Small molecules have demonstrated great potential in modulat

Small molecules have demonstrated great potential in modulating cellular fate and functions. A milestone work showed that mouse iPSCs were successfully induced from mouse embryonic fibroblasts (MEF) via six chemical molecules, in the absence of Yamanaka reprogramming factors (Hou et al., 2013). Recently, neuronal Q-VD(OMe)-OPh have been generated from MEF in the presence of small molecules (Hu et al., 2015; Cheng et al., 2014; Li et al., 2015). In the field of HSC, a few compounds, which show stimulating effects on expansion and proliferation of hematopoietic stem and progenitor cells (HSPCs) in vitro have also been screened (Boitano et al., 2010; Fares et al., 2014; Chaurasia et al., 2014). Of note, UM171 has the capacity to expand human umbilical cord blood HSCs, via an unknown mechanism. We hypothesized that UM171 might facilitate hematopoietic derivation from human pluripotent stem cells (hPSCs) in vitro. In this study, we investigated the effect of UM171 on hematopoietic specification using a stable monolayer differentiation protocol as described previously (Niwa et al., 2011). We observed that UM171 enhanced hematopoietic specification through key steps of hematopoietic induction in vitro: PSCs to hemogenic endothelium (D0–D6), as well as hemogenic endothelium to hematopoietic progenitors (D6–D14). In comparison to the control condition, hematopoietic progenitors from the UM171 group produced more and larger sized CFU-GM as well as larger sized CFU-Mix, implying UM171 as an ideal molecular additive to the derivation of HSPCs from hPSCs in vitro.
Results
Discussion Chemically defined mediums and factors are essential for establishing a stable protocol for hematopoietic differentiation, which can be easily repeated by many groups in the field of hematopoietic regeneration. In this study, we observed that UM171 enhanced hematopoietic derivation in monolayer induction protocol in vitro. Interestingly, hematopoietic derivatives in the presence of UM171 formed more and larger sized CFU-GM as well as larger sized CFU-Mix colonies in the CFU assay. Moreover, our preliminary data from the transplantation assay indicated that very rare human cells could be detected in the bone marrow of recipients four weeks after transplantation. Over decades, the hematopoietic regeneration field has been lagging behind due to the lack of a robust approach to derive hematopoietic cells with engraftment potential (Slukvin, 2013; Vo and Daley, 2015). We will further evaluate the engraftment potential of the hematopoietic progenitor cells collected at different time points in the presence of UM171. UM171 might be included as a beneficial small molecule for different approaches to obtain HPCs. It remains elusive whether UM171 has similar effects in other hematopoietic differentiation systems in vitro. Our current data also indicated the limits of UM171 in definitive hematopoiesis, though we observed robust definitive hematopoietic progenitors that formed CFU-G and CFU-GM. We are currently testing other differentiating approaches including stromal cell co-culture and embryoid bodies (EBs) method for hematopoietic derivation in the presence of UM171. Besides the known role of UM171 in the maintenance and expansion of human HSCs in vitro (Fares et al., 2014), the pathway(s) in which UM171 involves during hematopoietic specification in vitro are still unknown. Our ongoing experiments are investigating the underlying signaling pathway(s) modified by UM171 during hematopoietic derivation at multiple developmental steps in vitro, with the advantage of single-cell RNA-seq techniques.
Conclusion We show evidence that UM171 enhances hematopoietic derivation from pluripotent stem cells at multiple developmental phases in vitro, including PSCs to hemogenic endothelium, hemogenic endothelium to hematopoietic progenitor phases. Especially, UM171 promotes the production of hematopoietic progenitors that have the capacity to form more and larger sized CFU-GM as well as larger sized CFU-Mix colonies. In combination with other factors and protocols, UM171 might be broadly used for hematopoietic differentiation from hPSCs in vitro.