Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells

• TGF-β is a negative regulator of primitive HSCs expansion.
• MSCs can enhance the survival and self-renewal of HSCs.
• Downregulation of TGF-βRII and use of MSCs as a feeder layer could considerably increase the number of progenitors.

BackgroundUmbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression.Materials and methodsCD34+ cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34+ cells were expanded in four culture media with different conditions, including 1) expansion of CD34+ cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture.ResultsThe fold increase in CD34+ cells, TNCs, and colony numbers (71.8 ± 6.9, 93.2 ± 10.2 and 128 ± 10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0 day) before expansion was statistically significant (P < 0.05).ConclusionThe results showed that transfection of CD34+ cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion.