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

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

Three-dimensional tracking of cardiac catheters using an inverse geometry x-ray fluoroscopy system

Purpose: Scanning beam digital x-ray (SBDX) is an inverse geometry fluoroscopic system with high dose efficiency and the ability to perform continuous real-time tomosynthesis at multiple planes. This study describes a tomosynthesis-based method for 3D tracking of high-contrast objects and present the first experimental investigation of cardiac catheter tracking using a prototype SBDX system