br Results br Discussion br Experimental Procedures br

Results
Discussion
Experimental Procedures
Acknowledgments
Introduction
Results
Discussion
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Hematopoietic in vitro differentiation of pluripotent stem ep4 (PSCs) such as embryonic stem cells (ESCs) and induced PSCs (iPSCs) holds great promise for disease modeling, drug testing, and the development of novel cell- and gene-therapy strategies. In the past, interest has been directed primarily toward reconstituting stem cells, a cell type that is difficult to generate from PSC sources. Recently, however, long-lived, mature myeloid cells have been described (Guilliams et al., 2013), and the organotropic transplantation of such cells may allow for new therapeutic scenarios (Happle et al., 2014; Suzuki et al., 2014). During embryonic development, hematopoietic cells are generated by two distinct but partly overlapping programs termed primitive and definitive hematopoiesis. Both are orchestrated by a highly complex interaction of regulatory molecules, including transcription factors, cytokine-induced and intercellular signaling, and niche factors (Lancrin et al., 2009; Nostro et al., 2008; Sturgeon et al., 2014). Primitive hematopoietic development originates from distinct multipotent precursors known as hemangioblasts, which are able to generate both vascular and hematopoietic progeny via an intermediate, hemogenic endothelial stage (Lancrin et al., 2009). Subsequently, further hematopoietic specification and differentiation result in mature cells that are primarily of an erythroid and, to a lesser degree, myeloid lineage (Palis, 2014; Schulz et al., 2012). In a separate process originating in the dorsal aorta, definitive hematopoiesis allows for the generation of transplantable hematopoietic stem cells (HSCs) that are capable of repopulating the entire lympho-hematopoietic system long term. In this context, an important role for the cytokine interleukin-3 (IL-3) (Donahue et al., 1988; Robin et al., 2006; Wiles and Keller, 1991) as well as wnt signaling (Sturgeon et al., 2014) has been reported by a number of groups. Again, the fate of these repopulating HSCs, such as self-renewal, apoptosis, quiescence, and further differentiation and proliferation, is dependent on their exposure to other cells, matrix factors, or cytokines (Arai et al., 2004; Williams et al., 1991). For both programs, granulocyte-colony-stimulating factor (G-CSF) and monocyte-CSF (M-CSF) constitute the main driving forces for the generation and terminal differentiation of functional cells of a granulocytic or monocytic/macrophage lineage, respectively (Sengupta et al., 1988; Welte et al., 1985a, 1987). G-CSF originally was identified by its capacity to promote the differentiation of human bone marrow progenitor cells toward neutrophils and is a critical component of this process (Welte et al., 1985b, 1987). However, the G-CSF receptor (CSF3R) is not exclusive to myeloid cells and has also been identified on HSCs, thus explaining the profound stem cell defects observed in congenital neutropenia patients suffering from defects in G-CSF signaling (Panopoulos and Watowich, 2008). In contrast, M-CSF, the crucial cytokine for generating mononuclear phagocytes or macrophages (MΦ) from HSC sources, appears to be primarily involved in terminal lineage differentiation (Yoshida et al., 1990). M-CSF was the first hematopoietic cytokine to be identified and cloned, and acts by activating its type III protein tyrosine kinase family receptor (c-fms) (Clark and Kamen, 1987; Sieff, 1987). Generating MΦ by M-CSF exposure, similarly to priming with IL-4/IL-10, results in alternatively activated M2-type Φ, in contrast to the classical pro-inflammatory M1Φ, which is differentiated from monocytes by GM-CSF or interferon-gamma (IFN−γ) exposure (Martinez et al., 2008; Sica and Mantovani, 2012). To date, most protocols for hematopoietic differentiation of PSCs in vitro have utilized a multitude of cytokines or small molecules to mimic the modulation of signaling pathways at various stages of embryonic development (Choi et al., 2011; Kennedy et al., 2012; Sturgeon et al., 2014). However, many of the factors involved in this process remain ill defined. Therefore, this excessive priming may have unwanted effects on the differentiation or functionality of the desired target cells, hampering their use in disease modeling or cell- and gene-therapy applications. Thus, the development of simple but robust protocols for generating nonbiased and fully functional hematopoietic cells appears to be highly warranted.