[TGF-bReceptor2 knocked down by SiRNA increases cord blood CD34+ HSCs self-renewal]

ABSTRACT

Nowadays, cord blood Hematopoietic stem cells [HSCs] are known as a valuable source for bone marrow transplantation but unfortunately their insufficient number is a limiting factor for using them in adult bone marrow transplantation. Cord blood HCSs expansion is an approach to overcome this problem, by inducing their self-renewal. TGF-b signaling pathway is a key inhibitory agent for HSCs self-renewal. In this study, we tried to enhance self-renewal of long term culture initiating cell by inhibiting TGFbR2 expression. CD34+ HSCs were isolated from cord blood units with MACS column. SiRNA against TGFbR2 was transfected by Lipofectamine [TM] RNAiMAX as transfection reagent. HSCs were cultured in IMDM medium containing 10% FBS and early acting cytokines [Flt3L, SCF, Tpo] for 8 days. Then we evaluated TGFbR2 expression by QRT-PCR. The CD34+ subpopulation of cultured cells were examined by flow cytometry on the 8th day. Finally the expanded cells were evaluated for the presence of early hematopoietic stem cells by LT-CIC and clonogenic assays. According to our results, TGFbR2 down regulation increases CD34+ subpopulation of HSCs. In addition, LT-CIC assay showed an enhancement in primitive hematopoietic stem cell capable of self-renewal. All in all, it seems that positive regulators have attracted more attention in the field of HSCs expansion while negative regulators have same importance in self-renewal process of HSCs and their inhibition can be a beneficial tool for enhancement of HSCs self-renewa