Cardiac muscle patches containing four types of cardiac cells derived from human pluripotent stem cells improve recovery from cardiac injury in mice.

AIMS: We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells-cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)-significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-derived CMs (hPSC-CMs) are phenotypically immature, which may lead to arrhythmogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical [hCMP containing hPSC-CMs, -AECs, and -SMCs (3TCC-hCMPs)]. METHODS AND RESULTS: hCMPs were generated in a fibrin scaffold. MI was induced in severe combined immunodeficiency (SCID) mice through permanent coronary artery (left anterior descending) ligation, followed by treatment with cardiac muscle patches. Animal groups included: MI heart treated with 3TCC-hCMP; with 4TCC-hCMP; MI heart treated with no patch (MI group) and sham group. Cardiac function was evaluated using echocardiography, and cell engraftment rate and infarct size were evaluated histologically at 4 weeks after patch transplantation. The results from experiments in cultured hCMPs demonstrate that the inclusion of cardiac fibroblast in 4TCC-hCMPs had (i) better organized sarcomeres; (ii) abundant structural, metabolic, and ion-channel markers of CM maturation; and (iii) greater conduction velocities (31 ± 3.23 cm/s, P < 0.005) and action-potential durations (APD50 = 365 ms ± 2.649, P < 0.0001; APD = 408 ms ± 2.757, P < 0.0001) than those (velocity and APD time) in 3TCC-hCMPs. Furthermore, 4TCC-hCMPs transplantation resulted in better cardiac function [ejection fraction (EF) = 49.18% ± 0.86, P < 0.05], reduced infarct size (22.72% ± 0.98, P < 0.05), and better engraftment (15.99% ± 1.56, P < 0.05) when compared with 3TCC-hCMPs (EF = 41.55 ± 0.92%, infarct size = 39.23 ± 4.28%, and engraftment = 8.56 ± 1.79%, respectively). CONCLUSION: Collectively, these observations suggest that the inclusion of hPSC-CFs during hCMP manufacture promotes hPSC-CM maturation and increases the potency of implanted hCMPs for improving cardiac recovery in mice model of MI.

Alterations in heparan sulfate proteoglycan synthesis and sulfation and the impact on vascular endothelial function.

The glycocalyx attached to the apical surface of vascular endothelial cells is a rich network of proteoglycans, glycosaminoglycans, and glycoproteins with instrumental roles in vascular homeostasis. Given their molecular complexity and ability to interact with the intra- and extracellular environment, heparan sulfate proteoglycans uniquely contribute to the glycocalyx's role in regulating endothelial permeability, mechanosignaling, and ligand recognition by cognate cell surface receptors. Much attention has recently been devoted to the enzymatic shedding of heparan sulfate proteoglycans from the endothelial glycocalyx and its impact on vascular function. However, other molecular modifications to heparan sulfate proteoglycans are possible and may have equal or complementary clinical significance. In this narrative review, we focus on putative mechanisms driving non-proteolytic changes in heparan sulfate proteoglycan expression and alterations in the sulfation of heparan sulfate side chains within the endothelial glycocalyx. We then discuss how these specific changes to the endothelial glycocalyx impact endothelial cell function and highlight therapeutic strategies to target or potentially reverse these pathologic changes.

Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress-related AMPK/FoxO1 signaling.

Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress-mediated 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

Myocardial protection by nanomaterials formulated with CHIR99021 and FGF1.

The mortality of patients suffering from acute myocardial infarction is linearly related to the infarct size. As regeneration of cardiomyocytes from cardiac progenitor cells is minimal in the mammalian adult heart, we have explored a new therapeutic approach, which leverages the capacity of nanomaterials to release chemicals over time to promote myocardial protection and infarct size reduction. Initial screening identified 2 chemicals, FGF1 and CHIR99021 (a Wnt1 agonist/GSK-3ß antagonist), which synergistically enhance cardiomyocyte cell cycle in vitro. Poly-lactic-co-glycolic acid nanoparticles (NPs) formulated with CHIR99021 and FGF1 (CHIR + FGF1-NPs) provided an effective slow-release system for up to 4 weeks. Intramyocardial injection of CHIR + FGF1-NPs enabled myocardial protection via reducing infarct size by 20%-30% in mouse or pig models of postinfarction left ventricular (LV) remodeling. This LV structural improvement was accompanied by preservation of cardiac contractile function. Further investigation revealed that CHIR + FGF1-NPs resulted in a reduction of cardiomyocyte apoptosis and increase of angiogenesis. Thus, using a combination of chemicals and an NP-based prolonged-release system that works synergistically, this study demonstrates a potentially novel therapy for LV infarct size reduction in hearts with acute myocardial infarction.

