No abstract

Decellularized scaffolds are promising clinically translational biomaterials that can be applied to direct cell responses and promote tissue regeneration. Bioscaffolds derived from the extracellular matrix (ECM) of decellularized tissues can naturally mimic the complex extracellular microenvironment through the retention of compositional, biomechanical, and structural properties specific to the native ECM. Increasingly, studies have investigated the use of ECM-derived scaffolds as instructive substrates to recapitulate properties of the stem cell niche and guide cell proliferation, paracrine factor production, and differentiation in a tissuespecific manner. Here, we review the application of decellularized tissue scaffolds as instructive matrices for stem or progenitor cells, with a focus on the mechanisms through which ECMderived scaffolds can mediate cell behavior to promote tissue-specific regeneration. We conclude that although additional preclinical studies are required, ECM-derived scaffolds are a promising platform to guide cell behavior and may have widespread clinical applications in the field of regenerative medicine.

Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.

IntroductionHypoxia-inducible factor 1-alpha (HIF-1α) is the oxygen-sensitive subunit of the transcription factor HIF-1, and its expression is increased in placentas from pregnancies complicated by pre-eclampsia (PE). Fetal growth restriction (FGR) and PE often share a common pathophysiology; however, it is unknown whether increased placental HIF-1α occurs in FGR. We previously demonstrated that aberrant maternal inflammation in rats resulted in altered utero-placental perfusion and FGR, both of which were prevented by administration of the nitric oxide mimetic glyceryl trinitrate (GTN). Our aim here was to determine whether abnormal maternal inflammation causing FGR is linked to placental HIF-1α accumulation and whether GTN administration could prevent increases in placental HIF-1α.MethodsLevels of inflammatory factors in maternal plasma were measured using a multiplex assay after an injection of low-dose lipopolysaccharide (LPS) to rats on gestational day (GD) 13.5. Following three additional daily LPS injections from GD14.5–16.5, GD17.5 placentas were harvested for HIF-1α immunolocalisation; serial sections were also stained for the hypoxia marker pimonidazole. A subset of rats received LPS injections along with GTN delivered continuously (25 μg/h via a transdermal patch) on GD12.5-GD17.5.ResultsWithin two hours of LPS administration, levels of maternal pro-inflammatory cytokines were increased compared with saline-treated controls. GD17.5 placentas of growth-restricted fetuses exhibited increased HIF-1α accumulation; however, this did not correlate with pimonidazole staining for which no differences were observed between groups. Furthermore, the LPS-mediated increases in maternal inflammatory cytokine levels and placental HIF-1α accumulation did not occur in rats treated with GTN.DiscussionOur results demonstrate that inflammation-induced FGR is associated with increased placental HIF-1α accumulation; however, expression of this transcription factor may not correlate with regions of hypoxia in late-gestation placentas. The GTN-mediated attenuation of placental HIF-1α accumulation in LPS-treated rats provides insight into the mechanism by which GTN improves inflammation-induced complications of pregnancy.

No abstract

Adipose-derived mesenchymal stromal cells (MSC(AT)) display immunomodulatory and angiogenic properties, but an improved understanding of quantitative critical quality attributes (CQAs) that inform basal MSC(AT) fitness ranges for immunomodulatory and/or angiogenic applications is urgently needed for effective clinical translation. We constructed an in vitro matrix of multivariate readouts to identify putative CQAs that were sensitive enough to discriminate between specific critical processing parameters (CPPs) chosen for their ability to enhance MSC immunomodulatory and angiogenic potencies, with consideration for donor heterogeneity. We compared 3D aggregate culture conditions (3D normoxic, 3D-N) and 2D hypoxic (2D-H) culture as non-genetic CPP conditions that augment immunomodulatory and angiogenic fitness of MSC(AT). We measured multivariate panels of curated genes, soluble factors, and morphometric features for MSC(AT) cultured under varying CPP and licensing conditions, and we benchmarked these against two functional and therapeutically relevant anchor assays – in vitro monocyte/macrophage (MΦ) polarization and in vitro angiogenesis. Our results showed that varying CPP conditions was the primary driver of MSC(AT) immunomodulatory fitness; 3D-N conditions induced greater MSC(AT)-mediated MΦ polarization toward inflammation-resolving subtypes. In contrast, donor heterogeneity was the primary driver of MSC(AT) angiogenic fitness. Our analysis further revealed panels of putative CQAs with minimum and maximum values that consisted of twenty MSC(AT) characteristics that informed immunomodulatory fitness ranges, and ten MSC(AT) characteristics that informed angiogenic fitness ranges. Interestingly, many of the putative CQAs consisted of angiogenic genes or soluble factors that were inversely correlated with immunomodulatory functions (THBS1, CCN2, EDN1, PDGFA, VEGFA, EDIL3, ANGPT1, and ANG genes), and positively correlated to angiogenic functions (VEGF protein), respectively. We applied desirability analysis to empirically rank the putative CQAs for MSC(AT) under varying CPP conditions and donors to numerically identify the desirable CPP conditions or donors with maximal MSC(AT) immunomodulatory and/or angiogenic fitness. Taken together, our approach enabled combinatorial analysis of the matrix of multivariate readouts to provide putative quantitative CQAs that were sensitive to variations in select CPPs that enhance MSC immunomodulatory/angiogenic potency, and donor heterogeneity. These putative CQAs may be used to prospectively screen potent MSC(AT) donors or cell culture conditions to optimize for desired basal MSC(AT) immunomodulatory or angiogenic fitness.

