Research & Publications
Cardiovascular diseases are the number one cause of death worldwide, taking about 18 million lives each year. Our stem cell and regenerative medicine laboratory focuses on establishing novel cellular, tissue engineered, and animal models of human cardiovascular disease for the purpose of elucidating causative mechanisms and identifying therapeutic interventions to treat these diseases. Through a close collaboration with several clinicians at Yale, we are able to procure cells from healthy subjects and from those with clinically manifesting cardiovascular diseases. These cells include dermal fibroblasts derived from skin punch biopsies and peripheral mononuclear blood cells, which are reprogrammed into induced pluripotent stem cells (iPSCs) by introducing stem cell factors. iPSCs are self-renewable indefinitely and can differentiate into functional cardiovascular cells. Our unique position, made possible by Yale’s clinical resources, gives us the ability to derive unlimited numbers of patient specific cardiovascular cells for use in our investigations into cardiovascular disease mechanisms and the discovery of potential therapeutic treatments by performing high-throughput drug screening. This research paradigm also places our group in a great position to generate autologous, allogeneic or hypoimmunogenic “universal” stem cell based cardiovascular tissues for organ repair.
Specialized Terms: Cardiovascular; Heart; Stem cell; Regenerative Medicine; Tissue Engineering; Animal Models; ESC; iPSC; Physiology; Pathology; Patient; Small Molecule; Disease
Extensive Research Description
Our stem cell and regenerative medicine laboratory focuses on establishing novel cellular, tissue engineered, and animal models of human cardiovascular diseases for the purpose of elucidating causative mechanisms and identifying therapeutic interventions to treat these diseases. Our research covers the following areas:
1. Vascular tissue engineering and repair: Our group has used Sendai virus, integration-free technology to produce induced pluripotent stem cells (iPSCs) from somatic human donor cells via introduction of stem cell factors. iPSCs are self-renewable and can differentiate into functional vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), providing an unlimited source of vascular cells for generation of tissue-engineered vascular grafts (TEVGs) for treating narrowing/blockage of arteries-the largest cause of mortality in the developed world.
We have generated mechanically robust small-diameter (2-4 mm) TEVGs by seeding human iPSC-VSMCs onto biodegradable polyglycolic acid (PGA) scaffolds in custom made bioreactors. Coupled with decellularization and subsequent re-endothelialization strategies using human iPSC-derived ECs (hiPSC-ECs) in both rat and pig models, our studies lay the foundation for the future production of therapeutic, “off the shelf” ready TEVGs for clinical use. Additionally, we are developing culture strategies to create fully cellular “universal” endothelialized hiPSC-TEVGs that are immunocompatible and readily available to any patient recipient. Recent studies were reported in Gui et al., Biomaterials 2016, 102:120-129 and Luo et al., Cell Stem Cell 2020, 26:251-261 in collaboration with Dr. Laura Niklason. New scientists are welcome to join and learn stem cell biology, tissue engineering, and animal modeling.
We have developed “universal” human iPSCs by using CRISPR-Cas9 technology to knock-out the adaptive immune mediating MHC class I and II molecules, paired with the ectopic expression of the macrophage and natural killer (NK) cell suppressor molecule CD47 with TALEN-mediated insertion at the AAVSI "safe harbor" gene locus. We utilize RRGS rats, which are deficient in T, B, and NK cells and allow effective immune humanization with human peripheral blood mononuclear cells (PBMCs), to assess the immunogenicity of engineered tissues using this “universal donor” cell line. Immune-humanized rats enable the investigation of the efficacy of decellularized hiPSC-TEVGs endothelialized with universal hiPSC-ECs, establishing the foundation for future assessments of this TEVG system in non-human primates as a therapeutic. New scientists can join this project and learn CRISPR and TALEN gene editing, tissue engineering, and immunology.
2. Vascular disease mechanisms and drug screening: Patient-specific vascular smooth muscle cells (VSMCs) facilitate the study of clinically manifesting vascular disease and the development of novel therapeutic interventions. We were the first to describe the development of a human induced pluripotent stem cell (iPSC) line from a patient with the vascular condition supravalvular aortic stenosis (SVAS). SVAS iPSC-VSMCs recapitulate key pathological features of patients with SVAS and provide a promising strategy to study disease mechanisms and to develop novel therapies (Ge et al., Circulation 2012, 126: 1695-1704; Dash et al., Stem Cell Reports 2016, 7: 19-28). The objectives of our research program are to obtain mechanistic insights into how elastin inhibits the hyperproliferation of SVAS iPSC-VSMCs in addition to screening for clinically applicable small molecules that ameliorate the hyperproliferative defect in SVAS. By studying SVAS, a disease with an intrinsic defect in VSMC proliferation, we may ultimately be able develop novel therapies for multiple vascular proliferative diseases. New scientists are welcome to join this project and learn vascular disease mechanisms and drug screening using patient stem cell modeling.
