br Results br Discussion The experiments presented

Results

Discussion
The experiments presented here identify compounds that promote SKP self-renewal and proliferation in culture, and provide evidence that these compounds activate dermal precursors in vivo to promote skin repair. They also define the MEK-ERK pathway as a key self-renewal pathway for SKPs, and suggest that molecules targeting distinct cell surface receptors enhance self-renewal by converging on this intracellular signaling pathway. These data therefore provide support for the concept that pharmacological activation of endogenous tissue stem S63845 manufacturer provides a valid therapeutic approach (Miller and Kaplan, 2012).
How do alprostadil, TM, and SKP growth factors activate the MEK-ERK pathway to enhance SKP proliferation and self-renewal? FGF2 and EGF signal via receptor tyrosine kinases expressed by SKPs, and are well-known activators of the MEK-ERK pathway. Alprostadil (PGE1) binds to the EP1–4 isoforms of the EP family of receptors (Breyer et al., 2001), which are known to stimulate ERK1/2 (Yu et al., 2008). TM binds peripheral mu and kappa-opioid receptors (Kaneto et al., 1990), which also activate ERK1/2 (Gutstein et al., 1997). Thus, three distinct cell surface cues converge on a single intracellular pathway to enhance SKP self-renewal.
How do SKP self-renewal drugs promote skin repair? We previously showed that SKPs arise from, and are similar to, endogenous dermal precursors, and that transplantation of either population led to dermal reconstitution, dermal repair, and hair follicle morphogenesis (Biernaskie et al., 2009). We therefore propose that alprostadil and TM enhance skin repair by promoting proliferation and self-renewal of endogenous dermal precursors. Support for this idea comes from the data showing that SKPs isolated from drug-treated regenerating skin displayed a long-term increase in self-renewal. We suggest that alprostadil and TM enhanced the self-renewal and numbers of dermal precursors in vivo, and that this resulted in an increase in SKP sphere formation in culture. Precedent for this idea comes from studies where transient embryonic exposure to IL-6 led to an increase in adult forebrain neural precursors in vivo, and a persistent increase in sphere formation when these adult precursors were cultured (Gallagher et al., 2013). However, while our findings suggest that activation of dermal precursors underlies the enhanced skin repair, they do not exclude the possibility that alprostadil and/or TM also regulate other endogenous cells to promote wound healing.
Chronic skin wounds are a major medical problem (Eming et al., 2014), and there is a paucity of safe and effective approaches for promoting wound healing. Our findings suggest that targeting endogenous dermal precursors by repurposing of drugs already known to be safe in humans provides a new therapeutic approach for this largely unmet medical need.

Experimental Procedures

Author Contributions

Acknowledgments
This work was funded by CIHR grant MOP-64211 to F.D.M. and a Canadian Stem Cell Network Global Research grant to F.D.M. and D.R.K. S.A.Y. is funded by an Ontario Stem Cell Initiative fellowship. F.D.M. and D.R.K. hold Canada Research Chairs, and F.D.M. is an HHMI Senior International Research Scholar. We thank Darius Bagli for providing the human foreskin tissue, Tatiana Kroupnik, Natalie Grinshtein, Anastassia Voronova, and Guang Yang for advice and assistance.

Introduction
Culture conditions affect features of mouse embryonic stem cells (mESCs), such as their proliferation, gene expression, epigenetic status, self-renewal, and capacity for multi-lineage differentiation (Marks et al., 2012; Tesar et al., 2007). In culture medium with fetal calf serum, naive mESCs grown on mouse embryonic fibroblast feeder cells (here abbreviated as “serum”) transit between inner cell mass (ICM)-like and epiblast-like pluripotency states (Sasai et al., 2013; Trott and Martinez Arias, 2013). However, when cultured in serum-free conditions with inhibitors of mitogen-activated protein kinase and glycogen synthase kinase 3 signaling, also called “2i” medium, mESCs become more homogeneous and adopt the more ICM-like or “ground” state (Marks et al., 2012; Nichols et al., 2009; Ying et al., 2003). The observation that naive mESCs interconvert between pluripotent states while remaining uncommitted has raised the suggestion that such heterogeneity may allow the cells to respond differently to environmental cues. In agreement, subpopulations of naive mESCs show different potentials to differentiate (Graf and Stadtfeld, 2008; Hanna et al., 2009; Hayashi et al., 2008). How the metastable transcriptional and epigenetic diversity of cultured mESCs is regulated and maintained has remained elusive.