Several groups have reported the culture

Several groups have reported the culture and differentiation of fetal or adult neural stem finding molarity (NSCs) on polymer-based substrates (Park et al., 2014; Little et al., 2008; Bhang et al., 2007; Lundin et al., 2011; Saha et al., 2007). It is important to point out that the NSCs used in these studies, which can be isolated from numerous species and various regions in the fetal and adult nervous system, are biologically and developmentally distinct from the hNPCs used in this study (Kornblum, 2007; Temple, 2001). Specifically, the differentiation potential and, thereby, the therapeutic application of NSCs is much more limited than hNPCs, which can differentiate into all the neural lineages (i.e. neurons, astrocytes, and oligodendrocytes) that compromise the central nervous system (CNS) (Chambers et al., 2009; Elkabetz et al., 2008; Shin et al., 2006). Because of these inherent biological differences in NSCs and hNPCs, it is unclear if these substrates that have been previously used for the growth and differentiation of NSCs would have similar efficacy if used with hNPCs. To our knowledge, the synthetic matrix developed in this study is the first demonstration of the use of a defined polymer-based substrate for the culture and differentiation of hNPCs. In order to have a sufficient number of hNPCs for regenerative medicine and drug screening, efficient methods for their large-scale expansion and differentiation need to be developed. In fact, cell doses for stem cell-based neural therapies have been reported in excess of 6 billion cells per patient (Bretzner et al., 2011; Schwartz et al., 2012; Chen et al., 2013). Although in this study theoretical calculations suggested that 1×106 hNPCs cultured on P4VP could be expanded up to 1×109 cells in 10 passages (~50days), practical expansion and differentiation of hNPCs to these numbers are not feasible with current 2D culture systems. Alternatively, microcarriers (MCs), which provide a high-surface area to volume ratio, enable high density cell expansion and scale-up in stirred bioreactors (Reuveny, 1990; Sart et al., 2013). Recently, the use of protein-coated MCs in stirred suspension bioreactors has been reported for the expansion of several hPSC lines and their derivatives (Sart et al., 2013; Fan et al., 2013; Ting et al., 2014). In the future, the culture of hNPCs on polymer-coated MCs in suspension bioreactors may enable their expansion and differentiation to the numbers required for regenerative medicine purposes.
Conclusions In this study, we used a high-throughput screening process to identify a synthetic polymer, P4VP, which can support the long-term self-renewal and proliferation of hNPCs at a similar level to cells cultured on purified ECMPs. Moreover, neuronal differentiation of hNPCs was more efficient on P4VP substrates than these traditional ECMP-based substrates. P4VP is chemically defined and available off-the-shelf, thus overcoming the limitations associated with culture on purified ECMPs. Overall, the polymeric biomaterial developed in this study offers a cost-effective, scalable, and robust platform to support the in vitro expansion and neuronal differentiation of hNPCs to the quantities needed for disease modeling, drug screening, and cell-based therapies. The following are the supplementary data related to this article.
Acknowledgments
Introduction The ability of adult tissues and organs to maintain itself and repair after injury is dependent on the activity of tissue-resident stem cells. These tissue stem cells respond to exogenous cues from the surrounding environment, the niche, by quiescence, proliferation and/or differentiation. Understanding how this niche regulate the stem cell behavior is essential in order to be able to exploit stem cells for various therapeutic applications (Wagers, 2012). The lung is a complex organ with multiple functions and extensive branching. It is composed of three major distinct regions the tracheobronchial, bronchiolar airways and the alveolar regions. Each of these regions consists of distinct epithelial cell types with unique cellular physiologies and stem cell compartments. Several recent studies have identified and characterized the stem cells of these regions during homeostasis and after injury (Hegab et al., 2011, 2012; Rock et al., 2009; Barkauskas et al., 2013; Rawlins et al., 2009; Zheng et al., 2012; Tata et al., 2013; Giangreco et al., 2002). Cells lining the ducts of submucosal gland of the tracheobronchial airways have been shown to be responsible for maintaining and repairing submucosal glands after injury (Hegab et al., 2011, 2012). Basal cells are the stem cells of trachea as they self-renew and differentiate into club (previously called Clara) and ciliated cells (Rock et al., 2009) while Alveolar type II cells are the stem cells of the alveolar region (Barkauskas et al., 2013). Club cell is the main and well-characterized stem cell of the intrapulmonary airways and it can self-renew and differentiate into ciliated cells (Rawlins et al., 2009). Under severe injury conditions, club cells can differentiate into alveolar cells (Barkauskas et al., 2013; Zheng et al., 2012) or “de-differentiate” into basal cells (Tata et al., 2013). The presence of a subpopulation of club cells at branching points, close to neuroepithelial bodies and at terminal bronchioles close to bronchiolaveolar duct junction (BADJ) that are more resistant to injury and have more superior stem cell characteristics has been repeatedly described (Giangreco et al., 2002; Chen et al., 2012; Reynolds et al., 2000). Pulmonary neuroendocrine cells also self-renew and contribute to club and ciliated cells after injury (Reynolds et al., 2000; Song et al., 2012). However, to the contrary to several other organs like the heart (Christalla et al., 2012) and skin (Solanas and Benitah, 2013), in which extensive knowledge regarding their stem cell niche have accumulated over the past years, our knowledge regarding what niche components are influencing lung regenerative potential and what are the mechanisms regulating the various lung stem cells\' differentiation and repair are lacking. A stem cell niche would usually comprise several of these components: Cellular components, which may include epithelial, endothelial, mesenchymal (especially fibroblasts) and/or hematopoietic cells. These cells interact with the stem cell through signaling components which may be paracrine (e.g. sonic hedgehog, Wnt), autocrine or juxtacrine (contact) signals. In addition, substances present in the extracellular matrix represent another component (e.g. collagen, lamenin and fibronectin) that plays an active role in the interaction with stem cells (Wagers, 2012).