A Newly Cloned Lectin-FRIL Maintained Quiescence Of HSCs Through Regulation Of Cell Cycle Related Factors

Hematopoietic stem cell transplantation (HSCT) has been widely used in a variety of malignant and non-malignant hematologic tumors' therapy. Due to the rarity of HSCs, a lot of methods were tried to expand HSCs ex vivo. However, those culture conditions induce extensive proliferation and differentiation, resulting in the loss of repopulating capacity of primitive cells. At the same time, entering of cultured HSCs into active cell cycle would dramatically reduce their homing ability, and subsequently, their long-term repopulating potential. Maintenance of HSCs in vitro, which means to preserve their self-renewal and multi-differentiation potential, thus is very important for HCST. To maintain quiescence of HSCs is to keep them out of cell cycle, that is keep them in the GO phase.A newly cloned lectin, FRIL (Flt3 receptor-interacting lectin), was identified to be capable of preserving primitive progenitors in vitro without loss of repopulating capacity. FRIL could bind specifically to Flt3, a tyrosine kinase receptor (RTK) expressed by HSCs. Through activation of the RTK signaling pathway, FRIL could regulate interaction of some important cell cycle proteins, and keep HSCs in the quiescent state. We extracted FRIL from Dolichos lablab. Purified FRIL was able to agglutinate erythrocytes and has high affinity with 3-Oa-Dmannose. Cord blood CD34+ cells were cultured in suspension medium supplemented with FRIL. Results showed that it could prolong cell cycle in HSCs and reduce cell number in cell cycle, inhibit differentiation of HSCs, and preserve the HSCs' self-renewal. But the underlying mechanism for maintaining quiescence of HSCs is largely unknown at present, so in this study, we focused on the mechanism of how FRIL maintains quiescence of HSCs through regulation of cell cycle. FL (Flt3 ligand) is also the ligand for Flt3, and is one of the most commonly used cytokines in preserving HSCs in vitro. Together with other cytokines, it could induce expansion and differentiation of HSCs as well. Because FRIL and FL use the same receptor and FL could regulate early hematopoiesis, FL was used as control in our experiments.Factors involved in cell cycle regulation are classified mainly by their function, they are cyclin, CDK and CKI. Among them, cyclin and CDK are positive regulators of cell cycle; activation of CDK is dependent on its interaction with corresponding cyclin, while expression of different cyclin is cell cycle status dependent. Cyclin-CDK complex is activated by CAK and when the activity is high enough cell will pass through the Gl/S check point and goes into S phase. The CDK inhibitor (CKI) could inhibit activity of cyclin-CDK complex and serves as negative regulator of cell cycle. To investigate the underlying mechanism of how FRIL maintain HSCs in vitro, we detected the expression of various cell cycle regulators, especially those involved in G0/G1 phase regulation on mRNA and protein level. They are CDK4, CDK6; cyclin D1, cyclin D2, cyclin D3 cyclinE; p21, p27, p16, p53 and WT1. Though do not belong to the above three families, p53 and WT1 could activate or inactivate those cell cycle regulators and exert impact on cell cycle progression. Cord blood CD34+ cells were cultured in suspension medium supplemented with FRIL or FL or with no supplement, and total RNA was extracted from cells collected at different time point. RT-PCR was used to detect expression of mRNA and western blot to detect protein.CD34"1" cells cultured with FRIL showed lower expression of cyclin and CDK compared with FL or blank both on mRNA and protein level, which consistent with the former cell expansion and cell cycle analysis results. The lower expansion rate, the lower and slower expression of cyclin in FRIL group; the higher expansion rate, the higher and faster expression of cyclin in FL group; while the expansion rate and expression of cyclin are both moderate in blank. Western blot detected no expression of cyclin Dl, cyclin D3, CDK4 or CDK6 on freshly isolated CD34+ cells. On day 3, expression of cyclin D3 and CDK6 were d...