Understanding the role of miRNA expression in the differentiation of haematopoietic stem cells from pluripotent stem cells

Abstract

Hematopoietic diseases such as leukaemia can be treated by a haematopoietic stem cell transplant, but suitable donors are in short supply. Hematopoietic cells derived from pluripotent stem cells have been suggested as a possible alternative, however hematopoietic progenitors made this way have so far been unable to either engraft in sufficient numbers or differentiate into all mature blood lineage types in vivo. Recent published evidence suggests this may be due to a failure to down-regulate key miRNAs. Microarray data comparing hematopoietic progenitors derived from bone marrow or from pluripotent stem cells was studied and key miRNAs were selected for validation. This combined analysis showed that several miRNAs fail to down-regulate in pluripotent cell derived progenitors, unlike the equivalent cells in bone marrow. These miRNAs play a role in suppressing epithelial-mesenchymal transition and their high expression may be a cause of the observed failure to engraft. Five of these miRNAs were chosen as candidates for inhibition (miR-200b, miR-200c, miR-148a, miR-205, and miR-424). Methods of miRNA inhibition were investigated, and the effects of the inhibition of the chosen miRNAs on hematopoietic development from hESCs and hiPSCs were assessed using gene expression, marker expression and colony assay techniques. Although miRNAs were successfully inhibited using lipofection both individually and in multiplex these experiments did not significantly improve the efficiency of hematopoietic development, possibly due to high levels of redundancy among tumour suppressors. The limitations of the inhibition technique, i.e. the short timeframe of inhibition and inability to down-regulate more than 5 miRNA at once may be insufficient to overcome this problem. Differentiation in hypoxic conditions was tried in the hope of making more global changes to miRNA expression, and these experiments yielded an increase in CFU-GEMMs and cells expressing HSC markers, which may be helpful in future attempts at hematopoietic stem cell generation.