Lithium Chloride Mediated Wnt/β-catenin Signaling Regulating The Proliferation And Differentiation Of Menstrual Blood-derived Mesenchymal Stem Cells

Stem cells are unique cell populations with the ability of self-renewal, high proliferation, multipotent differentiation. Due to the unique biological characteristics of stem cells, it has becoming the the rapid development of modern biology and broad areas of concern. The potential of stem cells in regenerative medicine has also increasingly attracted attention. However, the bottleneck problems of stem cells are how to obtain adequate stem cells sources and quite amount of cells for clinical application, the directed differentiation also draws more attention.Recent years, mesenchymal stem cells existed in the menstrual blood which were called menstrual blood-derived mesenchymal stem cells (MMCs) were discovered in American and Japan. It's not only overcome the arguments of ethic issue of ESCs(Embryonic stem cells), but relieved the limitations of BMSCs(bone marrow stem cells) by traumatic approach and limited sources. MMCs could express the surface markers of stem cells, and had low immunogenicity. It was characteristic of rapid proliferation, self-renewal and multipotent differentiation. The MMCs could differentiate into neuron-like cells in vitro when exposed to some specific inducers. To a certain extent, it broughted hope to the patients with the central nervous system diseases. However, no reports had been anounced in China till now.Several signal pathways are involved in the proliferation and differentiation of stem cells. One major signal transduction pathway associated with stem cells development is the canonical Wnt signaling, which could regulate the downstream target genes and mediated cell cycle, cell survival and differentiation.Recently, considerable research had been applied to Wnt/β-catenin in the ESCs, BMSCs and UCBSCs (umbilical cord blood stem cells). It could active Wnt/β-catenin by recruiting Wnt protein or inhibitting the activation of GSK-3β, APC, no paper mentioned this signal pathway function in the MMCs.Lithium had been used to treat manic depression in clinic. Further studies showed lithium was also a potent stimulant of stem cells at the molecular level. Lithium directly inhibited of GSK-3β, and promoted the accumulation ofβ-catenin in the nucleus. These evidences proved that canonical Wnt signaling was actived in MSCs.In this experiment, we isolated and cultured MMCs successfully, adopted different concentration of lithium chloride on MMCs, detected the expression ofβ-catenin, explored the effect that lithium chloride regulated the proliferation and differentiation by Wnt/β-catenin signaling pathway.This paper is divided into three parts. Part one:Isolation, amplification and characterization of MMCs; Part two:To explore the potential of MMCs differentiation into neural-like cells in vitro; Part three:After exposured to lithium chloride, the expression ofβ-catenin was detected in MMCs.Part one:The study was carried out on menstrual blood. They were collected from healthy female volunteers. Mononuclear cells derived from menstrual blood were separated by Ficoll-Paque. CCK-8 kit was applied for cell proliferation assay, it aimed to observe expansion of MMCs after subculture in vitro. MSCs markers were explored by Flow Cytometry (FCM). The results showed that MMCs presented in menstrual blood, and could be subcultured steadily, the cell growth curves of P3, P10 demonstrated subcultured cells were not influenced MMCs proliferation rate at least at Passage 10. MMCs appeared to possess some markers of mesenchymal stem cells such as CD29, CD44, while lacking hematopoietic stem cell markers such as CD34, and the panleukocyte marker CD45, HLA-ABC had low level expression. HLA-DR was negative, indicated their immunogenity were very low. Part two:We further studied whether neural precursor cell present in MMCs. By culturing MMCs under the condition of bFGF (basic fibroblast growth) andβ-ME, DMSO, cells were induced to neuron-like morphology. RT-PCR and immunocytochemical methods were employed to detect the markers of nerve cells, nestin, NSE, NF, GFAP. This results showed neural precursor cell marker (nestin) were found in undifferentiated MMCs. The mRNA expression of neuron markers were increaseded in differentiated cells induced by bFGF,β-ME and DMSO. There were no expression of GFAP (glial fibrillary acidic protein). It clearly demonstrated MMCs had the capacity to develop into neuron cells under experimental conditions.Part three:Exposured MMCs to DMEM medium containing different concentration of lithium chloride. After 4 days the proliferation of MMCs were observed by morphologic changes and CCK-8 assay. The expression ofβ-catenin was determined by RT-PCR, western blot respectively in control and treated cells. We found that low levels (4 mmol/L) promoted the proliferation of MMCs. Oppositely, high level of lithium chloride (above 10 mmol/L) caused inhibition of proliferation.4 mmol/L LiCl increased the level ofβ-catenin in MMCs, which suggested lithium chloride could activate the canonical Wnt signaling pathway, but presenting dose-dependent effects.In brief, we first sucessfully isolated MSCs from menstrual blood, and could subculture stablely, and MMCs had the potential of neuronal differentiation. After exposured to different doses of LiCl, it demonstrated 4mmol/L LiCl was optimized for rapid cell proliferation. The expression ofβ-catenin increased by LiCl, which indicated LiCl could active Wnt signaling pathway in MMCs.