Long-term self-renewing human epicardial cells generated from pluripotent stem cells under defined xeno-free conditions.

The epicardium contributes both multi-lineage descendants and paracrine factors to the heart during cardiogenesis and cardiac repair, underscoring its potential for cardiac regenerative medicine. Yet little is known about the cellular and molecular mechanisms that regulate human epicardial development and regeneration. Here, we show that the temporal modulation of canonical Wnt signaling is sufficient for epicardial induction from 6 different human pluripotent stem cell (hPSC) lines, including a WT1-2A-eGFP knock-in reporter line, under chemically-defined, xeno-free conditions. We also show that treatment with transforming growth factor beta (TGF-β)-signalling inhibitors permitted long-term expansion of the hPSC-derived epicardial cells, resulting in a more than 25 population doublings of WT1+ cells in homogenous monolayers. The hPSC-derived epicardial cells were similar to primary epicardial cells both in vitro and in vivo, as determined by morphological and functional assays, including RNA-seq. Our findings have implications for the understanding of self-renewal mechanisms of the epicardium and for epicardial regeneration using cellular or small-molecule therapies

Functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors

Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The limited availability of human CFs from native heart impedes investigations of CF biology and their role in disease. Human pluripotent stem cells (hPSCs) provide a highly renewable and genetically defined cell source, but efficient methods to generate CFs from hPSCs have not been described. Here, we show differentiation of hPSCs using sequential modulation of Wnt and FGF signaling to generate second heart field progenitors that efficiently give rise to hPSC-CFs. The hPSC-CFs resemble native heart CFs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by TGFβ1 and angiotensin II. Furthermore, hPSC-CFs exhibit a more embryonic phenotype when compared to fetal and adult primary human CFs. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration.J.L.C. received funding from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Fundação de Amparo à Pesquisa do Distrito Federal. The work was funded by NIH R01 HL129798 (T.J.K.); NIH U01 HL134764 (T.J.K.); S10RR025644 (T.J.K.); and the UW Institute for Clinical and Translational Research, grant UL1TR000427, from the Clinical and Translational Science Award of the NCATS/NIH.S

Identifying molecular and functional similarities and differences between human primary cardiac valve interstitial cells and ventricular fibroblasts

Introduction: Fibroblasts are mesenchymal cells that predominantly produce and maintain the extracellular matrix (ECM) and are critical mediators of injury response. In the heart, valve interstitial cells (VICs) are a population of fibroblasts responsible for maintaining the structure and function of heart valves. These cells are regionally distinct from myocardial fibroblasts, including left ventricular cardiac fibroblasts (LVCFBs), which are located in the myocardium in close vicinity to cardiomyocytes. Here, we hypothesize these subpopulations of fibroblasts are transcriptionally and functionally distinct.Methods: To compare these fibroblast subtypes, we collected patient-matched samples of human primary VICs and LVCFBs and performed bulk RNA sequencing, extracellular matrix profiling, and functional contraction and calcification assays.Results: Here, we identified combined expression of SUSD2 on a protein-level, and MEOX2, EBF2 and RHOU at a transcript-level to be differentially expressed in VICs compared to LVCFBs and demonstrated that expression of these genes can be used to distinguish between the two subpopulations. We found both VICs and LVCFBs expressed similar activation and contraction potential in vitro, but VICs showed an increase in ALP activity when activated and higher expression in matricellular proteins, including cartilage oligomeric protein and alpha 2-Heremans-Schmid glycoprotein, both of which are reported to be linked to calcification, compared to LVCFBs.Conclusion: These comparative transcriptomic, proteomic, and functional studies shed novel insight into the similarities and differences between valve interstitial cells and left ventricular cardiac fibroblasts and will aid in understanding region-specific cardiac pathologies, distinguishing between primary subpopulations of fibroblasts, and generating region-specific stem-cell derived cardiac fibroblasts

