Recent reports have had success with promoting differentiati

Recent reports have had success with promoting differentiation of PSC first to NKX2-1+ lung progenitor eicosapentaenoic acid and further to airway (Gilpin et al., 2014; Gouon-Evans et al., 2006; Huang et al., 2014; Kubo et al., 2004; Mou et al., 2012; Wong et al., 2012) or alveolar (Ghaedi et al., 2013; Huang et al., 2014; Jensen et al., 2012; Kimura et al., 1996; Longmire et al., 2012; Minoo et al., 1999) epithelial cells using monolayer cultures and supplementation with inductive factors. However, these monolayer protocols achieved lung differentiation to a limited repertoire of functional epithelial cells with unclear efficiencies, potential contamination from other endodermal lineages, lacking relevant 3D structure and in some cases with undefined culture conditions using fetal bovine serum. To our knowledge, there have been no previous reports demonstrating the efficient generation of stem cell-derived mature airway structures with CFTR function differentiated with extended culture on decellularized lung scaffolds alone. The ability to repopulate decellularized scaffolds in culture has been used as an assay to assess the regenerative potential of differentiated cells in previous reports, where primary cells or predifferentiated cells derived from PSC have been seeded onto rat or human decellularized lungs (Bilodeau et al., 2014; Ghaedi et al., 2013; Gilpin et al., 2014; Huang et al., 2014; Jensen et al., 2012; Longmire et al., 2012; Ott et al., 2010; Petersen et al., 2010). Successful repopulation and maturation of seeded cells on lung scaffolds achieved by these studies reinforces the importance of a 3D ECM setting for supporting cell adherence, organization, and maturation. However, in these models, cells were seeded after specification to the lung lineage using inductive factors in monolayer cultures, and supplementation continued after seeding on scaffolds, therefore hindering assessment of the influence of lung ECM solely on promoting lineage-specific differentiation. Matrix-associated HS and CS proteoglycans are major modulators of growth factor binding and signaling on the ECM surface and function by stabilizing FGF/FGFR complexes, increasing local gradients, and promoting FGF internalization and processing (Izvolsky et al., 2003; Kimura and Deutsch, 2007; Shannon et al., 2003; Thompson et al., 2010). Specification to the airway lineage in our model was found to be dependent on HS proteoglycans and bound factors remaining on scaffolds. Using a proteome profiler antibody array as an initial screen, we showed that numerous proteins remain on scaffolds after decellularization. A list of potential candidates implicated in lung specification was identified using this method, although the array profile is not an exhaustive list and a more in-depth analysis is required to parse out the HS-bound proteins present on scaffolds that are essential for differentiation.
Experimental Procedures
Introduction Recent advances in the generation, propagation, and differentiation of pluripotent stem cells (PSCs) offer great promise in the field of regenerative medicine. Both embryonic stem cells (ESCs) and induced PSCs (iPSCs) provide limitless sources of self-renewing cells endowed with the potential to generate tissue-specific cell populations that can be used in transplantation therapy (Grabel, 2012; Keller, 2005). However, one major hurdle in realizing this potential is the lack of specific and efficient protocols for differentiating these PSCs to specific populations that can be used for therapeutic applications. Although stem-cell-based regenerative medicine is still a distant goal, outstanding progress has been made in generating and engrafting ESC-derived lineages such as dopamine neurones (Kriks et al., 2011) and cardiomyocytes (Shiba et al., 2012; Yang et al., 2008). In contrast, since the first report of blood cell generation from ESCs 30 years ago (Doetschman et al., 1985), progress in deriving hematopoietic cells that are able to engraft in vivo has been rather modest. To date, the most successful in vitro derivation of hematopoietic cells capable of repopulating mouse models has relied on the ectopic expression of transcription factors such as HOXB4 (Kyba et al., 2002), CDX4 (Wang et al., 2005b), LHX2 (Kitajima et al., 2011), and RUNX1a (Ran et al., 2013). However, although HOXB4 overexpression has been shown to confer reproducible engraftment capability in differentiating mouse ESCs (Bonde et al., 2008; Kyba et al., 2002; Lesinski et al., 2012; Matsumoto et al., 2009), this approach has not been successfully translated to human ESCs (Wang et al., 2005a). An alternative approach to the use of HOXB4 in differentiated human ESCs was recently reported by Doulatov et al. (2013), who showed that the ectopic expression of transcription factors (HOXA9, ERG, RORA, SOX4, and MYB) in differentiating ESCs promotes short-term erythroid and myeloid engraftment. Few reports have documented the in vitro generation of hematopoietic repopulating potential from unmanipulated ESCs (Burt et al., 2004; Hole et al., 1996; Müller and Dzierzak, 1993; Potocnik et al., 1997). However, these approaches have not been reproduced or pursued, suggesting that they involve serum-dependent conditions that cannot be easily replicated. The use of high serum concentrations (Wang et al., 2005a) and/or stroma cell lines (Ledran et al., 2008) to support the formation of repopulating hematopoietic cells derived from human ESCs has also shown promising results, but to date, no follow-up studies have further validated or extended these differentiation protocols. It is likely that the reported successes in deriving repopulating hematopoietic cells relied on specific factors present in rare batches of serum—parameters that are impossible to control for and thus are extremely difficult to reproduce.