Human Bone Marrow Mesenchymal Stem Cell (BM-MSC) Culture And The Expression Of Hematopoietic Cytokines

Pluripotent human bone marrow-derived mesenchymal stem cells (MSC) can differentiate into osteogenic, chondrogenic, adipogenic and tendonogenic cells, which are the cell components of bone marrow hematopoietic microenviroment. These cells represent a unique subpopulation of bone marrow stroma cells. Normally they are very rare in bone marrow. Recent studies show MSCs can grow in vitro and have the the capacity to differentiate along osteogenic, chondrogenic, adipogenic, and tendonogenic lineages when placed , both in vitro and in vivo, in an appropriate condition. Isolation and ex vivo expension of MSCs from human bone marrow, and distinguishment of MSCs from hematopoietic or endothelial cells with their specific cell surface markers have been reported. In addition several previous studies have shown that mesenchymal progenitor cells secrete a variety of cytokines and growth factors, and are able to support hematopoiesis in long-term bone marrow culture (LTBMC).In this study, we attempt to establish a stable MSC culture system and tostudy their biological features including cell morphology, cell-doubling time, colony-forming ability and their expression of hematopoietic cytokines in vitro. Since it was reported hydrocortisone was related to differentiation of MSCs, we also investigate its effect on the hematopoietic cytokine expression of MSCs oPart I BM-MSC culture in vitroTo culture bone marrow mesenchymal stem cells (BM-MSCs) in vitro, we collect human bone marrow samples from some healthy donors and primary diagnosed patients with hematological diseases and BM-MSCs were isolated, and cultured in DMEM/FBS or MesenCult serum media. BM-MSCs doubling time was determined at exponential stage. CD13^ CD105 and CD34 of cell surface marker s were tested with histoimmunochemistry assay. Colony forming potent of the expanded BM-MSCs were analyzed by CFU-F test. BM-MSCs grew as monolayer cells attached on the bottom of culture flasks. These cultured cells arranged regularly with a uniform radiation. At the exponential stage, cell doubling time was around 46 hours in serum free culture system . BM-MSCs were highly potential to proliferate. The colony-forming ability of the cultured BM-MSCs was 15000 folds higher than bone marrow mononuclear cell (MNC) according to the results of CFU-F assay. A great number of BM-MSCs with high purity could be obtained by culture in vitro. Histoimmuno-chemistry analysis indicated that all the cultured BM-MSCs were CD13(+), CD105(+) and CD34(-). BM-MSC is a sort of bone marrow stem cell different from HSC.Part II Human bone marrow mesenchymal stem cells express multiple hematopoietic growth factorsTo study the biological role of human cultrued bone marrow mesenchymal stem cell (BM-MSC) in hemaotpoiesis , RT-PCR was used to analyzeexpression of SCF, Flt3-ligand , TPO , LIF, G-CSF, GM-CSF, IL-3, IL-6, and IL-11 at mRNA level for human BM-MSCs from healthy donors and patients with hematologic diseases. BM-MSCs were incubated with or without hydrocortison (HC). It was clearly shown that the cultured BM-MSC at different passages of P3 up to PI5 expressed mRNAs of SCF,Flt3-ligand, TPO, LIF, IL-6 and IL-11 but not G-CSF^ GM-CSF and IL-3. The same pattern of above cytokine expression was also seen in the patient's BM-MSC. HC was able to induce BM-MSC to express G-CSF but not GM-CSF. BM-MSC seemed not to change morphologically after incubation with HC for up to 21 days. In conclusion, Both normal and patient's BM-MSCs are potential to promote hematopoiesis with their expression of multiple hematopoietic cytokines. And HC is one of the inducers to induce hematopoietic growth factor expression.