| The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the fate decisions of multipotent precursor cells. Notch often acts by inhibiting differentiation along a particular pathway while permitting or promoting self-renewal or differentiation along alternative pathways. Hematopoietic stem/progenitor cells express Notch receptors and stromal cells express their ligands. Notch signaling affects the survival, proliferation, and fate choices of precursors at various stages of hematopoietic development. Notch signal promotes the self-renewal and suppresses the differentiation of hematopoietic stem/progenitor cells. Thus, activation of Notch is a novel strategy for expansion of hematopoietic stem/progenitor cells in vitro. Bone marrow-derived mesenchymal stem cells (MSCs) also express Notch receptors, however, the role of Notch signal in the regulation of the proliferation and differentiation of MSCs remains unclear. In this study, the effect of Notch signaling on expansion of cord blood (CB) hematopoietic progenitors and osteogenic differentiation of marrow MSCs was evaluated.1. Activation of Notch in cord blood-derived CD34+ cells promotes their self-renewal and growth in vitroThe expression of Notchl, Dll-1 and Jaggedl detected by RT-PCR suggests Notch signals play a physiological role in regulation of proliferation and differentiation of cord blood-derived hemaopoietic cells. The intracellular domain of Notch (ICN) is the active form of Notch. Constitutively expression of ICN can activate Notch signals in cells without ligands' binding. The ICN was cloned from K562 cells and the retro viral vector containing ICN was constructed. CD34+ cells isolated from cord blood by MACS system were transfected with ICN by retrovirus. Activaiton of Notch in CB-derived CD34+ cells prolonged their lifespan in vitro. Some progenies of the ICN tranfected CD34+ cells cultured for 2~4 months were also characterized as immature blasts by their cell morphology, expression of CD34 antigen, and preservation of the ability to form CFU-GM and CFU-M. These results demonstrate that activation of Notch in hematopoietic stem/progenitor cells delays their differentiation and promotes their self-renewal.2. The molecular cloning, expression and purification of the extracellular domain of Dll-1 and its application in the expansion of CB cells in vitro.The effect of a soluble form of human Delta-like-1, one of Notch ligands, on expansion of hematopoietic stem/progenitor cells was investigated. Total RNA was isolated from human marrow mononuclear cells. An extracellular domain of human Delta-like-l(hDll-lext) was amplified by RT-PCR and cloned to T vector, then this gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin, and the expression of secreted hDll-lext in G418-resistant clones was assayed by Western Blot. The clone with high hDll-lext expression was cultured to collect supernatant. Fusion protein hDll-lext with Myc-His was purified from the supernatant by immobilized metal affinity chromatography (IMAC). Human umbilical blood CD34+ cells were cultured in serum-free medium containing cytokine cocktail, with or without purified hDll-lext, and the effect of hDll-lext on the expansion of progenitor cells was analyzed by clonogenie assays. The results showed that hDll-lext enhances cytokine-induced expansion of CPU-Mix and HPP-CFC in serum free expansion sytem (the number of CPU-Mix or HPP-CFC increased 1.5-2.0 folds compared with control). In the presence of hDll-lext, the number of HPP-CFC expanded 9.7 or 43 times after culture with SCF, IL-3 and IL-6 for 8 or 12 days, respectively. The results indicate that the recombinant hDll-lext enhances the expansion of primitive hematopoietic progenitors derived from cord blood .3. Notch signal promotes the differentiation of mesenchymal stem cells to osteoblastsWe examined the role of Notch signaling in differentiation of marrow mesenchyma...
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