Human Cardiac Progenitor Research Articles

From the Literature

From the Literature

Abstract 721: Human Neonatal Cardiac Progenitor Cells Derived Exosomes Induce Cardiomyocytes Proliferation

Background: Human neonatal cardiac progenitor cells (ckit+/CD45-/Lin-, CPCs) improve cardiac function and attenuate adverse left ventricular remodeling after myocardial infarction (MI) through their exosomes (nEXOs). Success of cell therapy to treat injured myocardium depends mainly on reactivation of endogenous quiescent cardiomyocytes to proliferate and recover the lost myocardium. Here, we investigated the mechanism of reactivating quiescent cardiomyocyte proliferation by exosomes. Hypothesis: RNAseq analysis of nEXOs identified miRs which may be responsible for modulation of Hippo signaling, thereby promoting cardiomyocytes proliferation. nEXOs effectively stimulate endogenous cardiomyocyte (CM) proliferation by targeting the Hippo pathway to restore cardiac function in an injured heart. Methods and Results: nCPCs were conditioned for 48 hours in serum free nutrition mix (Ham’sF12) and nEXOs were purified from supernatant using size exclusion chromatography (SEC; CL2B) coupled with ultracentrifugation. nEXOs were quantified by Nanosight (NS300) and characterized by transmission electron microscopy and flow cytometry for the presence of CD63 and CD9. Our results show recovery of cardiac function (ejection fraction = 63.4% vs 40.5%, n=10, p<0.001)) and generation of myocardial mass in a rodent MI model following nEXOs’ intra-myocardial injection. We further show that nEXOs are preferentially acquired by CMs in the border zone of the infarction (m-cherry-Alix labeled EXOs). Our In vitro experiments show that miR-582-3p and miR-7641 (25nM) are the most effective miRs to induce proliferation of quiescent cardiomyocytes (40.1% and 48.8%, respectively, n=3). Increased miR-7641 expression led to a profound increase in quiescent CMs proliferation, in part through repression of the Hippo signal transduction pathway (increased YAP/pYAP ratio). By immunoblotting we show that LATs1/2 (a protein in the Hippo pathway) is directly targeted by miR-7641. nEXOs enriched with miR 7641 promote CM proliferation by 3 folds as compared to non-enriched nEXOs. Conclusion: Our data for the first time demonstrates the ability of nEXOs derived miRNA based therapeutic approaches to activate cardiomyocyte proliferation.

Abstract 197: Transcription Factor Interactome in Human iPS-derived Cardiac Progenitors is Enriched for Proteins Associated With Congenital Heart Disease

Congenital heart disease (CHD) affects ~1% of live births and remains the leading cause of mortality in infants. While large-scale genetic studies have uncovered genes associated with CHD, distinguishing variants that confer risk from the background noise of inconsequential variants remains a challenge. Causative mutations in transcription factors (TF) essential for cardiovascular development, such as NKX2-5, GATA4 and TBX5, have been identified in familial cases of CHD, however they are rare. To expand our understanding of their molecular function and to test whether their interacting proteins may be enriched for variants associated with CHD. We defined the protein-protein interaction (PPI) network of NKX2-5, GATA4 and TBX5 using unbiased mass spectrometry in human iPSC-derived cardiac progenitors (iPS-CPs). This approach yielded a network of 172 proteins. An interdependent gene-regulatory role has been reported at the DNA-binding level for these 3 TF during cardiac development, and we also found interdependent protein interactomes where loss of one TF affected the interactome of the others. Interactomes for each were enriched in proteins involved in similar biological processes, such as chromatin remodeling and gene regulation, or previously unrelated processes such as splicing and mRNA transport. Integration of the iPS-CP-PPI network with the CHD-associated damaging variants found in the Pediatric Cardiac Genomics Consortium whole-exome sequencing cohort revealed statistically significant enrichment in the GATA4 interactome for de novo missense variants. In contrast, neither the TBX5 or NKX2-5 PPIs were enriched for either de novo missense or rare damaging variants. Finally, we developed a framework to rank PPIs with reported damaging variants for functional validation studies. Overall, this work identified novel protein interactors of TFs essential for cardiac development, offering new insights regarding their regulatory roles and the mechanisms through which they may cause CHD.

Progenitor Cells Derived from Drain Waste Product of Open-Heart Surgery in Children.

Human cardiac progenitor cells isolated from the same host may have advantages over other sources of stem cells. The aim of this study is to establish a new source of human progenitor cells collected from a waste product, pericardiac effusion fluid, after open-heart surgery in children with congenital heart diseases. The fluid was collected every 24 h for 2 days after surgery in 37 children. Mononuclear cells were isolated and expanded in vitro. These pericardial effusion-derived progenitor cells (PEPCs) exhibiting cardiogenic lineage markers, were highly proliferative and enhanced angiogenesis in vitro. Three weeks after stem cell transplantation into the ischemic heart in mice, cardiac ejection fraction was improved significantly without detectable progenitor cells. Gene expression profiles of the repaired hearts revealed activation of several known repair mechanisms including paracrine effects, cell migration, and angiogenesis. These progenitor cells may have the potential for heart regeneration.

Therapeutic Cell Protective Role of Histochrome under Oxidative Stress in Human Cardiac Progenitor Cells.

Cardiac progenitor cells (CPCs) are resident stem cells present in a small portion of ischemic hearts and function in repairing the damaged heart tissue. Intense oxidative stress impairs cell metabolism thereby decreasing cell viability. Protecting CPCs from undergoing cellular apoptosis during oxidative stress is crucial in optimizing CPC-based therapy. Histochrome (sodium salt of echinochrome A—a common sea urchin pigment) is an antioxidant drug that has been clinically used as a pharmacologic agent for ischemia/reperfusion injury in Russia. However, the mechanistic effect of histochrome on CPCs has never been reported. We investigated the protective effect of histochrome pretreatment on human CPCs (hCPCs) against hydrogen peroxide (H2O2)-induced oxidative stress. Annexin V/7-aminoactinomycin D (7-AAD) assay revealed that histochrome-treated CPCs showed significant protective effects against H2O2-induced cell death. The anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and Bcl-xL were significantly upregulated, whereas the pro-apoptotic proteins BCL2-associated X (Bax), H2O2-induced cleaved caspase-3, and the DNA damage marker, phosphorylated histone (γH2A.X) foci, were significantly downregulated upon histochrome treatment of hCPCs in vitro. Further, prolonged incubation with histochrome alleviated the replicative cellular senescence of hCPCs. In conclusion, we report the protective effect of histochrome against oxidative stress and present the use of a potent and bio-safe cell priming agent as a potential therapeutic strategy in patient-derived hCPCs to treat heart disease.

412-P: Oleate Prevents Palmitate-Induced Apoptosis and Autophagy in Human Cardiac Progenitor Cells by Inhibiting p38 MAPK and c-Jun Phosphorylation

Elevated circulating levels of saturated fatty acids (SFA; e.g., palmitate) are associated with increased cardiovascular risk in diabetes. In contrast, monounsaturated fatty acids (e.g., oleate) prevent the toxic effects of SFA in multiple cell types. The viability of cardiac progenitor cells (CPC) is essential for myocardial tissue homeostasis. In this study, the ability of palmitate and oleate to induce apoptosis and autophagy, as well as the effects of oleate on the palmitate-induced damage, were investigated in human CPC isolated from right auricle biopsies. Palmitate, but not oleate, induced apoptosis in a dose- and time-dependent manner in human CPC, as assessed by caspase-3 cleavage and ELISA assay for cytoplasmic oligonucleosomes (p<0.05). Exposure of CPC to 0.25 mM palmitate for 16 h also resulted in increased autophagy, evidenced by autophagosome labeling with monodansyl cadaverine and light chain 3 (LC3)-II immunoblotting (p<0.05). In contrast, oleate did not induce autophagy. Palmitate, but not oleate, also induced phosphorylation of both p38 MAPK and the JNK substrate, c-Jun (p<0.05). Pretreatment with the p38 MAPK inhibitors, SB202190 and SB203580, significantly inhibited palmitate-induced apoptosis and autophagy (p<0.05), whereas the JNK inhibitor SP600125 was able to prevent palmitate-induced apoptosis, but not autophagy (p<0.05). Interestingly, palmitate-induced apoptosis and autophagy were prevented when human CPC were co-treated with 0.1 mM oleate for 16 h (p<0.05). Co-incubation with oleate also abolished the ability of palmitate to induce p38 MAPK and c-Jun phosphorylation. In conclusion, oleate prevents the palmitate-induced increase in apoptosis and autophagy of human CPC by counteracting the activation of p38 MAPK and JNK. Hence, oleate supplementation might limit the lipotoxic damage of human heart by preserving the viability of myocardial progenitors. Disclosure R. Doria: None. R. Schipani: None. C. Caccioppoli: None. M. Incalza: None. A. Leonardini: None. A. Natalicchio: Other Relationship; Self; Novo Nordisk Inc., Sanofi-Aventis. S. Perrini: None. A. Cignarelli: Consultant; Self; Eli Lilly and Company. Speaker's Bureau; Self; Merck Sharp & Dohme Corp., Novo Nordisk A/S. L. Laviola: Advisory Panel; Self; Abbott, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk Inc. Board Member; Self; AstraZeneca, Roche Diabetes Care, Sanofi-Aventis. Speaker's Bureau; Self; Medtronic, Mundipharma, Takeda Pharmaceutical Company Limited. F. Giorgino: Advisory Panel; Self; Aegerion Pharmaceuticals, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, MedImmune, Merck Sharp & Dohme Corp., Novo Nordisk A/S, Roche Diabetes Care, Sanofi. Consultant; Self; Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Pharmaceutical Company Limited. Other Relationship; Self; AstraZeneca, Eli Lilly and Company, Sanofi.

