Person:

Ebina, Wataru

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine.

Hematopoietic stem cells (HSCs) sustain continuous turnover and maintenance of all blood lineages through organismal lifespan. The extensive regenerative potential of HSCs has been harnessed in transplantation medicine to enable curative therapies for numerous life-threatening conditions that require hematological reconstitution. However, the rarity of HSCs combined with the limited availability of immunologically matched donors have constrained the utility of HSC transplantation whose success and safety depend critically on the quantity donor HSCs; therefore, ex vivo expansion of HSCs has been a highly sought after goal in HSC research. In this thesis, I present a hypothesis driven approach toward identifying cocktails of small molecules that enable ex vivo maintenance and propagation of mouse and human HSCs. Specifically, using HSCs isolated from Fgd5ZsGreen HSC-specific fluorescent reporter mice along with previously identified immunophenotypic markers, I conducted a small scale combinatorial chemical screen of developmental signaling modulators to determine a condition that would preserve immunophenotypic HSCs ex vivo. The screen led to the discovery that murine HSCs can be maintained for at least 14 days ex vivo when the basal media was supplemented with cytokines and the minimal combination of a small molecule inhibitor of TGF-β signaling and two epigenetic inhibitors, namely LSD1 inhibitor and HDAC inhibitor, which were selected based on reports that they may derepress Notch target gene expression. Additionally, metabolic optimizations led to the identification of putrescine as a critical culture supplement for promoting HSC propagation. The three chemicals identified in the murine screen were conserved in their ability to promote the ex vivo preservation of primary human HSCs. However, as presence of the two epigenetic inhibitors caused substantial growth suppression, alternative, more specific means to activate the Notch pathway were sought. To this end, I hypothesized that inhibition of IKAROS transcription factor, a repressor of Notch target gene expression, would be able to derepress Notch pathway activity and hence replace the growth suppressive epigenetic inhibitors. Indeed, substitution of the epigenetic inhibitors with an IKAROS inhibitor rescued immunophenotypic HSC propagation, and additional supplementation with UM171, a recently identified small molecule enhancer of human HSC expansion, further improved HSC yield as well as the durability of immunophenotypic preservation over prolonged culture; in sum, three compounds, namely a TGF-β inhibitor, pomalidomide, and UM171, were found to be necessary for robust ex vivo maintenance and propagation of human HSCs. At the time of writing, xenotransplantation is underway to assess the in vivo function of ex vivo cultured human HSCs. Collectively, this body of work contributed to identifying chemically defined ex vivo culture conditions supportive of murine and human HSCs while underscoring the importance of combinatorial pathway modulation for maintaining HSC immunophenotype and function. Development of effective HSC culture conditions should not only benefit clinical medicine but also facilitate interrogation and manipulation of HSCs in vitro.