Osteoarthritis (OA) is a degenerative multifactorial disease with concomitant structural, inflammatory, and metabolic changes that fluctuate in a temporal and patient-specific manner. This complexity has contributed to refractory responses to various treatments. MSCs have shown promise as multimodal therapeutics in mitigating OA symptoms and disease progression. Here, we evaluated 15 randomized controlled clinical trials (RCTs) and 11 nonrandomized RCTs using culture-expanded MSCs in the treatment of knee OA, and we found net positive effects of MSCs on mitigating pain and symptoms (improving function in 12/15 RCTs relative to baseline and in 11/15 RCTs relative to control groups at study endpoints) and on cartilage protection and/or repair (18/21 clinical studies). We examined MSC dose, tissue of origin, and autologous vs. allogeneic origins as well as patient clinical phenotype, endotype, age, sex and level of OA severity as key parameters in parsing MSC clinical effectiveness. The relatively small sample size of 610 patients limited the drawing of definitive conclusions. Nonetheless, we noted trends toward moderate to higher doses of MSCs in select OA patient clinical phenotypes mitigating pain and leading to structural improvements or cartilage preservation. Evidence from preclinical studies is supportive of MSC anti-inflammatory and immunomodulatory effects, but additional investigations on immunomodulatory, chondroprotective and other clinical mechanisms of action are needed. We hypothesize that MSC basal immunomodulatory “fitness” correlates with OA treatment efficacy, but this hypothesis needs to be validated in future studies. We conclude with a roadmap articulating the need to match an OA patient subset defined by molecular endotype and clinical phenotype with basally immunomodulatory “fit” or engineered-to-be-fit-for-OA MSCs in well-designed, data-intensive clinical trials to advance the field.

Purpose: Osteoarthritis (OA) is a debilitating disease associated with chronic inflammation, metabolic dysregulation, and progressive cartilage degradation in the joint. Mesenchymal stromal cells (MSCs) have been investigated as a promising therapy for the disease; however, insufficient in vivo potency and MSC donor variability can limit their efficacy. To address this, the Viswanathan lab has pioneered a proprietary culture engineering strategy to culture MSCs in 3D aggregates to augment their anti-inflammatory and immunomodulatory functions (US62/397,572). The objective of this work is to assess the ability of 3D MSCs to reduce inflammation, fibrosis, and cartilage degradation relative to conventional 2D culture controls within in vitro and in vivo OA models. Methods: MSCs were isolated from human bone marrow (REB# 14-7909) and subject to a proprietary 3D cell aggregation protocol under serum-free conditions (US62/397,572) with conventional 2D culture in serum-free medium or with 10% fetal bovine serum serving as controls. As a standard assessment of MSC potency, licensing experiments were performed by stimulating the MSCs with the pro-inflammatory cytokines tumour necrosis factor-alpha (TNFa; 10 ng/mL for 24 h) or interferon-gamma (IFNg; 30 ng/mL for 24 h) and OA-related gene expression was evaluated by quantitative PCR. Lastly, co-culture experiments were performed using MSCs and human peripheral blood monocytes (REB#14-7483), and flow cytometry was used to assess the effects of MSCs on the expression of monocyte/macrophage phenotypic markers after 48 h of culture. In ongoing work, we are investigating extracellular vesicle release (isolated by ultracentrifugation) and microRNA expression (profiled through microRNA-sequencing) in 2D versus 3D MSCs and will examine the role of these factors in mediating monocyte polarization and synovial fibroblast proliferation/migration in vitro. Results: After licensing with TNFa, 3D MSCs (serum-free 3D culture group) displayed higher transcript levels of the anti-inflammatory marker TNFa-stimulated gene 6 (TSG6) versus serum-free 2D and serum 2D controls by 6.5-fold and 12-fold, respectively, despite somewhat lower basal levels observed in unstimulated serum-free 3D MSCs (N¼2 MSC donors, n¼3 technical replicates/condition; Fig. 1A). In IFNglicensing experiments, 3D MSCs displayed reduced transcript levels of the pro-inflammatory markers chemokine (C-X-C motif) ligand 8 (CXCL8) and cyclooxygenase-2 (COX2), as well as the pro-fibrotic marker transforming growth factor beta (TGFb) (N¼2, n¼3; Fig. 1B). Highest expression of the anabolic joint-lubricating marker proteoglycan 4 (PRG4) was observed in the serum-free 2D condition but serum-free 3D MSCs displayed higher mRNA levels relative to serum 2D controls, while no differences in the expression of the anti-inflammatory genes indoleamine 2,3-dioxygenase 1 (IDO) and TSG6 were observed across groups. As expected, co-culture of MSCs with monocytes promoted a shift toward a more homeostatic "M2-like" monocyte/macrophage phenotype ...