Epigenetic Priming of Human Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Accelerates Cardiomyocyte Maturation.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) exhibit a fetal phenotype that limits in vitro and therapeutic applications. Strategies to promote cardiomyocyte maturation have focused interventions on differentiated hPSC-CMs, but this study tests priming of early cardiac progenitor cells (CPCs) with polyinosinic-polycytidylic acid (pIC) to accelerate cardiomyocyte maturation. CPCs were differentiated from hPSCs using a monolayer differentiation protocol with defined small molecule Wnt temporal modulation, and pIC was added during the formation of early CPCs. pIC priming did not alter the expression of cell surface markers for CPCs (>80% KDR+/PDGFRα+), expression of common cardiac transcription factors, or final purity of differentiated hPSC-CMs (∼90%). However, CPC differentiation in basal medium revealed that pIC priming resulted in hPSC-CMs with enhanced maturity manifested by increased cell size, greater contractility, faster electrical upstrokes, increased oxidative metabolism, and more mature sarcomeric structure and composition. To investigate the mechanisms of CPC priming, RNAseq revealed that cardiac progenitor-stage pIC modulated early Notch signaling and cardiomyogenic transcriptional programs. Chromatin immunoprecipitation of CPCs showed that pIC treatment increased deposition of the H3K9ac activating epigenetic mark at core promoters of cardiac myofilament genes and the Notch ligand, JAG1. Inhibition of Notch signaling blocked the effects of pIC on differentiation and cardiomyocyte maturation. Furthermore, primed CPCs showed more robust formation of hPSC-CMs grafts when transplanted to the NSGW mouse kidney capsule. Overall, epigenetic modulation of CPCs with pIC accelerates cardiomyocyte maturation enabling basic research applications and potential therapeutic uses. Stem Cells 2019;37:910-923.

Coculture with noncardiac cells promoted maturation of human stem cell-derived cardiomyocyte microtissues.

Cardiomyocytes derived from human pluripotent stem cells (hPSC-CM) provided a promising cell source for cell therapy, drug screening, and disease modeling. However, hPSC-CM are immature and phenotypically more similar to fetal rather than adult cardiomyocytes in vitro. We explored the impact of coculture of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSC) and endothelial cells (ECs) with human embryonic stem cells-derived cardiac progenitor cells (hESC-CPC) on the gene expression and electrophysiological properties of hESC-CPC in 3D culture (microtissue spheroid). In this regard, hESC-CPC were cultured either alone (CM microtissue) or in coculture with EC and hESC-MSC (CMEM microtissue) on agar-coated 96-well round-bottomed plates for 1 week. Lumen-like structures were formed in CMEM but not in CM microtissue. Cardiac progenitor markers (TBX5, GATA4) were downregulated and cardiac sarcomeric transcripts (MLC2v and β-MHC) were upregulated in CMEM compared with CM microtissue. Furthermore, beating frequencies, beating cycles, and field potential durations of CMEM resided in the range of adult cardiomyocytes rather than fetal like phenotypes observed in CM microtissue. These findings demonstrated that CPC spheroids in coculture with EC and hESC-MSC may undergo greater maturation toward an adult-like cardiomyocyte.

