Study On Wharton’s Jelly Mesenchymal Stem Cells To Differentiate Into Cholinergic Neurons

Alzheimer’s disease (AD) patients present an irreversible decline in the cognitivefunction, which may result from the damage of the forebrain cholinergic projectionsystem innervating widespread regions of the cerebral cortex, and the loss ofcholinergic neurons. Unfortunately, so far no successful treatment for AD has beendeveloped. With the development of stem cell technology, stem cell-based therapy forAD has been recently proposed. However, this new therapy faces many difficulties.For example, embryonic stem cells (ESCs) suffer from a series of constraintsincluding ethical concerns, limited availability, potential for teratoma formation upontransplantation and immune rejection; adult stem cells such as mesenchymal stemcells derived from bone marrow have the problems of the number, proliferativecapacity and the differentiating potential decreasing with age. Hence there is a direneed to look for an alternative source of stem cells.Hence, an alternative source ofstem cells for cell replacement therapy is urgently needed, and Wharton’s jellymesenchymal stem cells (WJ-MSCs) derived from umbilical cord (UC) is focused inrecent years on cell replacement therapy.Wharton’s jelly of UC is the embryonic mucous connective tissue lying betweenthe amniotic epithelium and the umbilical vessels, which was first described byThomas Wharton in1656. In recent years, in parallel to the enormous effort to explorenovel and alternative sources of stem cells, WJ-MSCs appeared as a promisingcandidate for reservoir of fetal cells that could be readily used as multi-potential stemcells. These cells can be harvested by non-invasive means, and maintained in vitro asundifferentiated cells for more than10passages. In addition, freeze-thawed andfreshly isolated WJ-MSCs present similar cell viability and protein expressions. Asmulti-potential stromal cells, WJ-MSCs own plastic adherence and surface markers ofmesenchymal stem cells and non-hematopoietic cells. More important, thegraft-vshost disease markers are not detectable or weakly expressed in WJ-MSCs,indicating a transplantation without immunological suppression. Additionally, humanWJ-MSCs do not appear to form teratomas when transplanted. Thus, it has been postulated that WJ-MSCs could be used for cell transplantation therapy and representa more eligible source of MSCs. It has been increasingly recognized that WJ-MSCsmay differentiate into several cell lineages in all three germ layers such aschondrocytes, osteoblasts, adipocytes, cardiomyocytes, adenocytes, hepatocytes,gliocytes and neurocytes. However, the capacity of WJ-MSCs to differentiate intocholinergic-like neurons remains undetermined.The specific mechanism required for the differentiation of cholinergic neurons isnot completely defined in any model system. Data showed that bone morphogeneticprotein-4(BMP4) and fibroblast growth factor8(FGF8) correlated with developmentof cholinergic neuron. That conjunction of LIM homeodomain proteins formed “LIMcode” of transcription to define different neural cells on development of centralnervous system (CNS) has been known as common mechanism of CNS development.The mechanism has been specifically verificated by the study of embryonaldevelopment of dynamoneure. Study verificated that Lhx8was necessary LIMhomeodomain transcription factor of corpora striata cholinergic endaxoneurons andbasalis telencephal cholinergic projection neurons and lslet-1also was necessarytranscription factor of cholinergic neurons.Objective: To determine whether WJ-MSCs could differentiate intocholinergic-like neurons with neural stem cell conditioned medium (NSCcm), BMP4and FGF8.Methods: WJ-MSCs were isolated by planting method; NSCcm was preparatedby mtaking supernate of telencephali cell culture on embryonic day14; WJ-MSCs atpassage3were incubated in DMEM-Ham’s F-12medium supplemented with10%FBS for one day, and the half of the medium was changed to NSCcm for the next twodays; thereafter, the cells were cultured in NSCcm alone for one week; cells werecultured in NSCcm supplemented with BMP4and FGF8for10days;immunofluorescence and RT-PCR were used for CHAT, NF, Islet-1and Lhx8, andthe Acetylcholine Assay Kit was used for acetylcholine (Ach).Results: After the induction, the spindle-shaped or fibroblast-like WJ-MSCschanged into bulbous cells. Positive coexpression of CHAT and NF of phenotype ofcholinergic neuron was found in WJ-MSCs by immunofluorescence studies, and themRNA of CHAT and NF was significantly expressed (P<0.01). Ach secretion ininduced WJ-MSCs was significantly elevated compared with the control group (P<0.01). Positive coexpression of lslet-1and Lhx8expressing on development ofcholinergic neuron was found in the differentiated cells by immunofluorescencestudies, and the mRNA was significantly expressed (P<0.01).Conclusion: WJ-MSCs are cable of differentiating into cholinergic-like neuronswith neural stem cell conditioned medium, BMP4and FGF8. The differentiated cellsexpressed phenotype of cholinergic neuron CHAT and NF, secreted Ach, and expressedlslet-1and Lhx8expressing on development of cholinergic neuron.