br Experimental Procedures br Author Contributions br Acknowledgments br

Experimental Procedures

Author Contributions

Acknowledgments

Introduction
Adult mesenchymal stromal Protease Inhibitor Library (MSCs), defined in vitro by their potential to contribute to bone-cartilage-fat cell lineages, are currently used for cell-based bone and cartilage therapies because of their ready accessibility. However, as for other adult stem cells, it is difficult to obtain sufficient MSCs for treatment. Expansion culture is therefore necessary before transplantation; however, it tends to cause the loss Protease Inhibitor Library of long-term viability of the MSCs and their capacity to differentiate, especially into chondrocytes (Somoza et al., 2014). Different types of bone and cartilage are formed most actively during embryonic skeletogenesis from one of three precursor cell types: paraxial mesoderm, lateral plate mesoderm, and cranial neural crest. Such embryonic cells and their osteochondrogenic progeny may be as effective as or more effective than MSCs for the regeneration of adult bone and cartilage.
The early processes of in vitro differentiation of pluripotent embryonic stem cells (ESCs) mimic those of in vivo embryogenesis (Nishikawa et al., 2007). Therefore, ESCs and induced pluripotent stem cells (iPSCs) (collectively designated pluripotent stem cells or PSCs) would appear to be the practical source of embryonic precursor cells in humans. In fact, in vitro induction of osteogenesis and chondrogenesis from human PSCs (hPSCs) and mouse (mPSCs) has been demonstrated by many groups (Nakayama and Umeda, 2011). With the exception of recent reports, including ours (Craft et al., 2013; Diekman et al., 2012; Nakayama et al., 2003; Toh et al., 2009; Umeda et al., 2012; Zhao et al., 2014), many of the earlier reports described spontaneous differentiation of hPSCs followed by enrichment of mesenchymal cells by further culturing the progeny in MSC medium. As adult human tissue-derived MSCs or chondroprogenitors (Koelling et al., 2009; Pittenger et al., 1999), mesenchymal cells derived from mESCs/iPSCs were able to be expanded extensively; however, expansion occurred with the loss of their chondrogenic activity (Bakre et al., 2007; Diekman et al., 2012). Thus far, the potential benefits of bone and cartilage repair of hPSC-derived osteochondroprogenitors over those of adult MSCs, whether in quantity or in quality, have not been demonstrated, even in vitro (Nakayama and Umeda, 2011). In theory, the wealth of information on the signaling mechanisms involved in mouse skeletogenesis should be of great help in improving the expansion culture methods. However, the unclear embryonic origins of the chondrogenic activity developed from PSCs and the undefined conditions used for expansion have hampered full use of the information and thereby hindered progress.
A large portion of craniofacial bone and cartilage arises from osteochondrogenic progeny (i.e., ectomesenchyme) from cranial neural crest (Santagati and Rijli, 2003), generated from the junction between anterior neuroectoderm and surface ectoderm (Milet and Monsoro-Burq, 2012). Neural crest cells have been developed from hESCs in 12–28 days of differentiation culture either through neuroepithelial intermediates induced by suppression of Nodal/Activin/transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signaling in a defined medium (Chambers et al., 2009; Smith et al., 2008) or directly by activation of WNT signaling with suppression of Nodal/Activin/TGFβ signaling (Menendez et al., 2011). Further differentiation and expansion for 2–3 weeks of such neural crest cells or earlier neural cells in a serum-containing medium generate MSC-like cells with variable chondrogenic activity, but never sufficient to reproducibly form cartilage particles that accumulate proteoglycan-rich, mature matrices uniformly (hereafter designated “full-cartilage”).
Our group has focused on generating and characterizing paraxial mesodermal progeny from mPSCs/hPSCs, which are highly chondrogenic (Tanaka et al., 2009; Umeda et al., 2012; Zhao et al., 2014). In the current study, we report simple, effective methods for the specification of neural crest-like progeny from hPSCs and subsequent generation and expansion of chondrogenically committed ectomesenchymal cells without loss of their chondrogenic activity over 7–8 weeks in chemically defined media (CDM). The outcomes were achieved by the control of fibroblast growth factor (FGF) signaling and Nodal/Activin/TGFβ signaling. We have also defined the cellular developmental pathway from hPSCs to such ectomesenchymal cells using the neural crest markers the low-affinity nerve growth factor receptor (CD271) (Lee et al., 2007; Stemple and Anderson, 1992), and the platelet-derived growth factor receptor α (PDGFRα) (Morrison-Graham et al., 1992; Weston et al., 2004), and the MSC markers, CD73 and CD13 (Olivier et al., 2006; Pittenger et al., 1999).