Coculture of Endothelial Cells with Human Pluripotent Stem Cell-Derived Cardiac Progenitors Reveals a Differentiation Stage-Specific Enhancement of Cardiomyocyte Maturation.

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC-derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC-derived ECs with hPSC-derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC-derived CMs.

Fabrication of graphene‐silver/polyurethane nanofibrous scaffolds for cardiac tissue engineering

The graphene‐based nanocomposites are considered as great candidates for enhancing electrical and mechanical properties of nonconductive scaffolds in cardiac tissue engineering. In this study, reduced graphene oxide‐silver (rGO‐Ag) nanocomposites (1 and 2 wt%) were synthesized and incorporated into polyurethane (PU) nanofibers via electrospinning technique. Next, the human cardiac progenitor cells (hCPCs) were seed on these scaffolds for in vitro studies. The rGO‐Ag nanocomposites were studied by X‐ray diffraction (XRD), Raman spectroscopy, and transmission electron microscope (TEM). After incorporation of rGO‐Ag into PU nanofibers, the related characterizations were carried out including scanning electron microscope (SEM), TEM, water contact angle, and mechanical properties. Furthermore, PU and PU/nanocomposites scaffolds were used for in vitro studies, wherein hCPCs showed good cytocompatibility via 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay and considerable attachment on the scaffold using SEM studies. Real‐time polymerase chain reaction (PCR) and immunostaining studies confirmed the upregulation of cardiac specific genes including GATA‐4, T‐box 18 (TBX 18), cardiac troponin T (cTnT), and alpha‐myosin heavy chain (α‐MHC) in the PU/rGO‐Ag scaffolds in comparison with neat PU ones. Therefore, these nanofibrous rGO‐Ag–reinforced PU scaffolds can be considered as suitable candidates in cardiac tissue engineering.

Human Cardiac Progenitors Grown Under Microgravity Conditions Generate Myocytes With High Conduction Velocities

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSC) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiac myocytes or vasculature. Thus, reprogramming hADMSCs into mature myocytes may fare better in future investigations. To reprogram hAdMSCs into electrically conductive cardiac myocytes (CM), we pioneered a 3‐step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, beta 2‐adrenergic receptor (b2AR) and micro‐gravity signaling induced in 3D cardio‐spheroids. In stage 1, ETS2 and MESP1 activated Nkx2.5, Tbx5, Mef2C, dHAND and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells (CPCs). Next, in stage 2, b2AR activation repositioned CPCs into de novo myocytes, along with the appearance of RYR2, CAV2.1, CAV3.1, Nav1.5, SERCA2 and CX45 gene transcripts, myofibrils and action potentials. In stage 3, electrical conduction fostered by 3D cardio‐spheroids formed in a Synthecon®, Inc. rotating bioreactor that mimics micro‐gravity induced the appearance of hypoxic genes: HIF1a/b, PCG1a/b and NOS2 which coincided with the robust activation of adult contractile genes MYH6, MYH7 and TNNI3, ion channel genes and the appearance of hyperpolarization‐activated and cyclic nucleotide‐gated channels (HCN1‐4). Conduction velocities doubled to ~200mm/s after hypoxia, and doubled yet again after dissociation of the 3D cell clusters to ~400mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5–1m/s. Epinephrine stimulation of stage‐3 myocytes in patches resulted in an increase in amplitude of the electrical wave, indicative of mature CMs. Our efficient protocol converted hAdMSC into highly conductive CM and demonstrated the potential utilization of stage‐3 cells for tissue engineering applications for cardiac repair.Support or Funding InformationSupported by a grant from the Center for Advanced Science in SpaceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pluripotent stem cells derived cardiac progenitors cells graft improve right ventricular function in a porcine model of repaired Tetralogy of Fallot

