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    • Following fundamental analysis of pharmacological

    2018-11-08

    Following fundamental analysis of pharmacological and electrophysiological features of suspension-derived CMs, we have demonstrated direct applicability of bioreactor-produced EBs for tissue engineering. While BCT generation from undissociated, contracting spheres was recently shown, this required genetic enrichment of CMs before tissue formation (Kensah et al., 2012). On the other hand, enrichment to ∼99% CM purity was incompatible with direct BCT generation, but required 15% fibroblast addition to remodel the initial collagen I matrix and hence support tissue formation (Kensah et al., 2012). Here, we show that the ∼80% CM content resulting from our differentiation protocol was directly compatible with the production of functional tissues, suggesting that residual non-CMs within EBs provided structural support. A detailed assessment of the phenotype and features of respective non-CMs is currently in progress. Taken together, we show that bioreactor-controlled programs of hPSC culture can be used to direct the subsequent fate of hPSCs on differentiation. As recently noted, it is now necessary to focus on the improvement of mass suspension culture for hPSC production and differentiation (Chen et al., 2014). Our study provides a substantial step along this path.
    Experimental Procedures
    Acknowledgments
    Introduction Adipose precursor JQ1 (APCs) are a subpopulation of lineage (Lin)−SCA1+ adipose-derived stromal cells competent to undergo adipogenesis in vivo and generate a functional adipose depot after transplantation (Rodeheffer et al., 2008; Joe et al., 2009; Schulz et al., 2011). It is widely anticipated that these cells will have an important role in regenerative medicine (Ong and Sugii, 2013). Furthermore, the recent isolation and characterization of APCs from white adipose tissue (WAT) using cell surface markers (Rodeheffer et al., 2008) open new avenues of stem cell and metabolic research. These markers can be used to quantify precursor cells within the adipose depot or to isolate cells for transplant or ex vivo assays (Rodeheffer et al., 2008; Joe et al., 2010; Schulz et al., 2011; Berry and Rodeheffer, 2013). However, it would be of great utility to manipulate gene expression in APCs in their physiologic in vivo context. Such experiments would require the identification of promoter/enhancer sequences that can direct gene expression specifically to this population. Previously, the promoter regions of several genes expressed in mesenchymal progenitors have been isolated and their activity characterized (Logan et al., 2002; Roesch et al., 2008; Berry and Rodeheffer, 2013). These regulatory elements, when used to drive expression of Cre recombinase, may be useful for genetic manipulation of APCs. Here we analyze recombination directed by several Cre lines in stromal populations of WAT and brown adipose tissue (BAT). We find that the most commonly used line to direct adipose-specific expression, Fabp4-Cre (He et al., 2003), is not active in most WAT APCs. We confirm previous studies that PdgfRα-Cre (Roesch et al., 2008) is active in most WAT APCs (Berry and Rodeheffer, 2013) and also find activity in most BAT Lin−SCA1+ cells. However, we also detect activity in other adipose stromal cells. In contrast, a line previously reported to direct expression in uncommitted mesenchymal progenitors (Calo et al., 2010), Prx1-Cre, has activity restricted to adipose precursors of the subcutaneous inguinal fat pad, with little recombination in other cell types within the fat pads examined. Together, these data serve as a guide for the use of these tools in the manipulation of gene expression in adipose tissues in vivo.
    Results The stromal compartment of adipose depots includes lymphocytes, macrophages, endothelial cells (ECs), fibroblasts, and APCs and, when isolated from adipocytes, is collectively referred to as the stromal vascular fraction (SVF) (Sanchez-Gurmaches and Guertin, 2014). Using a combination of four markers (CD45, CD11b, CD31, and SCA1), SVF cells can be categorized into five populations (Figures 1A and 1B), including APCs (CD45−CD11b−CD31−SCA1+). In culture, these isolated Lin−SCA1+ cells are highly responsive to standard adipogenic induction cocktail (Figure 1C and Figure S1A available online). Although cells from BAT with the APC marker profile differentiate ex vivo (Sanchez-Gurmaches et al., 2012) and express markers of BAT (Schulz et al., 2011; Liu et al., 2013), these cells are less well studied than WAT APCs; therefore, we refer to these as Lin−SCA1+ cells. Cells not labeled by any of the cell surface markers in our scheme (CD45−CD31−SCA1−, hereafter referred to as SCA1−CD31− cells) are heterogeneous in size and morphology (Figures S1B and S1C). In order to thoroughly document expression in all stromal cells, we report the recombination data (as in Figure S1D) but have not further characterized this population.