Gene Expression Of Notch Signaling After UC-MSC Co-culture With HSC

Mesenchymal stem cells (MSCs) are bone marrow populating cells, differentfrom hematopoietic stem cells, which possess an extensive proliferative potential andability to differentiate into various cell types, including: osteocytes, adipocytes,chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in themaintenance of bone marrow homeostasis and regulate the maturation of bothhematopoietic and non-hematopoietic cells. The cells are characterized by theexpression of numerous surface antigens, but none of them appears to be exclusivelyexpressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like:adipose tissue, peripheral blood, cord blood, liver and fetal tissues. UC-MSC cellshave many advantages because of the immaturity of newborn cells compared withadult cells. Furthermore, UC-MSC provides no ethical problems for basic studies andclinical applications.The specific characteristics of MSCs, including their extensive proliferativepotential and the ability to differentiate into various cell types, like bone, fat andcartilage, make them an attractive tool in regenerative medicine. This is especiallyevident in such fields as cellular biology and gene therapy, resulting in considerableincrease in the number of clinical trials based on the use of MSCs. Due to suchcharacteristics, the number of clinical trials based on the use of MSCs increase. Thesecells have been successfully employed in graft versus host disease (GvHD) treatment,heart regeneration after infarct, cartilage and bone repair, skin wounds healing,neuronal regeneration and many others. Of special importance is their use in thetreatment of osteogenesis imperfecta (OI), which appeared to be the only reasonabletherapeutic strategy. MSCs seem to represent a future powerful tool in regenerativemedicine, therefore they are particularly important in medical field. Hematopoieticstem cell transplantation is the effective but only approach to treat some refractoryhematopoietic diseases, such as leukemia, aplastic anemia, immune imperfect, mostsevere radiation disease. The therapy effect can be improved after cotransplatationwith MSCs. The main object of the study is to investigate the hematopoietic supporting capacity of UC-MSCs and the relational molecular mechanism.The Notch pathway is a widely utilized, evolutionarily conserved regulatorysystem that plays a central role in the fate decisions of multipotent precursor cells.Notch often acts by inhibiting differentiation along a particular pathway whilepermitting or promoting self-renewal or differentiation along alternative pathways.Haematopoietic cells and stromal cells express Notch receptors and their ligands,andNotch signalling affects the survival, proliferation, and fate choices of precursors atvarious stages of haematopoietic development, including whether haematopoieticstem cells self-renew or differentiate, In mammals, four Notch receptors(Notch1–Notch4) and fives ructurally similar Notch ligands (Delta-like1[also calledDel-ta1], Delta-like3, Delta-like4, Jagged1, and Jagged2) have been identified. Notchproteins are synthesized as three-length unprocessed proteins composed ofextracellular, transmembrane, and intracellular domains.First, we insolated the adherent cells from umbilical cord. After adherenttreatment, we got UC-MSC from umbilical cord. The UC-MSC was characterized bymorphology, cell cycle, immune-phenotype, growth curve and pluri-potentiality. Theresult showed that the UC-MSC has typical biological characteristics of MSC. Firstly,the cell displays fibroblastic morphology. Secondly, immunophenotypic analysisshowed that CD73, CD90, CD105were positive, but negative expression of CD31,CD34, HLA-DR and CD14. Thirdly, the UC-MSC could be induced into adipocytes,osteocytes and chondrocytes. Fourthly, the expansion potential of UC-MSC wasdramatically increased in vitro. Fifthly, more than80%of the cells were G0/G1phasethat indicates the UC-MSC could be proliferated under the low nutritional culture.Second, UC-MSC were co-cultured with CD34+HSC for6days, real-time PCRwas applied to investigate the gene expression of notch ligands (Jagged1,2Delta1,3,4)，receptors(Notch1-4) and hes-1. After co-cultured of MSC and CD34+cells, Jagged-1and notch1showed a significantly change by real-time PCR assay. Theexpression of hes-1in CD34+cells increased, but there is no obvious change afterDAPT (50nM) was added in co-culture medium.In conclusion, we established a method of isolating UC-MSCs from the matureumbilical cord and found that UC-MSCs had the hematopoietc supporting capabilityin vitro. Notch signaling played an important role in the hematopoietic supportingcapability. The study provides the rich MSCs sources for tissue engineering, genetherapy and cell therapy, which can contribute to the therapeutic utilization of MSCs clinically.