miRNA 125b Enhances the differentiation and Functionality of in Vitro Generated Human Hematopoietic Progenitor Cells

Abstract 1217

The discovery of transcription factors and micro RNAs (miRNAs) capable of converting somatic cells, either into cells from different lineages or embryonic stem-like cells, has opened a road to differentiate and produce in vitro engraftable cells that could replace and fix damaged tissues. One of the most attractive and promising fields is the differentiation towards blood, considering it is a tissue without a complex tridimensional structure and that the phenotypes of the different sublineages are already well characterized. We previously reported the differentiation from induced pluripotent stem cells (iPS) towards hematopoietic cells and their derived lineages in in vitro assays. More recently, overexpression of Oct4 has been shown to allow for the transdifferentiation of human fibroblasts (HF) into blood progenitor cells albeit with compromised lymphoid differentiation capacity. However, there are no reports of successful differentiation into blood progenitors which are able to completely recover functionally immunodepleted mice.

Considering the emerging role of miRNAs controlling development and differentiation of diverse cell lineages, we hypothesized that the gap missing in the current protocols to obtain hematopoietic stem cells can be filled by the data obtained by miRNA profiling of hematopoietic progenitor cells found in human Cord Blood samples (CB-HPC), which could unveil the key players in the maintenance of blood stemness.

After in vitro expansion of different CB-HPC subpopulations, we have found a set of miRNAS specifically upregulated in the CD34+/CD90+ fraction, a population that has shown the highest capacity to repopulate immunodepleted mice. Upon serial testing, we found that miR-125b, previously reported to have a key role in human and murine hematopoiesis, is highly upregulated in CD34+/CD90+ CB-HPC. In particular, differentiation of iPS towards blood progenitors demonstrated that miR-125b overexpression, specifically increases the percentage and number of blood progenitors, as well as the ability to produce hematopoietic colonies after colony forming unit assay (CFU-A). In addition, following the same rationale previously reported for direct conversion of HF into blood progenitors, we overexpressed one of the transcription factors described by Yamanaka's group. The blood progenitors we obtained were deficient for the differentiation towards CD45+ cells, production of colonies in CFU-A, and showed no repopulation of immunodepleted mice. Most interestingly, the overexpression of miR-125b during the HF conversion, allowed us to obtain blood progenitors able to circumvent these deficiencies. Our preliminary results indicate that miR-125b overexpression causes a positive effect in the maintenance of blood progenitors through the downregulation of p53, a previously reported target for this miRNA in hematopoiesis. While several aspects remain to be addressed in terms of the prospective therapeutical approaches, our study proves that overexpression of miR-125b strongly enhances the differentiation and functionality of in vitro generated HPC.