The concept of neoblast specialization at wounds is importan

The concept of neoblast specialization at wounds is important in understanding planarian regeneration and opens many avenues for future inquiry. For example, how is the neoblast response tailored to the identity of missing tissues? Do specialized neoblasts amplify their population, or do specialized neoblasts rapidly cease division and differentiate? There is an ongoing need for differentiated nucleoside analog in planarians, and low levels of specialized neoblasts appear to exist in intact animals (Cowles et al., 2013; Lapan and Reddien, 2012). For example, the ovo eye progenitors are abundant near anterior-facing wounds at 48 hr following amputation, but a small number of them can also be observed near the head of intact animals (Lapan and Reddien, 2012). Similarly, we observed some X1 neoblasts expressing FoxA, klf, and pax3/7 from macerated prepharyngeal regions of uninjured animals, with an increased number present at 48 hr following wounding (Figure S6). Regeneration involves instructions that specify the identity of missing tissue to be replaced. To understand the connection between regenerative instructions and the production of appropriate cell types, it is essential to address the cellular step at which specification of the fate of regenerative cells occurs. We propose that cNeoblasts (directly, or via their descendants) begin expressing numerous transcription factors of specific lineages in distinct neoblast cells (Figure 7). In this model, almost all of the lineages formed during development could be reconstituted during regeneration, with progenitors that generate and comprise planarian blastemas being a heterogeneous patchwork of lineage-specified cells.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) provide valuable platforms for developmental biology, disease modeling, and regenerative medicine (Ludwig et al., 2006; Reubinoff et al., 2000; Thomson et al., 1998; Yu and Thomson, 2008). Technically, however, hESC/iPSCs are difficult to culture, displaying slow growth and poor survival, especially upon cellular detachment and dissociation. Thus, hESCs were originally cultured in clusters on supporting feeder layers (Reubinoff et al., 2000). Feeder-free culture is possible if hESC/iPSCs are grown in Matrigel with chemically defined medium (Ludwig et al., 2006). In addition, methods such as culturing hESC/iPSCs in suspension (Steiner et al., 2010), with microcarriers (Bardy et al., 2013), or on synthetic polymers (Villa-Diaz et al., 2013) have been introduced. These new techniques, however, are expensive, have limited scalability, and may have high batch-to-batch variability. Y-27632, a ROCK inhibitor, is used to prevent cell apoptosis after cell dissociation and to promote cell viability after plating (Ohgushi et al., 2010; Watanabe et al., 2007), but the benefit of this chemical is limited to a brief period after cell dissociation and its continued effects on cell survival and proliferation are questionable (Couture, 2010). Thus, culture methods that are low cost, robust, scalable, easy to use, and consistent remain to be further developed to allow widespread applications of hESC/iPSCs in basic research and clinical. Human neural stem cells (hNSCs) were derived by in vitro differentiation of hESC/iPSCs. The hESC/iPSC-derived NSCs (hES/iPS-NSCs) homogeneously expressed nestin, a representative NSC-specific marker (Park et al., 2005). These nestin+ hNSCs can be expanded with basic fibroblast growth factor (bFGF) and differentiate toward neuronal cells upon withdrawal of bFGF and in the presence of combinatory neurotrophic factors (Park et al., 2005; Perrier et al., 2004). In an experiment involving a doxycycline-inducible expression system, we accidentally observed that doxycycline (1 μg/ml) itself promoted hNSC survival; the cell survival effect was not associated with exogene expression. These observations prompted us to scrutinize the doxycycline action further in undifferentiated hESC/iPSC cultures.