Adult individuals with a cardiac congenital disease are emerging as a major patient population in hospitals. Right ventricular (RV) failure with limited pharmacological therapeutic approaches is often the cause of premature death and morbidity. Cell therapy turns out to be an alternative approach in order to regenerate the RV and/or to improve mechanical function through other biological mechanisms. We used an immune-suppressed porcine model of repaired Tetralogy of Fallot that features within 5 months a RV pressure and volume overload leading to a defect in its mechanical function. Pluripotent stem cells derived cardiac progenitors were seeded in a collagen/alginate/gelatin hydrogel (stiffness as low as 1 kPa) printed and polymerized on a collagen membrane patch with the size of the pig's RV. The elastic patch was then grafted on the surface of the pig's epicardium without any suture. Myocardial function was assessed by echocardiography using standard (TAPSE, s’wave, FAC) and strain parameters before and after cell therapy. Pigs were euthanized 2 months post-graft, the heart explanted and the RV used for histology and immunostaining to track the fate of human cells. After cell therapy, RV dilatation decreased, overall RV function and RV myocardial contractility were improved on echocardiography. A significant number of human cardiac progenitors were found in the patch and within the myocardium in fibrotic interstitial space. Cells were still differentiating after 2 months post-graft while others were differentiated as assessed by the presence of sarcomeric actinin. Markers of cell proliferation and senescence were further used to better characterize the origin of the RV function improvement. Cell therapy of RV failure could thus be a therapeutic option for adults cardiac congenital patients.

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.

Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model

BackgroundNumerous studies from different labs around the world report human cardiac progenitor cells (hCPCs) as having a role in myocardial repair upon ischemia/reperfusion (I/R) injury, mainly through auto/paracrine signaling. Even though these cell populations are already being investigated in cell transplantation-based clinical trials, the mechanisms underlying their response are still poorly understood.MethodsTo further investigate hCPC regenerative process, we established the first in vitro human heterotypic model of myocardial I/R injury using hCPCs and human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs). The co-culture model was established using transwell inserts and evaluated in both ischemia and reperfusion phases regarding secretion of key cytokines, hiPSC-CM viability, and hCPC proliferation. hCPC proteome in response to I/R was further characterized using advanced liquid chromatography mass spectrometry tools.ResultsThis model recapitulates hallmarks of I/R, namely hiPSC-CM death upon insult, protective effect of hCPCs on hiPSC-CM viability (37.6% higher vs hiPSC-CM mono-culture), and hCPC proliferation (approximately threefold increase vs hCPCs mono-culture), emphasizing the importance of paracrine communication between these two populations. In particular, in co-culture supernatant upon injury, we report higher angiogenic functionality as well as a significant increase in the CXCL6 secretion rate, suggesting an important role of this chemokine in myocardial regeneration. hCPC whole proteome analysis allowed us to propose new pathways in the hCPC-mediated regenerative process, including cell cycle regulation, proliferation through EGF signaling, and reactive oxygen species detoxification.ConclusionThis work contributes with new insights into hCPC biology in response to I/R, and the model established constitutes an important tool to study the molecular mechanisms involved in the myocardial regenerative process.

Toward Second-Generation Cardiomyogenic and Anti-cardiofibrotic 1,4-Dihydropyridine-Class TGFβ Inhibitors.

Innovative therapeutic modalities for pharmacological intervention of transforming growth factor β (TGFβ)-dependent diseases are of great value. b-Annelated 1,4-dihydropyridines (DHPs) might be such a class, as they induce TGFβ receptor type II degradation. However, intrinsic drawbacks are associated with this compound class and were systematically addressed in the presented study. It was possible to install polar functionalities and bioisosteric moieties at distinct sites of the molecules while maintaining TGFβ-inhibitory activities. The introduction of a 2-amino group or 7-N-alkyl modification proved to be successful strategies. Aqueous solubility was improved by up to seven-fold at pH 7.4 and 200-fold at pH 3 relative to the parent ethyl 4-(biphenyl-4-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate. The therapeutic potential of the presented DHPs was further underscored in view of a potential dual mode of action: The differentiation of committed human iPSC-derived cardiac progenitor cells (CPCs) was potently stimulated, and the rescue of cardiac fibrosis phenotypes was observed in engineered heart tissue (EHT) constructs.

