Hierarchical Approaches to Study Human Hematopoiesis from hESCs Towards the Characterization and Validation of Transplantable Monocyte Progenitors.

Human pluripotent stem cells (hPSCs) carry big promises for the development of regenerative therapies. However, the understanding on how to exploit them imposes challenges, as their direct use is deleterious due to uncontrolled differentiation and tumorigenesis. Also, there are few defined methods to differentiate them towards progenitors that could contribute to tissues in vivo, and there is a lack of pre-clinical models to evaluate their future use in humans. Animal model studies have shown the importance of microenvironments and the relationship between multiple tissue systems to get correct lineage progression. This interdependency is crucial in hematopoiesis, a process that relies on niches integrating signals from bone, nervous system and blood vessels. Optimal transplantation of hematopoietic stem cells (HSCs) should consider the availability of enough progenitors and how they would incorporate within the niche. Ultimately, it would be desirable to develop strategies to generate hematopoietic progenitors and to modulate their microenvironment. We hypothesize that both goals are possible using hPSCs. First, using human embryonic stem cells, a method to recapitulate early human development generating mesenchymal, vascular, hematopoietic, and neuro-ectoderm lineages was developed, validated for in vitro lineage commitment and used to develop new reagents by way of monoclonal antibodies. Second, a method to generate early hematopoietic progenitors that subsequently progressed to myeloid progenitors was designed and used to define progenitor phenotypes comparing them with equivalent populations in blood. Through this, a new monocyte progenitor was revealed in human peripheral blood. Third, methods to evaluate the in vivo capability of derivatives were initiated by generating progenitors expressing fluorescent reporters for vascular, mesenchymal, and monocytic cells. Finally, a new mouse model was developed using immunodeficient mice expressing a transgenic trait allowing the conditional ablation of osteoblast lineage resulting in opening of the hematopoietic niche without pleiotropic effects. This new mouse model accepts allogeneic HSCs and is serving as the platform to test the incorporation of hPSC derived fluorescently labeled myeloid progenitors in conjunction with supporting vascular and mesenchymal progenitors. In summary, this work provides new strategies to study development of hPSCs, and to implement protocols for their use in regenerative approaches