Effects Of Hypoxia On Proliferation And Differential Protein Expressions Of Human Umbilical Mesenchymal Stem Cells

Objective The therapeutic efficacy of mesenchymal stem cells (MSCs) for hypoxic-ischemic diseases is closely related to the hypoxic environment of the damaged tissues. The study is to explore the effects of hypoxia on the morphology, ultrastructures, proliferation and the differential protein expressions of human umbilical cord mesenchymal stem cells (UCMSCs), then to search the new therapy targets of MSCs in hypoxic-ischemic diseases, and their further clinical applications.Methods (1) MSCs were isolated from human umbilical cords by collagenase digestion and adherence screening methods. Adipogenic and osteogenic differentiation potentials were confirmed by oil red O and von Kossa staining methods, respectively. (2) Normal human umbilical cord MSCs immunophenotypes and cell cycle ratios were determined by flow cytometry. (3) Human umbilical cord MSCs were treated with hypoxia-mimetic agents, desferrioxamine (DFO) and cobalt chloride (CoCl2). Their morphologies and ultrastructures were observed by atomic force microscope (AFM) and transmission electron microscopy (TEM). (4) The impact of DFO and CoCl2 on human umbilical cord MSCs proliferation were measured by MTT assay. (5) The impact of DFO and CoCl2 on human umbilical cord MSCs cell cycle ratio were measured by flow cytometry. (6) 2-DE was performed to separate proteins from human umbilical cord MSCs treated or untreated with CoCl2. Differential proteins were analyzed by ImageMaster 2D Platinum software 6.0. The differential proteins were identified by MALDI-TOF-MS. The functions of differential proteins were identified and classified.Results (1) The human umbilical cord MSCs G0/G1 phase were 89.4%. They overexpressd CD29, CD44 and CD105, while lowexpressed or not expressed CD106, CD40, CD34, CD45 and HLA-DR. (2) Following adipogenic induction, Oil Red O staining was positive. Upon osteogenic differentiation, human umbilical cord MSCs tested positive after von Kasso staining. (3) After treatment with DFO and CoCl2, human umbilical cord MSCs were elongated. Adjacent cells were not in proper contact with each other and gaps appeared between cells. In addition, vacuole-like structures were observed in cytoplasm, the rough endoplasmic reticulum appeared to expand, and mitochondria exhibited an expanded ridge. (4) Human umbilical cord MSCs proliferation were significantly inhibited by DFO and CoCl2. The proliferation inhibition rates gradually increased with concentrations of DFO and CoCl2 increased. Cell growth significantly decreased after treatment with 120μmol/1 DFO,10 and 100μmol/1 CoCl2 as compared with controls (all P<0.05). (5) DFO and CoCl2-induced hypoxia increased the ratio of GO/G1 phase cells and decreased the ratio of G2/M/S phase cells. (6) Differential protein expressions of human umbilical cord MSCs treated and untreated with CoCl2 were established. A total of twenty-six differential proteins were identified, among which eleven proteins were up-regulated and fifteen proteins were down-regulated.Conclusions (1) The hypoxia-mimetic agents, DFO and CoCl2, made morphologies and ultrastructures of human umbilical cord MSCs change in hypoxia, and inhibited their proliferation through influencing the cell cycle. (2) Multiple proteins and functional pathways were involved in the effects of CoCl2 on differential protein expressions of human umbilical cord MSCs. The biological function involved in carbohydrate metabolism, nucleic acid and lipid metabolism, protein metabolism and modification, coenzyme and prosthetic group metabolism, cell cycle, immunity and defense, cell structure and motility, signal transduction, protein targeting and localization, intracellular protein traffic, neuronal activities, and muscle contraction.