Feasibility of Atrial Delivery and Tracking of Stem Cells in a Porcine Model

Background: Many patients undergoing open heart surgery have sinus node dysfunction and atrial fibrillation, leading to adverse outcomes. Mesenchymal stem cells (MSC) delivered at the time of surgery may have a reparative effect on atrial tissue, thereby improving sinus node function and reducing or preventing atrial fibrillation. Stem cell delivery to the atrium is entirely unstudied. This is a significant gap in medical research, as atrial disease contributes significantly to health care costs. Purpose: The purpose of this pilot study is to establish a technique to deliver MSC to the atria through an open-chest model, to assess the safety of this technique, and to evaluate the acute retention of the delivered cells. Methods: All in vivo animal experimentation was approved by the University of Wisconsin Animal Care and Use Committee and took place in the Cardiovascular Physiology Core Facility at UW-Madison. MSC (3-5×106 in 50 μl per site) were injected intramyocardially during an open-chest procedure in anesthetized pigs. To track the cells in vivo, MSC were labeled with 18FDG then visualized at 1 and 6 hours postinjection by PET/CT. Pigs were monitored for intraoperative arrhythmia, bleeding and hypotension. Results: By gently repositioning the heart, both atria were accessible for the injections. The thickest part of each atrium was isolated and stabilized briefly for the injection using a hemostat. The injected cells were visible by PET/CT 1 and 6 hours postinjection. However, when the MSC were labeled with 10mCi 18FDG, the signal was too high, causing a bloom around the areas of injection. So the dose was lowered to 5mCi 18FDG, which resulted in a clear signal at 1 hour in both atria. At 6 hours, the right atrial injection was still easy to read, but the left injection was difficult to resolve from background signal. All injections resulted in cell leakage from the injection site and uptake of the signal into the lungs. However, pulmonary function as measured by SpO2 and EtCO2 was unchanged. Intraoperative arrhythmias detected during the injections were caused by manipulation of the heart. No additional arrhythmias were detected. No bleeding or hypotension was observed as a result of the injections. Conclusion: This pilot study demonstrated that atrial delivery of MSC is feasible and safe in an open-chest porcine model and that MSC are retained for at least 6 hours postinjection. Subsequent studies will determine the ability of MSC to downregulate inflammation, decrease scarring and prevent sinus node dysfunction

Impact of statins on cellular respiration and de-differentiation of myofibroblasts in human failing hearts

AIMS: Fibroblast to myofibroblast trans-differentiation with altered bioenergetics precedes cardiac fibrosis (CF). Either prevention of differentiation or promotion of de-differentiation could mitigate CF-related pathologies. We determined whether 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors-statins, commonly prescribed to patients at risk of heart failure (HF)-can de-differentiate myofibroblasts, alter cellular bioenergetics, and impact the human ventricular fibroblasts (hVFs) in HF patients. METHODS AND RESULTS: Either in vitro statin treatment of differentiated myofibroblasts (n = 3-6) or hVFs, isolated from human HF patients under statin therapy (HF + statin) vs. without statins (HF) were randomly used (n = 4-12). In vitro, hVFs were differentiated by transforming growth factor-β1 (TGF-β1) for 72 h (TGF-72 h). Differentiation status and cellular oxygen consumption rate (OCR) were determined by α-smooth muscle actin (α-SMA) expression and Seahorse assay, respectively. Data are mean ± SEM except Seahorse (mean ± SD); P \u3c 0.05, considered significant. In vitro, statins concentration-dependently de-differentiated the myofibroblasts. The respective half-maximal effective concentrations were 729 ± 13 nmol/L (atorvastatin), 3.6 ± 1 μmol/L (rosuvastatin), and 185 ± 13 nmol/L (simvastatin). Mevalonic acid (300 μmol/L), the reduced product of HMG-CoA, prevented the statin-induced de-differentiation (α-SMA expression: 31.4 ± 10% vs. 58.6 ± 12%). Geranylgeranyl pyrophosphate (GGPP, 20 μmol/L), a cholesterol synthesis-independent HMG-CoA reductase pathway intermediate, completely prevented the statin-induced de-differentiation (α-SMA/GAPDH ratios: 0.89 ± 0.05 [TGF-72 h + 72 h], 0.63 ± 0.02 [TGF-72 h + simvastatin], and 1.2 ± 0.08 [TGF-72 h + simvastatin + GGPP]). Cellular metabolism involvement was observed when co-incubation of simvastatin (200 nmol/L) with glibenclamide (10 μmol/L), a K CONCLUSIONS: This study demonstrates the de-differentiating effect of statins, the underlying GGPP sensitivity, reduced OCR with potential activation of