchymal stem cells (Mesenchymal Stem cells, MSCs) are a kind of adult stem cells with self-renew and multilineage differentiation.MSCs can be isolated from a variety of tissues such as bone marrow, fat, umbilical cord, umbilical cord blood, placental, and the MSCs isolated from different sources have similar immune regulation, hematopoietic support and tissue repair properties. The immunosuppression of MSCs on T cells is the theoretical basis for clinical application of MSCs on autoimmune disease. However, the immunomodulatory of MSCs on B cells still need further research.Objective:To investigate the immune regulation of the human umbilical cord mesenchymal stem cells on the proliferation and terminal differentiation of B lymphocytes, and to reveal the main mechanism.Methods:(1) To detect the immune phenotype of the human umbilical cord mesenchymal stem cells (hUC-MSCs) and the omnipotence markers of the embryonic stem cell by flow cytometry.(2) Three lines of differentiation-inducing culture of mesenchymal stem cells:hUC-MSCs were induced for adipocytes, osteoblasts and chondrocytes in the specific-induction medium. Oil red O staining, Von Kossa staining, safranin O staining to observate lipid droplet formation and calcium deposition and chondrocytes identification of the human umbilical cord derived mesenchymal stem cell.(3) Human umbilical cord mesenchymal stem cells and CD19+B lymphocytes were cocultured in vitro. B lymphocyte proliferation was assessed by Brdu ELISA (Brdu incorporation assay) and B cell terminal differentiation markers CD138expression was detected by flow cytometry; B cells secretion IgM, IgA, and IgG secretion was detected by ELISA kit, so as to reflect the immune modulation of human umbilical cord mesenchymal stem cells on the proliferation and terminal differentiation of activated B lymphocytes.(4) Transwell24well culture plates were used to block the direct contact between hUC-MSC and B lymphocyte to study if the direct contact of cells is necessary on the immunosuppressive effects of human umbilical cord mesenchymal stem cells on B cells.(5) Enzyme-linked immunosorbent assay detection kit were used to detect in the secretion levels of PGE2and IL-6in the co-culture system, and added PGE2synthase inhibitor indomethacin and IL-6neutralization antibody into the co-culture system to detect the proliferation and IgM, IgA, and IgG secretion to verify the main mediator.(6)6-8weeks of BALB/c mices were divided into8groups labled A-H,5mices per groups, group A and D were injected with the T cell-dependent antigen I (TNP-the Ficoll), group B and E were injected with the T cell-dependent antigen II (TNP-KLH), and group C and F were injected with the T-cell-independent antigens (TNP-LPS). Mouse bone marrow mesenchymal stem cells were cultured for three passage, group A,C,E,G were injected with1×106mouse bone marrow the MSCs, groupB,D,F were injected with PBS, and H group was negtive control. The serum was collected to detect the level of IgM secretion7days later, and serum collected again14days later to detect the secretion level of IgG. The modulation of bone marrow MSCs on immunoglobulin secretion were detected by ELISA in vivo.Results:(1)Human umbilical cord MSCs expressed CD13, CD29, CD49e, CD44, CD90, CD105, CD166, and of HLA-ABC (HLA-I); not expressed the hematopoietic cells related markers CD3, CD11b, CD14, CD19, CD31, CD34, CD45, CD80, CD86, CD133and HLA-DR (HLA-II) and so on.(2) hUC-MSCs were able to differentiate to adipogenic, osteogenic and chondrogenic lineages in lineage-specific inductive condition, indicating that hUC-MSC possessed multilineage differentiation potential.(3)The human umbilical cord cells promoted the proliferation of B cells.(4) The human umbilical cord MSCs promoted the terminal differentiation of B cells.(5) The human umbilical cord MSCs modulated the B cells by soluble factors, and PGE2not IL-6played the important role.(6) The human umbilical cord MSCs promoted the T cell-dependent antibody and non-dependent antibody production in vivo.Conclusion:Human umbilical cord MSCs promote proliferation and terminal differentiation of B cells, and PGE2partially mediate the immunosuppressive activity of human UC-MSC but not IL-6. Background:The initial research on the Mesenchymal stem cells (MSCs) focuses on its plasticity and regenerative capacity of differentiation to other tissues, but more attention focused on the immunomodulatory of MSCs, and it is clear that MSCs inhibit the cytokines secretion especially TH1-type cytokines of peripheral blood mononuclear cells. Although the mechanism of immunomodulatory is not very clear, more and more studies have shown that the soluble cytokines mediated the immunomodulatory of mesenchymal stem cells. Therefore, mesenchymal stem cells are ideal cells for the treatment of immune diseases especially autoimmune diseases. Mesenchymal stem cells derived from different tissues and even different individual sources are not exactly the same in immunological activity. It lacks the quantitative methods to detect