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Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues.

ABSTRACT: We investigated whether the developmental stage of mouse cardiac fibroblasts (CFs) influences the formation and function of engineered cardiac tissues made of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs). Engineered cardiac tissue patches were fabricated by encapsulating pure mESC-CMs, mESC-CMs?+?adult CFs, or mESC-CMs?+?fetal CFs in fibrin-based hydrogel. Tissue patches containing fetal CFs exhibited higher velocity of action potential propagation and contractile force amplitude compared to patches containing adult CFs, while pure mESC-CM patches did not form functional syncytium. The functional improvements in mESC-CM?+?fetal CF patches were associated with differences in structural remodeling and increased expression of proteins involved in cardiac function. To determine role of paracrine signaling, we cultured pure mESC-CMs within miniature tissue "micro-patches" supplemented with media conditioned by adult or fetal CFs. Fetal CF-conditioned media distinctly enhanced CM spreading and contractile activity, which was shown by pathway inhibitor experiments and Western blot analysis to be mediated via MEK-ERK signaling. In mESC-CM monolayers, CF-conditioned media did not alter CM spreading or MEK-ERK activation. Collectively, our studies show that 3D co-culture of mESC-CMs with embryonic CFs is superior to co-culture with adult CFs for in vitro generation of functional myocardium. Ensuring consistent developmental stages of cardiomyocytes and supporting non-myocytes may be a critical factor for promoting functional maturation of engineered cardiac tissues.

SUBMITTER: Liau B

PROVIDER: S-EPMC5299411 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues.

Liau Brian B Jackman Christopher P CP Li Yanzhen Y Bursac Nenad N

Scientific reports 20170209

We investigated whether the developmental stage of mouse cardiac fibroblasts (CFs) influences the formation and function of engineered cardiac tissues made of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs). Engineered cardiac tissue patches were fabricated by encapsulating pure mESC-CMs, mESC-CMs + adult CFs, or mESC-CMs + fetal CFs in fibrin-based hydrogel. Tissue patches containing fetal CFs exhibited higher velocity of action potential propagation and contractile force amplitude ...[more]

PMID: 28181589

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Project description:BACKGROUND:Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS:Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0?Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS:The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75?Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (-?0.687?Hz-?1; p?=?0.013 vs. zero) and hCSC-supplemented (-?0.233?Hz-?1;p?=?0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p?=?0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS:For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Project description:Complex heterocellular interactions between cardiomyocytes and fibroblasts in the heart involve their bidirectional signaling via cell-cell contacts, paracrine factors, and extracellular matrix (ECM). These interactions vary with heart development and pathology leading to changes in cardiac structure and function. Whether cardiac fibroblasts of different ages interact differentially with cardiomyocytes to distinctly impact their function remains unknown. Here, we explored the direct structural and functional effects of fetal and adult cardiac fibroblasts on cardiomyocytes using a tissue-engineered 3D co-culture system. We show that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of co-cultured tissues. In particular, in vitro expanded adult, but not fetal, cardiac fibroblasts significantly deteriorated electrical and mechanical function of the co-cultured cardiomyocytes, as evidenced by slower action potential conduction, prolonged action potential duration, weaker contractions, higher tissue stiffness, and reduced calcium transient amplitude. This functional deficit was associated with structural and molecular signatures of pathological remodeling including fibroblast proliferation, interstitial collagen deposition, and upregulation of pro-fibrotic markers. Our studies imply critical roles of the age of supporting cells in engineering functional cardiac tissues and provide a new physiologically relevant in vitro platform to investigate influence of heterocellular interactions on cardiomyocyte function, development, and disease.Previous studies have shown that cardiomyocytes and fibroblasts in the heart interact through direct contacts, paracrine factors, and matrix-mediated crosstalk. However, whether cardiac fibroblasts of different ages distinctly impact cardiomyocyte function remains elusive. We employed a tissue-engineered hydrogel-based co-culture system to study interactions of cardiomyocytes with fetal or adult cardiac fibroblasts. We show that the age of cardiac fibroblasts is a strong determinant of the structure, function, and molecular properties of engineered cardiac tissues and that key features of fibrotic myocardium are replicated by supplementing cardiomyocytes with expanded adult but not fetal fibroblasts. These findings relate to implantation of stem cell-derived cardiomyocytes in adult myocardium and warrant further studies of how age and source of non-myocytes impact cardiac function and maturation.

Dataset Information

Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues.

Publications

Developmental stage-dependent effects of cardiac fibroblasts on function of stem cell-derived engineered cardiac tissues.

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