Research On Biological Properties Of Umbilical Cord Derived Mesenchymal Stem Cells Based On Cell Banking For Clinical Use

Background:Stem cells re of great therapeutic potential due to their capacity for self-renewal, multilineage differentiation, including different cell type. However, the ethics issues and other controversies around the embryonic cells make it a less interesting candidate for clinical applications. Adult stem cell types represent an archetype of multipotent somatic stem cells that hold promise for application in regenerative medicine. Among them, human umbilical cord derived MSCs, also known as hUCMSCs, are self-renewing cells that can be found in umbilical cord (UC), may be ideal sources due to their capacity for self-renewal, multilineage differentiation, hypoimmunogenicity, immune modulation, stromal support, paracrine, migration, and genetic stability. Moreover, they may be ideal sources due to their accessibility, painless procedures to donors, promising sources for autologous cell therapy, and lower risk of viral contamination. Regardless of the success achieved in isolation and expansion of MSCs at laboratory scale, manufacturing a therapeutic cell-based product is and will continue to be challenging. Because MSC manufacture is currently an open, time consuming, and labor intensive process. Recently, GMP (Good Manufacturing Practice) based cell banks for clinical use offer the standardized process for manufacture of the therapeutic UC-MSC. which provide the promise for future patients. The establishment of a stem cell bank having well-characterized and "ready-to-use" allogeneic MSCs could be promising and attractive to clinical researchers. However, appropriate maintenance of biological characteristics and uniformity of the cells is impossible using traditional cryopreservation methods. The cryopreserved cells is mainly damaged due to the solute reaction, the latter cause osmotic pressure and pH value leakage changes in cell membranes, resurrected water into the cells, which cause the cells to edema degeneration and death. Additionally, hUCMSCs from donors of various ages are of great importance and determined their therapeutic potential.Ecdysterone (ecdysterone, EDS) is also known as β-Ecdysone in animals. It is an insect metamorphosis hormone, which could stimulate the insect dermal cell division to generate new skin, result in insect metamorphosis and regulate insect metabolism. But it is found that ecdysterone exists in plants more than in animal. At present, ecdysterone is mostly extracted from plants. Ecdysterone in vertebrates is also the necessary substance. Studies indicate that it also showed a strong pharmacological activity in higher animals, and its side effects were minor. Nowadays, we know its actions include:1) promote nucleic acid and protein synthesis;2) adjust glucose metabolism;3) modulate lipid metabolism;4) regulate gene expression;5) improve immune function;6) posses antioxidan activity;7) promote angiogenesis and establishment of collateral circulation in ischemic area;8) promote proliferation of varied cells.Because Ecdysterone has various activities, including stimulating protein synthesis, promoting carbohydrate and lipid metabolism, alleviating hyperglycemia and hyperlipemia, immunologic modulation, and protecting endothelial cells from apoptosis and inducing their proliferation. Ecdysterone can obviously promote wound healing in rabbitsand. EDS has a proliferative effect on the growth of epidermal stem cell in vitro. It is a major component of several Chinese herbal medicines, such as Achyranthes bidentata BL. and Cyanotis arachnoidea C. B. CLARKE, various activities of Ecdysterone in human being, especially stimulating protein and RNA synthesis. So we considered that it may enhances proliferation and differentiation of hMSCs.In this study, we have studied the comparative characteristics of cryopreserved hUCMSCs by EDS influence and comparative biological properties of hUCMSCs from donors of various ages, which may provide the basement of cell banking establishment.Objective:To investigate the influence of EDS on bioactivity of cryopreserved hUCMSCs. And to investigate the comparative biological properties of hUCMSCs from donors of various ages.Methods:Collection and transport of hUCMSCs were obtained from full-term cesarean section deliveries with informed consent of the mother. Tissue collection was approved by the Institutional Medical Research Ethics Committee of the local maternity hospital. HUCMSCs were isolated and cultured from pregnant rat and expression of cell surface antigens was analyzed by flow cytometry (CD34, CD45, CD29<CD105. After3passages, hUCMSCs were preserved in liquid nitrogen by lowering the temperature gradually.6months later, cells were thawed and divided into3groups:cultured by common medium(blank control), cultured by common medium with lower concentration of EDS(100μg/mL) and cultured by common medium with higher concentration of by EDS (200μg/mL). The morphology and cloning formation are observed microscopically after10d cultivation; The cell growth curve was determined, and expression of cell surface antigens was analyzed by flow cytometry (CD34、CD45、CD29、CD105); The osteogenic and adipogenic differentiation of hUCMSCs were investigated with alizarin red and Oil Red O staining respectively. This study also compare the biological properties of hUCMSCs from donors of various ages by using2different range of donor ages and by measuring indices of expression of cell surface antigens and proliferation rates, osteogenic and adipogenic differentiation as well as karyotype analysis in vitro.Results:The results of morphology, survival and proliferation reveal that lower EDS group and higher EDS group are better than that of control group(P<0.05). But there was no significant differences in these results between lower EDS group and higher EDS group (P>0.05). Cells in all groups could differentiate into osteogenic and adipogenic direction while only higher EDS group could induce those cells to osteogenic differentiation without the induction medium. RT-PCR test showed hUCMSCs are continuously express Oct-4, Nanog, Rex-1and SCF genes. HUCMSCs from younger donors of2different age range showed the higher expression of cell surface antigens and proliferation ratesand similar karyotype, flow cytometry analysis show that the positive expression rate of CD29、CD105were higher in A group than in B group and the negative expression rate of CD34、CD45were lower in A group than in B group (P<0.05). they could differentiate into osteogenic and adipogenic direction, while the cells of younger donors showed the higher capability(P<0.05).Conclusions:EDS with lower concentration (100μg/mL) used in cryopreserved have the positive effect on bioactivity and survival of hUCMSCs, and EDS with higher concentration (200μg/mL) used in cryopreserved could induce those cells to osteogenic differentiation. HUCMSCs from younger donors have the obvious advantages on biological properties.