A microRNA-mediated approach towards generating functional hematopoietic stem cells from human pluripotent stem cells in vitro

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PROJECT SUMMARY Producing transplantable hematopoietic stem cells (HSCs) in vitro could benefit thousands of patients currently unable to receive treatment for hematological disorders. The long-term goal is to be able to generate clinically viable HSCs in vitro from human pluripotent stem cells (PSCs). This achievement would revolutionize the way numerous blood diseases could be treated, and could theoretically alleviate patients from having to wait to find a bone marrow transplant ‘match’, since a well-established clinical roadmap already exists for the use of HSCs in a therapeutic setting. Our central hypothesis is based on our strategic transcriptional profiling analysis of genetically identical in vivo HSCs (functional) and in vitro-derived ‘pre-HSCs’ (not functional). By eliminating genetic background as a source of variability, to identify the key molecular differences that underlie the functional deficiencies present in in vitro-generated HSCs, we were able to discover novel differences in regulatory pathways modulated by miRNAs. Guided by strong preliminary data, the overall objective of this application is to modulate key miRNAs during in vitro differentiation to thus provide a novel approach to obtaining functional HSCs from human PSCs. This objective will be pursued under two specific aims: (1) To identify miRNA changes during human PSC-mediated hematopoietic differentiation. In this aim, key miRNA expression levels will be analyzed during PSC-hematopoietic differentiation assays; and (2) To examine the potential of miRNAs to influence hematopoietic potential in vitro and in vivo. In this aim, key miRNA expression levels will be modified during PSC-hematopoietic differentiation assays, and analyzed for their ability to successfully generate fully functional HSCs in vitro. It is our expectation that our strategic approach will have a positive translational impact by examining a novel pathway to generate HSCs in vitro that leads to long-term hematopoietic transplantation capability, while also providing a much-needed comprehensive dataset resource for the hematopoietic field as a whole towards the successful derivation of clinically viable HSCs in vitro.