immunological activity of mesenchymal stem cells, so the clinical application of mesenchymal stem cells is messy and lacks of uniformity of standards.Objective:Investigate to find a method of quantitative detection the immunological activity of umbilical cord mesenchymal stem cells, and to lay the foundation for clinical quantitative applications.Methods:(1) Human umbilical cord mesenchymal stem cells and healthy adult peripheral blood mononuclear cells were cocultured in vitro, IFN-y secretion was detected by enzyme-linked immunosorbent assay kit, so as to reflect the immunosuppressive effects of the human umbilical cord mesenchymal stem cells on the activated peripheral blood mononuclear cells.2×105hUC-MSCs were seeded in6-well plates, the medium was changed after2hours, and then added1ng/mL or5ng/mL IL-1β, the supernatant was collected after some time incubation. PGE2secretion of mesenchymal stem cells was assessed by enzyme-linked immunosorbent assay.(2) Added different concentrations of exogenous PGE2to healthy adults activated peripheral blood mononuclear cell culture system, detected IFN-y secretion by ELISA to determine the exogenous PGE2on the peripheral blood mononuclear cells IFN-y secretion inhibition curve.(3) added50μ L umbilical cord mesenchymal stem cells culture supernatant to150μ L adult peripheral blood mononuclear cell culture systems, culture supernatant was collected after3days, IFN-y secretion were detected by ELISA.Results:(1) Umbilical cord mesenchymal stem cells inhibited the activated peripheral blood mononuclear cell IFN-γ secretion, and mainly mediated by PGE2.(2)the maximum PGE2secretion was at24hours, and the secrection of PGE2decreased with time after24hours. Umbilical cord mesenchymal stem cells from different individual sources secreted different PGE2.(2) Exogenous PGE2inhibited peripheral blood mononuclear cells IFN-y secretion in a dose dependent and the most obvious changes was in the lng/mL-50ng/mL concentration range.(3) PGE2secretion in the human umbilical cord mesenchymal stem cells supernatant shared the similar inhibition on peripheral blood mononuclear cells IFN-y secretion as exogenous PGE2, and different individual sources of umbilical cord mesenchymal stem cells possessed different inhibition ability on IFN-y secretion.Conclusion:The immunomodulatory capacities of umbilical cord mesenchymal stem cells from different individual sources are different. we define that the concentration of PGE2secretion reach to20ng/mL as critical point of immunological activity in the105umbilical cord mesenchymal stem cells supernatant which were cultured for24hours in6-well plates and2mL of the culture system,≥20ng/mL with strong immunological activity,<20ng/mL with weak immunological activity. Background:As one of bone marrow microenvironment cells, bone marrow mesenchymal stem cells are former cells of hematopoietic microenvironment cells, it support the proliferation and differentiation of the hematopoietic stem cells. However, the application of bone marrow mesenchymal stem cells are limited by volume, invasive procedures of their harvest and the age-dependent decline in the absolute number, differentiation and proliferation capacity. And the research shows that other sources of mesenchymal stem cells with the same hematopoietic support as bone marrow mesenchymal stem cells. Umbilical cord mesenchymal stem cells have emerged as a promising candidate for cellular therapeutics because of easily accessible, less troublesome in ethical controversy, readily long-time preparation and can be scaled up to large numbers in short doubling time and keeping stemness in lone time culture. However, its hematopoietic support capacity is still limited, it is necessary to look for the method to improve the hematopoietic support.Objective:To investigate the impact of IL-1β on hematopoietic support of human umbilical cord mesenchymal stem cells (hUC-MSCs).Method:2×106hUC-MSCs were seeded in75cm2flasks, after2hours, added10ng/mL IL-1β, hUC-MSCs were cultured for36hours, and then the culture supernatants and cells were harvested. To observe the conditioned medium to the colony-forming unit-granulocyte(CFU-G)、colony-forming unit-macrophage (CFU-M) colony-forming unit-granulocyte macrophage (CFU-GM)、burst forming unit-erythroid(BFU-E)、colony-forming unit-erythroid (CFU-E)and colony-forming unit-granulocyte/erythroid/macrophage/monocyte(CFU-GEMM) of CD34+cell with/without IL-1β; to compare the hematopoietic factor G-CSF、GM-CSF、M-CSF、 IL-6、SOD2、CDK、TRIM22mRAN expression of hUC-MSCs by real time PCR with/without IL-1β; To detect differences of hematopoietic factors G-CSF、GM-CSF、 M-CSF、IL-6by ELISA.Results:①The conditioned culture media of hUC-MSCs with IL-1β enhanced the CD34+cells differentiation into colony formation unity,especially CFU-G and CFU-GM in vitro;②IL-1β promoted GM-CSF, M-CSF, IL-6mRNA expression of MSCs;③IL-1β promoted GM-CSF, and G-CSF, IL-6protein secretion of MSCs. Conclusion:IL-1β enhances hematopoietic support capacity of MSCs to myeloid differentiation.