During the last decade significant progress has been made in

During the last decade, significant progress has been made in achieving hematopoietic differentiation from hPSCs. Multiple protocols for hematopoietic differentiation have been developed and have made it possible to routinely produce blood stavudine for experimentation. However, generating HSCs with long-term reconstitution potential from hPSCs remains a significant challenge. Hematopoietic cells and HSCs arise from a specific subset of endothelium (HE) in the embryo (Bertrand et al., 2010; Boisset et al., 2010; Jaffredo et al., 2000; Kissa and Herbomel, 2010; Zovein et al., 2008). Therefore, the ability to interrogate the signaling pathways that induce HE specification and the endothelial-to-hematopoietic transition in a completely chemically defined environment is essential in order to identify the factors required for HSC specification. Although the original protocols for hematopoietic differentiation employed xenogeneic feeder cells and/or serum, several serum- and feeder-free systems for hematopoietic differentiation have been described recently (Ng et al., 2008; Salvagiotto et al., 2011; Smith et al., 2013; Wang et al., 2012). However, these protocols still require serum components (albumin) and it remains unclear whether these protocols reproduce the distinct waves of hematopoiesis, including the generation of HE with definitive lymphomyeloid potential, observed in the original differentiation systems. Recently, Kennedy et al. (2012) developed a feeder- and stroma-free condition for EB-based hematopoietic differentiation in a proprietary medium with undisclosed nutrient supplements from hPSCs expanded on mouse embryonic fibroblasts. These conditions reproduced primitive and definitive waves of hematopoiesis and generated HE with T lymphoid potential. Here, we developed a protocol that enables the efficient production of blood cells in completely chemically defined conditions, free of serum and xenogeneic proteins, from a single-cell suspension of hPSCs maintained in chemically defined E8 medium (Chen et al., 2011). Our protocol eliminates the variability associated with animal- or human-sourced albumins, xenogenic matrices, clump size variation, and asynchronous differentiation observed in EB systems. It also reproduces the typical waves of hematopoiesis, including the formation of HE and definitive hematopoietic progenitors, observed in hPSCs differentiated on OP9. Importantly, based on molecular profiling of OP9 and stromal cell lines with different hematopoiesis-inducing activity, we found that the TenC matrix protein, which is uniquely expressed in OP9 with robust hemato-inducing potential, strongly promotes hematoendothelial and T lymphoid development from hPSCs. TenC is a disulfide-linked hexameric glycoprotein that is mainly expressed during embryonic development. Although TenC mostly disappears in adult organisms, its expression is upregulated during wound repair, neovascularization, neoplasia (Hsia and Schwarzbauer, 2005), and limb regeneration (Stewart et al., 2013). TenC is found in adult bone marrow, where it is expressed predominantly in the endosteal region (Klein et al., 1993; Soini et al., 1993). TenC supports the proliferation of bone marrow hematopoietic cells (Seiffert et al., 1998) and erythropoiesis (Seki et al., 2006). TenC-deficient mice were shown to have lower bone marrow CFC potential (Ohta et al., 1998), failed to reconstitute hematopoiesis after bone marrow ablation, and showed a reduced ability to support engraftment of wild-type HSCs (Nakamura-Ishizu et al., 2012). In addition, TenC is expressed in the thymus (Hemesath and Stefansson, 1994) and plays an important role in T cell development, as evidenced by decreased T lymphoid progenitors in the thymus and an increased proportion of T cells in the bone marrow of TenC-deficient mice (Ellis et al., 2013). Interestingly, high levels of TenC expression were also detected in the human and chicken AGM region (Anstrom and Tucker, 1996; Marshall et al., 1999), the site where the first HSCs emerge, and in hematopoietic sites of human fetal liver (Papadopoulos et al., 2004). Because TenC expression is highly enriched in the subaortic mesenchyme directly underneath hematopoietic clusters, it was suggested that TenC plays a pivotal role in HSC development during embryogenesis (Marshall et al., 1999). TenC is also involved in the regulation of angiogenesis and cardiac endothelial progenitors (Ballard et al., 2006). Our studies demonstrated the superior properties of TenC for promoting hematoendothelial development from hPSCs. The positive effect of TenC was obvious at all stages of differentiation, including the enhancement of hematovascular mesoderm, HE, and CD43+ hematopoietic progenitors. Importantly, TenC was able to support the development of definitive hematopoietic cells with T lymphoid potential, whereas we were not able to obtain such cells in cultures on ColIV. The TenC molecule is composed of an amino-terminal oligomerization region followed by heptad repeats, EGF-like and fibronectin type III repeats, and a fibrinogen globe (Hsia and Schwarzbauer, 2005). Each of these domains interacts with different surface receptors, including integrins α9β1, αvβ3, and αvβ6, and toll-like receptor 4 (TLR-4) (Midwood et al., 2011). It is believed that the effect and interaction of TenC with cells requires the integrated action of multiple domains (Fischer et al., 1997), although several unique mitogenic domains capable of inducing the proliferation of hematopoietic cells were identified within this molecule (Seiffert et al., 1998). The interaction of TenC with α9β1 integrin plays a central role in TenC-mediated expansion of hematopoietic stem and progenitor cells (Nakamura-Ishizu et al., 2012) and may be required for normal T cell development (Ellis et al., 2013). Several signaling mechanisms implicated in cell interaction with TenC have been identified, including the suppression of fibronectin-activated focal adhesion kinase signaling, Rho-mediated kinase signaling, and stimulation of Wnt signaling pathways (reviewed in Orend, 2005). Further studies to identify the mechanism of TenC signaling on hPSCs and their hematopoietic derivatives would help elucidate the role of this matrix protein during development. It is also important to determine the developmental stages that are most affected by TenC and clarify whether TenC simply enhances the expansion of hemogenic populations or promotes hematoendothelial commitment.