Expansion Of Human Hematopoietic Stem/Progenitor Cells Using GST-hDSL Fusion Protein

In recent years, hematopoietic stem cells (HSCs) transplantation is becoming more and more important in cure of leukemia, malignant tumor and some genetic diseases. HSCs are derived from bone marrow, umbilical cord blood (UCB) and mobilized peripheral blood. Due to limitated HSC donors and low number of HSCs, it is crucial to expand HSCs in vitro. The Notch pathway is a widely utilized, evolutionarily conserved regulatory system that plays a central role in the determination of cell fates in many developmental systems. Activation of the pathway is initiated by the interaction between Notch receptors and their ligands on cell surface. The expression of Notch receptors and their ligands respectively on HSCs and stromal cells suggest the potential role for Notch-mediated regulation in hematopoiesis.Notch ligands are single-pass transmembrane proteins consisting of a highly conserved region called DSL domain that is an essential unit for binding and activation of Notch receptor. Through structural and functional analysis of hDelta-like-1(hDll-1), one of the human Notch ligands, we choose the minimal protein sequence required for activation of Notch pathway. It includes the DSL domain and the N-terminal sequences and is named hDll-1NDSL(hDSL). In our laboratory, the hDSL sequence was cloned into expression vectors pGEX2T, then transformed into E. coli DH5α. The protein hDSL was expressed in E. coli via IPTG induction as a glutathione S-transferase (GST) fusion protein and then were purified using DEAE anion exchange column. Our work first applied this GST-hDSL to the expansion of human HSCs in vitro.CD34~+ cells were isolated from UCB using a high-gradient magnetic activated cell sorting (MACS) system and cultured with GST-hDSL and different combination of cytokines. The results of our work is as follows: 1) CD34~+ cells were cultured in medium with SCF, FL, IL-3, TPO and with or without GST-hDSL for a long term liquid culture of 21 days. The output of total cells, CD34~+ cells, colony forming cells (CFC), myeloid differentiation, and cell apoptosis were evaluated at the 7th, 14th, 21st day. We found that the culture time of 14 days was the best for cell expansion in the presence of SCF, FL, IL-3, and TPO. GST-hDSL had no effects on the output of total cells, CD34~+ cells, CFC, monocytic differentiation and cell apoptosis. However, GST-hDSL promoted granulocytic differentiation; 2) In the absence of other cytokines, GST-hDSL alone did not affect the output of total cells, CD34~+ cells, CFC and monocytic differentiation after culture for two weeks; 3) In the presence of SCF and GM-CSF, GST-hDSL did not affect the output of total cells, myeloid differentiation, cell apoptosis and cell cycle. However, the number of CD34~+ cells, CFC and HPP-CFC (high proliferative potential colony forming cells) in the culture system with GST-hDSL were 1.7, 1.2, 5.3 times of the control groups respectively. Together, we concluded that first, GST-hDSL did not affect the expansion of total cells; Second, cytokines might modulate the effects of Notch signaling; Third, GST-hDSL in combination with SCF and GM-CSF promotes expansion of cord blood CD34~+ hematopoietic stem/progenitor cells(HS/PC). The results established the future use of recombinant GST-hDSL in culture expansion of human hematopoietic stem/progenitor cells.