Baicalin induces the differentiation of human umbilical cord blood-derived mesenchymal stem cells towards neurons-like cells in vitro / 中国组织工程研究

ABSTRACT

BACKGROUND: Both antioxidant and cytokine can induce the differentiation of umbilical cord blood-derived mesenchymal stem calls (UCB-MSCs) towards neuron-like cells in vitro. It remains unclear regarding how to select an inductor that has the ability of either neuro-protection like cytokines or powerful antioxidation. After wide screening, baicalin has been included in this study. OBJECTIVE: To observe the effects of baicalin on in vitro purification, amplification, and differentiation towards neuron-like cells of hUCB-MSCs.DESIGN, TIME AND SETTING: A randomized, controlled, cytological in vitro observation was performed at the laboratory of Department of Neurology, First Affiliated Hospital of Nanchang University between February and April 2005. MATERIALS Ten portions of UCB were collected from healthy full-term normal delivery pregnant women aged 23-25 years old. Baicalin with purity > 95% was purchased from Department of Pharmaceutics, Xiangya Medical College of Central South University. Antioxidant additive J -mercaptoethanol was provided by Sino-American Biotechnology Company, China. METHODS: The collected UCB was anticoagulated with heparin to separate mononuclear cells. After concentration adjustment (1 ×109/L), UCB mononuclear cells were purified and amplified with dulbecco's modified eagle's medium containing fetal bovine serum (0.2 volume fraction), glutamine, B27, granulocyte colony-stimulating factors, and stem cell factors. According to antioxidant additive application, 4 groups were set baicalin, blank control, β -mercaptoethanol, and baicaUn+ β -mercaptoethanol. Cells in each group were cultured for a total of 4 consecutive weeks. MAIN OUTCOME MEASURES: (1) Detection of CD 34 and CD 29 immunoreactive expression on days 7, 14, 21, and 28 after cryopreservation. (2) Cellular morphology observation. (3) Detection of surface antigen expression of MSCs by flow cytometry. (4) Detection of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP) expression after 4 weeks of culture by immunocytochemistry. RESULTS: O Compared with prior to cryopraservation, trypan blue exclusion rate of UCB-MSCs was significantly reduced on days 7, 14, 21, and 28 after cryopreservation (P < 0.05). (2) Morphological observation UCB mononuclear cells adhered to the wall 2-3 days after culture, reached a peak level at 2 weeks, and formed a confluence of approximately 80%-90% 3 weeks after culture; at this time, all UCB-MSCs displayed a spindle shaped appearance. Four weeks later, in the baicalin group, some spindle-shaped UCB-MSCs began to present shrinkage, with slender processes on the cell edge, and some UCB-MSCs tended to be spherical-, conical-, and triangle-shaped appearance, with many slender processes on the pseudopodia. In the β-mercaptoethanol and baicalin+β -mercaptoethanol groups, an increasing number of cells defoliated and died with culture time in addition to above-mentioned appearances. (3) Four weeks after culture, cells were positive for CD45 in the blank control group, while cells in the remaining groups were positive for CD29 and CD 83, in particular in the baicalin+ β -mercaptoethanol group, followed by the baicalin group, and lastly the β -mercaptoethanol group. Significant difference in CD29 and CD 83 immunoreactivity exhibited between groups (P < 0.01). No CD34 immunoraactive calls were found in each group. (4) Four weeks after culture, NSE and MAP-2 immunoreactive expression was significantly lower in the blank control and β -mercaptoethanol groups than in the baicalin group (P < 0.01). The percentage of cells expressing GFAP was lower than 1% in each group. CONCLUSION: 100 μmol/L baicalin can promote the in vitro amplification of UCB-MSCs in a time-dependent manner and also can induce the differentiation of UCB-MSCs towards neuron-like cells in vitro to some extent.