CHIR99021 and fibroblast growth factor 1 enhance the regenerative potency of human cardiac muscle patch after myocardial infarction in mice.

BACKGROUND: We have shown that genetic overexpression of cell cycle proteins can increase the proliferation of transplanted cardiomyocytes derived from human induced-pluripotent stem cells (hiPSC-CMs) in animal models of myocardial infarction (MI). Here, we introduce a new, non-genetic approach to promote hiPSC-CM cell cycle activity and proliferation in transplanted human cardiomyocyte patches (hCMPs). METHODS: Mice were randomly distributed into 5 experimental groups (n = 10 per group). One group underwent Sham surgery, and the other 4 groups underwent MI induction surgery followed by treatment with hCMPs composed of hiPSC-CMs and nanoparticles that contained CHIR99021 and FGF1 (the NPCF-hCMP group), with hCMPs composed of hiPSC-CMs and empty nanoparticles (the NPE-hCMP group); with patches containing the CHIR99021/FGF-loaded nanoparticles but lacking hiPSC-CMs (the NPCF-Patch group), or patches lacking both the nanoparticles and cells (the E-Patch group). Cell cycle activity was evaluated via Ki67 and Aurora B expression, bromodeoxyuridine incorporation, and phosphorylated histone 3 levels (immunofluorescence); engraftment via human cardiac troponin T or human nuclear antigen expression (immunofluorescence) and bioluminescence imaging; cardiac function via echocardiography; infarct size and wall thickness via histology; angiogenesis via isolectin B4 expression (immunofluorescence); and apoptosis via TUNEL and caspace 3 expression (immunofluorescence). RESULTS: Combined CHIR99021- and FGF1-treatment significantly increased hiPSC-CM cell cycle activity both in cultured cells (by 4- to 6-fold) and in transplanted hCMPs, and compared to treatment with NPE-hCMPs, NPCF-hCMP transplantation increased hiPSC-CM engraftment by ~4-fold and was associated with significantly better measurements of cardiac function, infarct size, wall thickness, angiogenesis, and hiPSC-CM apoptosis four weeks after MI induction. CONCLUSIONS: Nanoparticle-mediated CHIR99021 and FGF1 delivery promotes hiPSC-CM cell cycle activity and proliferation, as well as the engraftment and regenerative potency of transplanted hCMPs, in a mouse MI model.

DNA damage-free iPS cells exhibit potential to yield competent cardiomyocytes.

DNA damage accrued in induced pluripotent stem cell (iPSC)-derived cardiomyocytes during in vitro culture practices lessens their therapeutic potential. We determined whether DNA-damage-free iPSCs (DdF-iPSCs) can be selected using stabilization of p53, a transcription factor that promotes apoptosis in DNA-damaged cells, and differentiated them into functionally competent DdF cardiomyocytes (DdF-CMs). p53 was activated using Nutlin-3a in iPSCs to selectively kill the DNA-damaged cells, and the stable DdF cells were cultured further and differentiated into CMs. Both DdF-iPSCs and DdF-CMs were then characterized. We observed a significant decrease in the expression of reactive oxygen species and DNA damage in DdF-iPSCs compared with control (Ctrl) iPSCs. Next-generation RNA sequencing and Ingenuity Pathway Analysis revealed improved molecular, cellular, and physiological functions in DdF-iPSCs. The differentiated DdF-CMs had a compact beating frequency between 40 and 60 beats/min accompanied by increased cell surface area. Additionally, DdF-CMs were able to retain the improved molecular, cellular, and physiological functions after differentiation from iPSCs, and, interestingly, cardiac development network was prominent compared with Ctrl-CMs. Enhanced expression of various ion channel transcripts in DdF-CMs implies DdF-CMs are of ventricular CMs and mature compared with their counterparts. Our results indicated that DdF-iPSCs could be selected through p53 stabilization using a small-molecule inhibitor and differentiated into ventricular DdF-CMs with fine-tuned molecular signatures. These iPSC-derived DdF-CMs show immense clinical potential in repairing injured myocardium.NEW & NOTEWORTHY Culture-stress-induced DNA damage in stem cells lessens their performance. A robust small-molecule-based approach, by stabilizing/activating p53, to select functionally competent DNA-damage-free cells from a heterogeneous population of cells is demonstrated. This protocol can be adopted by clinics to select DNA-damage-free cells before transplanting them to the host myocardium. The intact DNA-damage-free cells exhibited with fine-tuned molecular signatures and improved cellular functions. DNA-damage-free cardiomyocytes compared with control expressed superior cardiomyocyte functional properties, including, but not limited to, enhanced ion channel signatures. These DNA-intact cells would better engraft, survive, and, importantly, improve the cardiac function of the injured myocardium.

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.