Retraction of: Age-Associated Defects in EphA2 Signaling Impair the Migration of Human Cardiac Progenitor Cells.

Retraction of: Age-Associated Defects in EphA2 Signaling Impair the Migration of Human Cardiac Progenitor Cells.

Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants

Human cardiomyocytes (CM) differentiated from pluripotent stem cells (PSCs) are relatively immature when generated in two-dimensional (2D) in vitro cultures, which limits their biomedical applications. Here, we devised a strategy to enhance maturation of human CM in vitro by assembly of three-dimensional (3D) cardiac organoids (CO) containing human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs), endothelial cells (ECs), and mesenchymal stem cells (MSCs). In contrast to corresponding 2D cultures, 3D CO not only developed into structures containing spontaneously beating CM, but also showed enhanced maturity as indicated by increased expressions of sarcomere and ion channel genes and reduced proliferation. Heterotopic implantation of CO into the peritoneal cavity of immunodeficient mice induced neovascularization, and further stimulated upregulation of genes coding for the contractile apparatus, Ca2+ handling and ion channel proteins. In addition, CM in implanted CO were characterized by a more mature ultrastructure compared to CM implanted without CO support. Functional analysis revealed the presence of working cardiomyocytes in both in vivo and ex ovo chorioallantoic membrane implanted CO. Our results demonstrate that cultivation in 3D CO and subsequent heterotopic implantation enhance maturation of CM towards an adult-like phenotype. We reason that CO-derived CM represent an attractive source for drug discovery and other biomedical applications.

Cardiac Progenitor Cell\u2013Derived Extracellular Vesicles Reduce Infarct Size and Associate with Increased Cardiovascular Cell Proliferation

Cell transplantation studies have shown that injection of progenitor cells can improve cardiac function after myocardial infarction (MI). Transplantation of human cardiac progenitor cells (hCPCs) results in an increased ejection fraction, but survival and integration are low. Therefore, paracrine factors including extracellular vesicles (EVs) are likely to contribute to the beneficial effects. We investigated the contribution of EVs by transplanting hCPCs with reduced EV secretion. Interestingly, these hCPCs were unable to reduce infarct size post-MI. Moreover, injection of hCPC-EVs did significantly reduce infarct size. Analysis of EV uptake showed cardiomyocytes and endothelial cells primarily positive and a higher Ki67 expression in these cell types. Yes-associated protein (YAP), a proliferation marker associated with Ki67, was also increased in the entire infarcted area. In summary, our data suggest that EV secretion is the driving force behind the short-term beneficial effect of hCPC transplantation on cardiac recovery after MI.

Mitochondrial Dysfunction and Senescence of Human Cardiac Progenitor Cells Are Prevented by Hypoxic Culture

Rationale: Therapeutic potential of c-Kit+ cardiac progenitor cells (CPCs) is modest in autologous cell therapy clinical trials and could benefit from optimization to maximize functional activity. Early proliferation arrest limits expansion potential of human CPCs in vitro. In contrast, self-renewal is preserved and mitochondrial reactive oxidative species (mtROS) minimized within the CPC hypoxic niche in vivo. Expansion under oxygen tension comparable to the hypoxic niche may therefore improve CPC reparative function.

A Bioprinted Cardiac Patch Composed of Cardiac-Specific Extracellular Matrix and Progenitor Cells for Heart Repair.

Congenital heart defects are present in 8 of 1000 newborns and palliative surgical therapy has increased survival. Despite improved outcomes, many children develop reduced cardiac function and heart failure requiring transplantation. Human cardiac progenitor cell (hCPC) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Decellularized cardiac extracellular matrix hydrogel (cECM) improves heart function in animals, and human trials are ongoing. In the present study, a 3D-bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac patches are printed with bioinks composed of cECM, hCPCs, and gelatin methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homogeneous distribution of cECM and hCPCs. hCPCs maintain >75% viability and incorporation of cECM within patches results in a 30-fold increase in cardiogenic gene expression of hCPCs compared to hCPCs grown in pure GelMA patches. Conditioned media from GelMA-cECM patches show increased angiogenic potential (>2-fold) over GelMA alone, as seen by improved endothelial cell tube formation. Finally, patches are retained on rat hearts and show vascularization over 14 d in vivo. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium.