Isolation, Identification And Optimization Of Culture Condition Of Human Umbilical Cord Mesenchymal Stem Cells

Objective:1. To isolate, purify and identify mesenchymal stem cells (MSCs) derived from human umbilical cord.2. To explore the optimal culture conditions of human umbilical cord mesenchymal stem cells (UC-MSC) by investigating the effects of hypoxia, penicillin and streptomycin, platelet-derived growth factor (PDGF) on proliferation, apoptosis and components of extracellular matrix (ECM) expression of human UC-MSC.Methods:1. Cells were isolated from human umbilical cord by digestion of collagenase and purified by using attachment method. Morphology and growth characteristics were observed under microscope. The cell proliferation was analyzed by MTT assay and the growth curve was drawn. Their surface markers were detected by flow cytometry. The fifth-passage cells were respectively cultured in osteogenic and adipogenic induction medium, and confirmed by using the alkaline phosphatase (ALP) staining, type II collagen immunohistochemical staining and oil red O staining.2. Cultured UC-MSC was divided into experimental group and control group, which were cultured in hypoxia and normoxia conditions, respectively. MTT assay was used to detect the hypoxia effects on cell proliferation after 24 hours and 48 hours, respectively. Their surface markers were detected by flow cytometry after 2 days. The expressions of ECM mRNA and apoptosis-related genes (bcl-2, bax) were detected by real-time quantitative polymerase chain reaction (real-time Q-PCR) after 24 hours.3. Cultured UC-MSC was divided into 3 groups:control group, penicillin-treated groups (100,500 and 1000 U/mL) and streptomycin-treated groups (100,500 and 1000 U/mL). Cell proliferation was detected by using MTT assay after 24 hours. Real-time Q-PCR was used to detect the expressions of ECM mRNA and apoptosis-related genes after 12 hours.4. Cultured UC-MSC was divided into 6 groups: the control group, and the PDGF-treated groups (2,5,10,20 and 50ng/mL), Morphological changes of the cells were observed under an inverted microscope. MTT assay was used to detect the PDGF effects on cell proliferation after 24 hours. Expressions of ECM mRNA and apoptosis-related genes were detected by real-time Q-PCR after 12 hours.Results:1. The adherent cells derived from human umbilical cord were spindle-shaped adherent cells which have strong proliferation ability. They were strongly positive expression for CD 105, CD90, CD73, CD 166 and CD44, but negative expression for CD34, CD45 and CD31. The expression rate of CD29 was only 47.45%. Under appropriate induction conditions, all these cells could differentiate into bone, cartilage and fat cells.2. In further experiments, The OD value of UC-MSC and colony forming ability in hypoxic group was significantly higher than that in normoxia group. No significant difference was observed in surface antigen expression between the two groups, except CD 105 expression in hypoxia group was slightly higher than that in normoxia group. Expression of ECM in hypoxia group increased compared with normoxia group. Expression of bcl-2 and bax was up-regulated in hypoxic condition, with bcl-2 upregulating more obviously.3. Penicillin and streptomycin with low concentrations promoted UC-MSC proliferation, with the most effective concentration of 100U/mL. Expressions of ECM were down-regulated in addition of penicillin and streptomycin. Furthermore, apoptosis-related factor (bcl-2/bax) in low concentration penicillin and streptomycin groups was higher than that in the control group.4. With an increased concentration of PDGF, UC-MSC proliferation was gradually promoted compared with the control group.20ng/mL PDGF greatly promoted UC-MSC proliferation, however,50ng/mL showed inhibitive effects. Expressions of ECM mRNA and apoptosis-related factor were down-regulated in the 2ng/mL PDGF group but up-regulated when PDGF≥5ng/mL. 10ng/mL PDGF greatly up regulated ECM mRNA expression and apoptosis-related factor.Conclusion:1. The adherent cells isolated from the umbilical cord have biological characteristics of MSCs. They could be used for further experiments.2. Hypoxia condition could improve the proliferation and colony forming ability of UC-MSC. The expression of ECM and anti-apoptotic ability improved in hypoxia.3. Low concentrations penicillin and streptomycin might promote the proliferation and reduce the rate of apoptosis. However, high dose might inhibit the ECM component synthesis of UC-MSC.4. PDGF could promote the proliferation of UC-MSC, stimulate ECM synthesis and improve the ability to withstand apoptosis of UC-MSC in vitro. 10ng/mL PDGF was the most effective concentration.