Three-Dimensional Culture And Immunosuppressive Effects Of Mesenchymal Stem Cells In Vitro

ObjectiveMesenchymal stem cells are a class of non-hematopoietic stem cells which possess the capacity of self-renewal and multi-lineage differentiation. MSCs are viewed as a clinically promising progenitor/stem cell source because of their differentiation capacity, their haematopoietic support, their low immunogenicity, their immunomodulatory and pro-regenerative features.In recent years, immunosuppressive function of MSCs is a major concern. Numerous studies showed that MSCs derived from different tissues, such as adult bone marrow, adipose, umbilical cord, had immunosuppressive function. MSCs act on both the adaptive and innate immune systems by suppressing T cells, suppressing dendritic cell maturation, reducing B cell activation and proliferation, inhibiting proliferation and cytotoxicity of NK cells. MSCs had significant therapeutic effects on GVHD and autoimmune diseases in vivo.As a source of fetal tissue, fetal liver mesenchymal stem cells and fetal bone marrow mesenchymal stem cells accompanied with the gradual formation of bone marrow microenvironment during fetal development. FL-MSCs and FBM-MSCs are similar to adult bone marrow mesenchymal stem cells in some basic biological characteristics such as self-renewal, multi-lineage differentiation potential and proliferation. However, immunological properties of these two kinds of cells rarely reported at present. To investigate the immunological characteristics of these two kinds of cells may help us better understand the role of FL-MSCs and FBM-MSCs in hematopoietic microenvironment and hematopoietic regulation.Hematopoiesis occurs in the bone marrow, where primitive hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment composed of stromal cells. These studies have found that stromal cells regulate the proliferation and differentiation of HSCs through the production of diffusible hematopoietic regulatory factors and extracellular matrix, and through physical cell-cell interactions involving adhesion molecules and gap junction-mediated cell communication. However, the ability of stromal cells to support the expansion of HSCs and to maintain their self-renewal potential has generally been investigated in long-term, two-dimensional bone marrow culture systems, and most of the reports have shown a decline in HSCs within4-8weeks in culture. Whether this hematopoiesis support defects can be improved in three-dimensional culture system also lacks further study. To confirm the expansion and differentiation of hematopoietic stem cells in three-dimensional system, the change of cellular phenotype and the expression of cytokines associated with hematopoiesis in umbilical cord mesenchymal stem cells (UC-MSC) were studied in three-dimensional system.Methods1. The primary culture and identification of MSCs:Human FBM-MSCs and FL-MSCs were primary cultured. The general characteristics of these two cells were identified through morphological observation and flow cytometry. Using chemical induction methods, these two cells were induced into osteoblasts and adipocytes. Functional differentiations were evaluated using Alizarin Red S, Von Kossa, Oil Red O staining methods and specific markers expression respectively.2. Growth characteristics of FBM-MSCs and FL-MSCs:Growth rate and changes in cell morphology of these two cells were observed after serially passaged. The phenotype of MSCs was detected by flow cytometry.3. Effects of FBM-MSCs and FL-MSCs on proliferation of PBMC:Mononuclear cells were isolated from peripheral blood. FBM-MSCs and FL-MSCs co-cultured with PBMC to assesse effects of these two cells on proliferation and inflammatory cytokines expression of PBMC, and changes of inhibitory cytokines which secreted by FBM-MSCs and FL-MSCs. Proliferation of PBMC was detected by CFSE. mRNA expression of inflammatory cytokines and inhibitory cytokines were measured by Real-Time PCR. Concentrations of inflammatory cytokines in the supernatants were determined by using ELISA.4. Cell-cell interactions involved in immunosuppression:The role of cell-cell interactions were measured by Transwell assay. mRNA expression of adhesion molecules ICAM1, ICAM3and VCAM1were detected by Real-Time PCR.5. Three-dimensional culture of UC-MSCs:MSC were isolated from umbilical cord, and then cultured in two-dimensional and three-dimensional system individually. The phenotype of MSC was detected by flow cytometry. The angiogenic capability of two cell types was assessed using an in vitro capillary formation assay. The cytokines expression of MSC in two kinds of culture conditions was measured by real-time PCR.Results1. FBM-MSCs and FL-MSCs were successfully isolated from fetal bone marrow and fetal liver respectively. Both of MSCs can be exuberant proliferated with typical fibroblast cell morphology. They can express typical phenotype of MSC such as CD29, CD44, CD49e, CD73, CD90and CD105. No hematopoietic or endothelial cell markers such as CD14, CD34, CD45and CD31can be detected. CD80, CD86, CD106and HLA-DR which related to GVHD also can not be detected. These two cells low expression of CD146and HLA-ABC. Osteoblasts and adipocytes differentiation can be achieved after induction with the expression of specific markers respectively.2. The proliferation of FL-MSCs was markedly higher than FBM-MSCs. These two cells remained some basic biological characteristics of MSC even after serially passaged.3. FBM-MSCs and FL-MSCs can significantly inhibit proliferation and inflammatory cytokines IFN-γ/TNF-α secretion of PHA activated PBMCs in a dose-dependent manner. Meanwhile, the expression of IDO, TSG-6and TGF-β from FBM-MSCs and FL-MSCs increased significantly after co-culturing with PBMCs.4. Cell-cell interactions played an important role in immunosuppression of both cells. The expression of ICAM1from FBM-MSCs and FL-MSCs increased significantly after co-culturing with PBMCs.5. We successfully isolated MSC from umbilical cord. Flow cytometry results showed that the percentage of CD31, CD133and CD271which expressed in endothelial cells, endothelial progenitor cells and primitive mesenchymal stem cells increased significantly in three-dimensional culture conditions, compared to two-dimensional system. Capillary formation assay showed that the angiogenic capability of UC-MSCs had a great enhancement. Quantitative PCR results showed that the expression of β-Actin was upregulated in three-dimensional system. Some cytokines associated with hematopoiesis, such as G-CSF, LIF, SCF, IL-1α, IL-1β, IL-3, IL-7and IL-11, increased, especially for LIF, IL-3, IL-7. The IL-10expression which was associated with immune regulation was also increased. The expression of SDF-1, IL-6had a slight decrease, but no significant difference.Conclusion1. MSCs can be derived from both of fetal bone marrow and fetal liver and in line with international standard for MSCs. Compared with FL-MSCs, FBM-MSCs mediated more osteogenic and adipogenic differentiation. However, the proliferation of FL-MSCs was markedly higher than FBM-MSCs. These two cells remained some basic biological characteristics of MSC even after serially passaged.2. FBM-MSCs and FL-MSCs have low immunogenicity. FBM-MSCs and FL-MSCs have great immunosuppressive effects on PBMC in vitro. Inhibitory cytokines, IDO, TSG-6, TGF, and cell-cell interactions may be critical for these two cells mediated immunosuppression.3. The expression of CD31, CD133and CD271increased, the angiogenic capability of UC-MSC enhanced and the expression of cytokines associated with hematopoiesis on UC-MSC increased in three-dimensional system.