Adenylyl Cyclase

[PMC free article] [PubMed] [Google Scholar]Li L, Klim JR, Derda R, Courtney AH, Kiessling LL

[PMC free article] [PubMed] [Google Scholar]Li L, Klim JR, Derda R, Courtney AH, Kiessling LL. hMSC/hHT co-culture secretome using a reporter cell line for TGF-bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-1. These results suggest a potential for TGF–mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/ turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies. has been widely reported to be a potent inducer of tenogenic regeneration [Gafni et al., 2004; Lui et al., 2011]. Proteins of the TGF- superfamily are considered pleiotropic cytokines that play a prominent role during wound healing and musculoskeletal tissue development [Leask and Abraham, 2004; Schiller et al., 2004]. More specifically, during T/L development, TGF- has been reported to be a key mediator Fumonisin B1 of a panel of genes that are responsible for the anabolic and catabolic maintenance of ECM in vitro and in vivo [Massague, 1998; Li et al., 2011]. FzE3 Molecular changes evidenced in the altered expression of anabolic markers such as collagens and proteoglycans are known to accompany the healing of T/L [Kuo and Tuan, 2008]. Additionally, changes in the expression patterns of catabolic markers such as the collagen-degrading MMP family (matrix metalloproteinases) and proteoglycan-cleaving ADAMTS family (a disintegrin and metalloproteinase with thrombospondin motifs) have also been reported [Jones et al., 2006; Corps et al., 2008; Kuo and Tuan, 2008; Wylie et al., 2012; Maeda et al., 2013]. The balance between the regulation and production of these markers has significant implications in the extent of matrix remodeling during regeneration [Jones et al., 2006; Smith et al., 2008]. The objective of this study was to determine the effect of the paracrine signaling, or Fumonisin B1 cross-talk, between primary human hamstring tenocytes (hHT) and hMSC on cell response and the expression of T/L markers in both cell types in vitro and screen the co-culture for TGF- bioactivity. We hypothesize that this co-culture of hMSC with hHT will lead to enhanced tenogenic cell function when compared to populations cultured separately. We postulate that this exchange of soluble factors will facilitate the maintenance of ECM produced by both cell types, ultimately leading to enhanced tenogenic regeneration in vivo. To test this hypothesis, we employed an indirect cell co-culture model to investigate the effects of co-culture on cell metabolic activity, ECM production, and gene expression of anabolic and catabolic tenogenic markers. Additionally, we indirectly investigated TGF-bioactivity in the secretome of each cell type and during co-culture via a TGF-reporter bioassay. Lastly, we directly assayed for the effect of hMSC and hHT secretome on tenocyte morphology via immunostaining. MATERIALS Fumonisin B1 AND METHODS TISSUE HARVEST, CELL ISOLATION, AND hMSC CHARACTERIZATION The experimental overview summarizing the experimental design and all cell and secretome analyses conducted is presented in Physique 1. All experiments were conducted in accordance with recommendations and approval from the Medical Ethical Research Committee at the Utrecht Medical Center and MST Twente. Following standard written informed consent, hamstring tendon (hHT) samples were harvested from four adult patients undergoing anterior cruciate ligament reconstruction. The tendons were isolated, rinsed with phosphate buffered saline (PBS), and extra muscle tissue was carefully removed prior to dissection and mincing into smaller pieces. Next, tendon pieces were cultured in growth medium of Dulbeccos altered Eagles medium (PAA Laboratories, Australia) supplemented with 10% fetal bovine serum (FBS) (Lonza, Basel, Switzerland), 100 U/mL penicillin and 100 mg/mL streptomycin, and 0.2 mM ascorbic acid (SigmaC Aldrich, St. Louis, MO, USA) to allow the cells to migrate out from the tissue pieces. Open in a separate window Fig. 1 Experimental design showing co-culture configuration and non co-culture control groups. Experiments were performed in biological triplicate. Bone marrow aspirates were obtained from four additional adult patients following written informed consent. Donor information for each cell type is usually presented in Table I. hMSC were isolated and cultured in hMSC basic medium consisting of alpha minimal essential medium (aMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Lonza), 100 U/mL penicillin and 100 mg/mL streptomycin (Life Technologies),.