| Human umbilical cord blood (HUCB), as a replaceable source of hematopoietic stem cells, could be used to reconstruct bone marrow (BM) hematopoiesis and immunity, The hematopoietic stem/progenitor cells (HPSCs) from cord blood have obvious effects on reconstruction of hematopoietic function of short time and long time of the patients after chemotherapy. HPSCs also have the multipotentiality for differentiation; In addition, human umbilical cord blood has a rich resource and is convenient to collect. Therefore, There is much attention in the study of transplantation of human umbilical cord blood in the recent years and ex vivo expansion of HSPCs is highly valuable in clinic application.BM-derived mesenchymal stem cells are cradle of hematopoietic stem/progenitor cells. The adherent cell feeder layer consisted of BM-derived mesenchymal stem cells provide the microenvironment for maintenance, proliferation and differentiation of hematopoietic stem cells. This is probably due to the synthesis and excretion of many sorts of positive and negative regulatory factors of hematopoiesis (cytokine and adhesion molecule). Therefore, many labs nowadays are using BM-derived stromal cells or mesenchymal stem cells to establish adherent cell feeder layers and co-cultured with hematopoietic stem/progenitor cells to increase the expansion efficiency of hematopoietic stem/progenitor cells. There are primitive cells in HUCB similar to stromal cells or mesenchymal stem cells from BM. This study indicated that an adherent stromal layer from HUCB could be established with specific long-term culture conditions. We confirm the type of cells by the morphological and immunophenotypical characteristics of HUCB-derived mesenchymal stem cells. Besides, HUCB-derived mesenchymal stem cells were used as adherent cell feederlayer to support ex vivo expansion of HSPCs. We evaluated the ability of HUCB stromal cells to form a functional feeder layer and to support hematopoiesis over a long-period culture, in a groping for a new approach of ex vivo expansion of HUCB-derived (HSPCs). Then, HUCB-derived mesenchymal stem cells were induced to form chondrocyte, and the expression of articular cartilage matrix-procollagen II mRNA was examined to understand the biological and differentiation characteristic of HUCB-derived mesenchymal stem cells.HUCB-derived mesenchymal stem cells were isolated and cultured. Two weeks later, numerous fibroblast-like cells could be observed. Subsequently, they formed colonies, and then expanded. By the end of the fourth week, a homogeneous layer of fibroblastoid cells occupied the whole bottle surface. When mesenchymal stem cells were confluent, some of parallel cultures were sacrificed for analysis of Flow cytometry. Other cultures were used as the cell feeder layers to culture HUCB-derived HSPCs. Flow cytometry analysis indicated that HUCB-derived mesenchymal stem cells were universally positive for CD13, CD166 and CD29, and negative for CD14. It can be inferred that HUCB-derived fibroblastic mesenchymal stem cells showed the characteristics of MSCs from bone marrow. It is proper for the cells plated at the density of 2x 106/ml for propagation.Low-density MNCs from HUCB were isolated by Ficoll density centrifugation. MNCs from the gradient interface were collected and washed three times in IMDM and then half of the mononuclear cells (MNCs) was resuspended in culture medium. CD34+ cells were isolated from the other half of the MNCs preparations using a MACS laboratory separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to' the manufacturer's instructions. MNCs in the culture system with mesenchymal stem cell feeder layer had an expansion of 2.09-fold higher than that in the culture system without mesenchymal stem cell feeder layer. CD34+ cell in the culture system with mesenchymal stem cell feeder layer had an expansion of 2.94-fold higher than that in the culture system without mesenchymal stem cell feeder layer. Expanded cells in the culture system with mesenchymal stem cell feeder layer had hig...
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