Given the emerging role of
Given the (1) emerging role of IL-3 in early primitive as well as definitive hematopoietic specification (Donahue et al., 1988; Robin et al., 2006; Yang et al., 1986), (2) the fundamental importance of G-CSF and M-CSF in terminal granulocyte and monocyte/macrophage differentiation (Clark and Kamen, 1987; Sengupta et al., 1988; Sieff, 1987; Welte et al., 1987), and (3) the synergy reported between IL-3 and late-acting factors such as G-CSF, M-CSF, and granulocyte-macrophage CSF (GM-CSF) (Donahue et al., 1988; Wiles and Keller, 1991), we sought to investigate the combined use of IL-3 with either M-CSF or G-CSF, employing embryoid body (EB)-based hematopoietic in vitro differentiation. For this purpose, we developed an innovative protocol that allows for the prolonged and large-scale production of functional granulocytes as well as monocytes/macrophages. Generation of immature myeloid hif pathway was mediated by an intermediate myeloid-cell-forming complex (MCFC) containing CD34+ clonogenic progenitor cells, which upon continued G-CSF or M-CSF exposure generated terminally differentiated myeloid cells for ≥2 months. As generation of these cells was accomplished by exposure of PSCs to IL-3 and one additional cytokine only, this protocol can be expected to closely recapitulate many aspects of physiologic hematopoietic development. Thus, it may be particularly suitable for studying human hematopoietic development in vitro and generating mature functional cells for cell and gene therapy.
Discussion Hematopoietic differentiation of human PSCs represents a highly complex and finely tuned process in which the interaction of different cytokines, matrix factors, cell types, signaling pathways, and transcription factors results in specialized cells of the hematopoietic lineage (Clarke et al., 2013; Sumi et al., 2008). In this study, we provide experimental evidence that a combination of IL-3/G-CSF or IL-3/M-CSF in vitro is sufficient to drive differentiation of hiPSCs toward functional cells of granulocyte or monocyte/macrophage lineage. As a matter of fact, when only G-CSF was used, lower cell shedding from the MCFC was observed (data not shown). This implies that IL-3 is critical for the early part of this process. In the context of adult hematopoiesis, IL-3 has been demonstrated to support myelopoiesis, specifically when combined with other growth factors such as G-CSF or GM-CSF, and later work established its efficacy on the stem and early progenitor cell level (Donahue et al., 1988; Krumwieh et al., 1990). Similarly, a cooperative effect of IL-3 with cytokines such as IL-1, M-CSF, GM-CSF, and erythropoietin on both myeloid and erythroid in vitro differentiation of ESCs has been described (Wiles and Keller, 1991). More recently, IL-3 was also demonstrated to promote the development of HSCs in the yolk sac and the aorta-gonad-mesonephros (AGM) region of murine embryos, and a role of this cytokine in proliferation or survival of early HSCs was postulated (Robin et al., 2006). Given these results, our finding that IL-3 has a profound effect on the hematopoietic differentiation process comes as no surprise. Moreover, our own unpublished data suggest that IL-3 also cooperates with IL-4 in generating dendritic cells from hiPSCs, further supporting a pivotal role of IL-3 during hematopoietic in vitro differentiation of human PSC sources. Clearly, in our hands, the formation of an endothelial-like stromal cell layer by the MCFC was predictive for successful hematopoietic differentiation later on. Although the requirement of endothelial support and even endothelial origin (hemogenic endothelium) for definitive hematopoiesis has been firmly established (Choi et al., 2012; Clarke et al., 2013; Kennedy et al., 2007; Sturgeon et al., 2014), it appears unlikely that the endothelial-like stromal cells that anchored the EBs to the plates in our cultures directly interact with the primitive cells within the MCFC that is responsible for the continuous output of hematopoietic cells. A much more likely explanation is that also within the MCFC, endothelial stromal cell support is required for efficient hematopoietic differentiation, and the endothelial-like stromal cell layer on the outside functions as an indicator of the efficacy of this interaction (Sandler et al., 2014).