3. Mechanistic cardiac disease modeling: Human iPSCs provide a unique resource for generating patient-specific cardiomyocytes to study cardiac disease mechanisms for new treatments. We were the first group to report small molecule Wnt inhibitor IWP1 or IWP4-based, highly efficient production of functional cardiomyocytes from embryonic stem cells (ESCs) or iPSCs (Ren et al., J Mol Cell Cardiol 2011, 51: 280-7). We have used iPSC and tissue engineering approaches to study the functional consequences of sarcomeric and stretch-sensing mutations in hypertrophic cardiomyopathy (HCM; thickening of the heart tissue, affecting 1 out of 500 people). We have derived iPSCs from patients with MYH7 (sarcomeric) and MLP (stretch-sensing) mutations and are investigating mechanotransduction mediated disease mechanisms by producing engineered heart tissues (EHTs) that can be precisely stretched in collaboration with Dr. Stuart Campbell (manuscript under review). New scientists can join this project, gain experience in studying cardiac disease mechanisms, and learn how to design potential drug screening approaches to treat this devastating heart disease.
4. Congenital heart repair using human iPSC-derived ventricular cardiomyocytes: Single ventricle congenital heart defects (SVCHD) affect approximately 1 in 1000 live births and pose a prominent medical issue. Children born with these defects have a 70% mortality rate if there is no appropriate surgical intervention. The Fontan procedure consists of three surgeries spanning the first 2-3 years of life, which provides ample time to produce a personalized iPSC-based therapeutic. We have validated and optimized a modular design strategy for producing a contractile Fontan conduit that incorporates engineered heart tissues (EHTs) made by seeding iPSC-derived ventricular cardiomyocytes (iPSC-VCMs) into decellularized porcine heart matrices with a native fiber alignment that is crucial for force generation. Novel contractile Fontan conduits, currently named tissue engineered pulsatile conduits (TEPCs), have been developed by wrapping EHTs around decellularized human umbilical arteries (HUAs) to produce functional tissues capable of supporting blood flow and creating driving pressures (Park et al., Acta Biomater. 2020, 102:220-230). Importantly, TEPCs will further be subjected to biomimetic mechanical and electrical stimulation to induce maximal force production in bioreactors. New scientists can join this project and learn iPSC culture, cardiac differentiation, tissue engineering, and animal modeling.
5. Ischemic heart repair based on cardiac progenitor cells: We have established robust cardiac differentiation approaches in human embryonic stem cells (ESCs) and iPSCs to derive ISL1+ cardiovascular progenitor cells (ISL1-CPCs), a CPC population representative of an authentic cardiac origin, for repairing ischemic cardiac injury. We show that ISL1-CPCs have important physiological effects to improve heart contractile function, reduce scar size, and increase blood vessel formation in mouse models (Bartulos et al in Qyang group, JCI Insight 2016, 1:e80920). Thus, our findings indicate that the ISL1-CPC approach may represent a significant advance in the heart repair field. Future efforts will be made to translate ISL1-CPC heart repair from the mouse to large animal models. New scientists joining this project will learn how to isolate ISL1-CPCs from human ESCs and iPSCs, generate cardiac progenitor-based tissues, and gain experience in cardiac repair and regeneration.
6. Preclinical modeling of cardiovascular repair and regeneration: We have generated robust iPSC lines from pigs for the purpose of preclinical studies (Luo et al., Biomaterials 2017, 147:116-132). The availability of pig iPSCs will enable us to engineer the same kind of cardiovascular tissues we want to see used in the clinic, and then test them in pigs, which closely mimic human cardiovascular physiology. Our pig-to-pig studies will provide critical preclinical knowledge for the eventual application of human-to-human autologous and allogeneic stem cell-based cardiovascular repair and regeneration therapies. New scientists joining this project will learn how to derive functional cardiovascular cells from pig iPSCs for cardiovascular tissue engineering and investigate the therapeutic efficacy of engineered cardiovascular tissues in preclinical porcine models.
Animal Diseases; Cardiovascular Diseases; Heart; Pathology; Physiology; Stem Cells; Drugs, Investigational; Tissue Engineering; Regenerative Medicine; Embryonic Stem Cells; Induced Pluripotent Stem Cells
Public Health Interests
Aging; Bioinformatics; Biomarkers; Cardiovascular Diseases; Chronic Diseases; Clinical Trials; Genetics, Genomics, Epigenetics; Immunology; Obesity; Viruses; Women's Health; Toxicology