Supportive Effects Of Long-term Culture Of Human Umbilical Cord Derived Mesenchymal Stem Cells On Hematopoiesis In Vitro

BACKGROUND:Mesenchymal stem cells (MSCs) are a kind of multipotent stem cells which primally derived from mesoderm and ectoderm. At present, researchers have isolated MSCs from sorts of tissue, such as bone marrow, adipose, skeletal muscle, synovium, umbilical cord, placenta et al. Among all of these, human umbilical cords are the best and the optimal sources of MSCs currently. It reported that MSCs are the important components of hematopoietic microenvironment in bone marrow, which play a vital role in regulating and supporting hematopoiesis. MSCs could promote the transplantation of hematopoietic stem cells and accelerate reconstruction of hematopoietic microenvironment. Otherwise, MSCs have the unique physiological ability to differentiate into multi-lineages and modulate immune. Hence, MSCs become the focus of experimental study and clinical application. However there are low frequency of MSCs in human tissue, and expansion in vitro is a prerequisite for clinical application. Therefore, the effects of serial long-term culture in vitro should be studied.OBJECTION:This study was aimed to isolate and propagate hUC-MSCs in vitro, and explore whether the conditioned culture medium of hUC-MSC has supportive effects on hematopoiesis in vitro. Compare the biologic characteristic, especially the ability for supporting hematopoiesis, of the early and late hUC-MSCs.METHODS:After collecting the umbilical cords of neonate, we purified hUC-MSCs from the umbilical cords by collagenase digestion method and attachment culture. In our study, we analyzed morphology, immunophenotyping by using immunofluorescent assay and differentiation under inductive condition of cells to identify whether they are MSCs. The early and late hUC-MSCs growth kinetics were assessed by MTT, and cells cycle were observed by flow cytometry. To compare the ability of supporting hematopoiesis of different passages, we collected the conditioned culture media of hUC-MSCs, then performed the colony-forming assay in vitro. Meanwhile real-time PCR assay was used to examine the relative expression of hematopoietic cytokines. To define the concentration of the cytokines in the conditioned culture media, the culture media were detected by ELISA kit.RESULT:The cells we isolated were fibroblast like or spindle-shaped cells, and grew adherently to the culture substrate. As flow cytometry analyzed the mean percent of CD expression, the cells stained positively for CD29、CD44、CD73、CD90、CD105、CD166, while CD19、CD31、 CD34、CD45、CD106、HLA-DR were negative. In lineage-specific inductive condition, the cells could be differentiated along adipogenic and osteogenic. These revealed that the cells we isolated were MSCs. The results showed that the conditioned culture media of hUC-MSCs has the ability to support hematopoiesis separately in vitro, which supported the CD34+cells differentiation into CFU-G(47.67±0.58)、CFU-GM (48.67±4.73) and CFU-M (3.00±2.00) in vitro, while the CFU-E、BFU-E or CFU-GEMM were absent. After long-term culture in vitro, the morphological characteristics of late passages(passage30) cells changed that cells become slightly larger than the early ones(passage3). The cell cycle analysis showed that more than85%cells were in G0/G1phase. The proliferation profile by MTT assay demonstrated that, compared with early passage cells, the late ones grew slower. The hematopoietic supporting capacity of hUC-MSCs was examined by CFU-assay with conditioned culture media. The late cells formed more CFUs than the early ones (251.67±25.27vs.60.00±3.61,P<0.05). Further more, the relative expression of hematopoietic cytokines(such as G-CSF、GM-CSF、M-CSF、 IL-6et al) of the late cells were significantly higher than the early ones. Generally speaking, the hematopoietic supporting capacity of hUC-MSCs enhanced after long-term culture.CONCLUSION:Successfully, we have purified and identified hUC-MSCs. Their conditioned culture media could support hematopoiesis in vitro, and this media could promote hematopoietic stem cells to differentiate into myeloid cells, but not into erythroid or lymphoid cells. After passaged for a long term in standard system in vitro, there are series changes of biologic characteristic of hUC-MSCs. Although the ability of proliferation decreased, the ability of supporting hematopoiesis enhanced. With the long-term culture, the expression of hematopoietic cytokines up-regulate.