Thus hPSC derived chondrogenic ectomesenchymal cells are ame

Thus, hPSC-derived chondrogenic ectomesenchymal fak pathway are amenable to large-scale production in CDM without loss of activity. Although some hESC-derived neural crest stem cells are known to self-renew in culture for an extended period, during which they maintain the capacity to produce multipotential MSC-like activity (Menendez et al., 2011), no previous reports have described a method as simple and effective as ours for generating large quantities of (osteo)chondrogenically committed mesenchymal cells under clinically applicable, defined conditions. Therefore, the chondrogenic ectomesenchymal cells produced by the culture technology described are set to become a competitive alternative to adult MSCs, especially for craniofacial regenerative therapy. Moreover, taking advantage of high yields of chondroprogenitor cells, we have successfully modeled neonatal-onset multisystem inflammatory disease by applying our method to patient-derived iPSCs (Yokoyama et al., 2015).
Experimental Procedures
Acknowledgments
Introduction Cellular identity can be guided by ectopic expression of master regulators (Graf, 2011). Deriving induced pluripotent stem cells (iPSCs) through the activities of OCT3/4, SOX2, KLF4, and c-MYC (Takahashi and Yamanaka, 2006) provides a potent model in which to study the role of transcription factor coordination in driving somatic cells toward pluripotency. Early mechanistic studies using mouse embryonic fibroblasts (MEFs) were conducted through de novo introduction of viral vectors, each expressing an individual (monocistronic) reprogramming factor (Brambrink et al., 2008; Stadtfeld et al., 2008a), where modulation of factor levels by viral titration led to altered reprogramming characteristics (Yamaguchi et al., 2011). Monocistronic reprogramming allows for variation in copy number and integration site, and as a result, stoichiometry is inconsistent on a cell-to-cell level. Therefore, this method was succeeded by the development of polycistronic factor cassettes that can produce multiple proteins from one single transcript (Kaji et al., 2009; Sommer et al., 2009). Although such fixed polycistronic stoichiometry revealed the importance of relative factor ratios in determining the quality of reprogramming (Carey et al., 2011), the principles that establish optimal stoichiometry remain undefined. Studies of the mechanisms that underlie somatic cell reprogramming have revealed multi-step processes involving proliferation and cell-cell adhesion, along with molecular changes such as downregulation of lineage-specific genes and eventual upregulation of pluripotency markers (Plath and Lowry, 2011). Cell-surface markers were associated with reprogramming stages such as emergence of the embryonic stem cell (ESC) marker SSEA-1 (stage-specific embryonic antigen 1) (Polo et al., 2012; Stadtfeld et al., 2008a). Secondary reprogramming systems (Woltjen et al., 2009) helped define initiation, maturation, and stabilization as key stages in reprogramming toward pluripotency (David and Polo, 2014). Proliferation, colony formation, and a mesenchymal-to-epithelial transition (MET) define the initiation phase (Samavarchi-Tehrani et al., 2010), while stabilization is characterized by transgene independence and activation of pluripotency reporters such as Nanog and Dppa4 (Golipour et al., 2012). Thus, changes in global gene expression and epigenetics were associated with the progression of reprogramming through these stages (Theunissen and Jaenisch, 2014). However, discrepancies in reprogramming platforms influence reprogramming hallmarks, the severity of MET responses, lineage-specific gene repression and ectopic activation, the timing of cell-surface marker presentation, and the frequency of partial and complete reprogramming (Golipour et al., 2012; Mikkelsen et al., 2008; O’Malley et al., 2013; Polo et al., 2012; Samavarchi-Tehrani et al., 2010; Wernig et al., 2008).