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Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.

In vivo hematopoietic generation occurs in waves of primitive and definitive cell emergence. Differentiation cultures of pluripotent embryonic stem cells (ESCs) offer an accessible source of hematopoietic cells for blood-related research and therapeutic strategies. However, despite many approaches, it remains a goal to robustly generate hematopoietic progenitor and stem cells (HP/SCs) in vitro from ESCs. This is partly due to the inability to efficiently promote, enrich, and/or molecularly direct hematopoietic emergence. Here, we use Gata2Venus (G2V) and Ly6a(SCA1). GFP (LG) reporter ESCs, derived from well-characterized mouse models of HP/SC emergence, to show that during in vitro differentiation they report emergent waves of primitive hematopoietic progenitor cells (HPCs), definitive HPCs, and B-lymphoid cell potential. These results, facilitated by enrichment of single and double reporter cells with HPC properties, demonstrate that in vitro ESC differentiation approximates the waves of hematopoietic cell generation found in vivo, thus raising possibilities for enrichment of rare ESC-derived HP/SCs. Kauts et al. demonstrate that Gata2Venus, Ly6aGFP, and double reporter expression in differentiating mouse embryonic stem cells (ESCs) discriminates and facilitates enrichment of most hematopoietic progenitors generated in vitro. Sequential waves of ESC-derived reporter cell emergence show increasing hematopoietic lineage potency and approximate the in vivo waves of hematopoietic cell generation found in the mouse embryo.

The mammalian hematopoietic system is maintained by hematopoietic stem cells (HSC). Whereas in the adult, they reside in the bone marrow, the first HSCs are generated in the main vasculature of the midgestation embryo as a result of tightly regulated extrinsic and intrinsic molecular signals. Transcriptional regulation is pivotal to the establishment of HSC fate during development and the Gata2 transcription factor is central to this process. Genetic deletion of Gata2 shows that it is essential for the embryonic generation of HSCs, hematopoietic progenitor cells (HPC), and also for the establishment of some mature blood lineages, such as mast cells. Although previous studies with HPC/HSC enriched populations indicate the importance of Gata2 in their generation, there has been no direct way for studying Gata2-expressing cells in normal embryonic development, and in in vitro hematopoietic differentiation assays. In this thesis research, I use a novel Gata2Venus reporter embryonic stem cell (ESC) system and mouse model to dissect the role of Gata2 in hematopoietic differentiation and development. I show that Venus expression discriminates progressive stages of hematopoietic cell generation in vitro that are highly analogous to the in vivo waves of hematopoietic cell generation. My results demonstrate the involvement of the Gata2 downstream target, Gpr56 (a putative HSC regulator), in the development of HPCs, and reveal the redundant expression and function of Gpr56 with another G-protein coupled receptor, Gpr97. This redundancy may explain the previous contradictory data in the field regarding the requirement for Gpr56 in HSCs, and opens the way for new strategies to delineate the role of Gpr56 in HSC emergence. In addition to HSCs and HPCs, the mast cell lineage is one of the few mature blood cell types that expresses Gata2. Taking advantage of the Gata2Venus ESC reporter line and human induced pluripotent stem cell (iPSC) lines, I found a novel and powerful approach

The development of the hematopoietic system during early embryonic stages occurs in spatially and temporally distinct waves. Hematopoietic stem cells (HSC), the most potent and self-renewing cells of this system, are produced in the final ‘definitive’ wave of hematopoietic cell generation. In contrast to HSCs in the adult, which differentiate via intermediate progenitor populations to produce functional blood cells, the generation of hematopoietic cells in the embryo prior to HSC generation occurs in the early waves by producing blood cells without intermediate progenitors (such as the ‘primitive’ hematopoietic cells). The lineage relationship between the early hematopoietic cells and the cells giving rise to HSCs, the genetic networks controlling their emergence, and the precise temporal determination of HSC fate remain topics of intense research and debate. This Review article discusses the current knowledge on the step-wise embryonic establishment of the adult hematopoietic system, examines the roles of pivotal intrinsic regulators in this process, and raises questions concerning the temporal onset of HSC fate determination.

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, therehasbeen nodirect study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not allHPCs in the aorta, vitellineand umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.