br Methods br Results br Discussion

Methods
Results
Discussion Our method for osteoprogenitor cell culture is similar to protocols for mice (Meirelles and Nardi, 2003; Soleimani and Nadri, 2009; Nardi and Camassola, 2011; Abdelmagid et al., 2014; Fig. 1). Marrow and cortex-derived cell populations of bats and mice shared similar proliferation rates. However, proliferation was greater in phospholipase a2 inhibitor harvested from cortical bone of bats compared to mice (Fig. 2). Histological staining and quantification of mineral deposition showed cells harvested from bats deposited significantly less mineral compared to murine controls, with the lowest amount deposited by bone marrow-derived bat cells (Fig. 4). Significantly lower transcript numbers of genes associated with bone formation (ALPL and SP7), osteoblast and osteoclast differentiation (RUNX2, OPG and SP7) and extracellular matrix formation and interaction (BGLAP and ON) were detected in bats-derived cells relative to murine controls (Stein and Lian, 1993; Yao et al., 1994; Bailey et al., 1999; Delany et al., 2000; Harada and Rodan, 2003; Byers and García, 2004; Tai et al., 2004; Cao et al., 2005; Gregory et al., 2005; Stiehler et al., 2009; Zhang et al., 2009; Gramoun et al., 2010; Korostishevsky et al., 2012; Masrour Roudsari and Mahjoub, 2012; Sardiwal et al., 2012; Sroga and Vashishth, 2012; Pekovits et al., 2013; Koide et al., 2013; Krӓmer et al., 2014; Krege et al., 2014; Fig. 3). These differences in gene expression were associated with a less mineralized extracellular matrix in bats (Fig. 5). Deposition of a less mineralized extracellular matrix, even in vitro, is suggestive of intrinsic, naturally occurring differences in the auto-regulation of bat osteoprogenitor cell function and performance. These physiological differences suggest regulation of osteoprogenitor cell matrix synthesis differs in bats compared to mice. The wing bones of bats, including the radius studied here, are elongated, complaint bones (Papadimitriou et al., 1996; Swartz, 1997; Swartz and Middleton, 2008; Bergou et al., 2015). The proximal forelimb elements of bats, relative to terrestrial mammals such as mice, display thinner cortices and the greatest mineral content compared to distal elements (Papadimitriou et al., 1996; Dumont, 2010; Cooper and Sears, 2013). These modifications in length, mineral concentration and extracellular matrix may increase flexibility and create specialized skeletal microenvironments. It may be that the unusually flexible bones of bats impart unique micro-loads on constituent osteoprogenitor and stem cells, as documented in other taxa, e.g., rodents, etc. (Gilbert et al., 2010; Miller et al., 2015). Surprisingly, even in a 2D culture system with equivalent treatments and lacking the stressors associated with locomotion, our study shows the performance of osteoprogenitor cells harvested from bats differed significantly from mice both in their gene expression patterns and matrix production. Taken together, these results suggest that the osteoprogenitor cells of bats display different autoregulation of matrix secretion, compared to that of mice, regardless of microenvironment. Until now, the methods required for the study of bat osteoprogenitor cell activity and/or cell-cell interactions in a culture system were unknown. This study establishes a protocol to successfully isolate and differentiate osteoprogenitor cells from bat cortical bone and bone marrow. Furthermore, this study also partially lays the foundation for broader comparative studies of the molecular cross-talk between bone cells (osteoclasts, osteoblasts). Imbalances in osteoblast and osteoclast crosstalk lead to imbalanced cell activity that ultimately negatively impacts skeletal health, integrity and repair (Sims and Martin, 2014; Weivoda et al., 2015). Some of these imbalances are associated with senescence-related changes that negatively impact osteoprogenitor and stem cell differentiation rates and decrease stem cell populations leading to age-related skeletal fragility (Muraglia et al., 2000; Janzen et al., 2006; Raggi and Berardi, 2012; Yu and Kang, 2013). Our results suggest that osteoprogenitor cells of bats are intrinsically different from mice in their biology and performance. Future work may extend to quantifying similar characteristics in the hematopoietic stem cells (HSCs) of bats and mice, and therefore allow for comparative studies of age-specific bone cell cross-talk. Results may provide novel insights into potential therapeutic targets for human age-related skeletal disorders including osteoporosis, etc.