Comparative computational RNA analysis of cardiac-derived progenitor cells and their extracellular vesicles.

Stem/progenitor cells, including cardiac-derived c-kit+ progenitor cells (CPCs), are under clinical evaluation for treatment of cardiac disease. Therapeutic efficacy of cardiac cell therapy can be attributed to paracrine signaling and the release of extracellular vesicles (EVs) carrying diverse cargo molecules. Despite some successes and demonstrated safety, large variation in cell populations and preclinical/clinical outcomes remains a problem. Here, we investigated this variability by sequencing coding and non-coding RNAs of CPCs and CPC-EVs from 30 congenital heart disease patients and used machine learning methods to determine potential mechanistic insights. CPCs retained RNAs related to extracellular matrix organization and exported RNAs related to various signaling pathways to CPC-EVs. CPC-EVs are enriched in miRNA clusters related to cell proliferation and angiogenesis. With network analyses, we identified differences in non-coding RNAs which give insight into age-dependent functionality of CPCs. By taking a quantitative computational approach, we aimed to uncover sources of CPC cell therapy variability.

Bidirectional relationship between cardiac extracellular matrix and cardiac cells in ischemic heart disease.

Ischemic heart diseases (IHDs), including myocardial infarction and cardiomyopathies, are a leading cause of mortality and morbidity worldwide. Cardiac-derived stem and progenitor cells have shown promise as a therapeutic for IHD but are limited by poor cell survival, limited retention, and rapid washout. One mechanism to address this is to encapsulate the cells in a matrix or three-dimensional construct, so as to provide structural support and better mimic the cells' physiological microenvironment during administration. More specifically, the extracellular matrix (ECM), the native cellular support network, has been a strong candidate for this purpose. Moreover, there is a strong consensus that the ECM and its residing cells, including cardiac stem cells, have a constant interplay in response to tissue development, aging, disease progression, and repair. When externally stimulated, the cells and ECM work together to mutually maintain the local homeostasis by initially altering the ECM composition and stiffness, which in turn alters the cellular response and behavior. Given this constant interplay, understanding the mechanism of bidirectional cell-ECM interaction is essential to develop better cell implantation matrices to enhance cell engraftment and cardiac tissue repair. This review summarizes current understanding in the field, elucidating the signaling mechanisms between cardiac ECM and residing cells in response to IHD onset. Furthermore, this review highlights recent advances in native ECM-mimicking cardiac matrices as a platform for modulating cardiac cell behavior and inducing cardiac repair.

A modified gelatin zymography technique incorporating total protein normalization.

Gelatinase zymography is a commonly used laboratory procedure; however, variability in sample loading and concentration reduce the accuracy of quantitative results obtained from this technique. To facilitate normalization of gelatinase activity by loaded protein amount, we developed a protocol using the trihalocompound 2,2,2-trichloroethanol to allow for gelatin zymography and total protein labeling within the same gel. We showed that detected protein levels increased linearly with loading, and describe a loading concentration range over which normalized gelatinase activity was constant. We conclude that in-gel total protein detection is feasible in gelatin zymography and greatly improves comparison of gelatinase activity between samples.

The Effect of Parent Cell Type on Small Extracellular Vesicle-Derived Vehicle Functionality.

Cell therapies involving c-kit+ progenitor cells (CPCs) and mesenchymal stem cells (MSCs) have been actively studied for cardiac repair. The benefits of such therapies have more recently been attributed to the release of small extracellular vesicles (sEVs) from the parent cells. These sEVs are 30-180 nm vesicles containing protein/nucleic acid cargo encapsulated within an amphiphilic bilayer membrane. Despite their pro-reparative effects, sEV composition and cargo loading is highly variable, making it challenging to develop robust therapies with sEVs. Synthetic alternatives have been developed to allow cargo modulation, including prior work from the laboratory, to design sEV-like vehicles (ELVs). ELVs are synthesized from the sEV membrane but allow controlled cargo loading. It is previously shown that loading pro-angiogenic miR-126 into CPC-derived ELVs significantly increases endothelial cell angiogenesis compared to CPC-sEVs alone. Here, they expand on this work to design MSC-derived ELVs  and study the role of the parent cell type on ELV composition and function. It is found that ELV origin does affect the ELV potency and that ELV membrane composition can affect outcomes. This study showcases the versatility of ELVs to be synthesized from different parent cells and highlights the importance of selecting ELV source cells based on the desired functional outcomes.

Customized Loading of microRNA-126 to Small Extracellular Vesicle-Derived Vehicles Improves Cardiac Function after Myocardial Infarction.

Small extracellular vesicles (sEVs) are promising for cell-based cardiac repair after myocardial infarction. These sEVs encapsulate potent cargo, including microRNAs (miRs), within a bilayer membrane that aids sEV uptake when administered to cells. However, despite their efficacy, sEV therapies are limited by inconsistencies in the sEV release from parent cells and variability in cargo encapsulation. Synthetic sEV mimics with artificial bilayer membranes allow for cargo control but suffer poor stability and rapid clearance when administered in vivo. Here, we developed an sEV-like vehicle (ELV) using an electroporation technique, building upon our previously published work, and investigated the potency of delivering electroporated ELVs with pro-angiogenic miR-126 both in vitro and in vivo to a rat model of ischemia-reperfusion. We show that electroporated miR-126+ ELVs improve tube formation parameters when administered to 2D cultures of cardiac endothelial cells and improve both echocardiographic and histological parameters when delivered to a rat left ventricle after ischemia reperfusion injury. This work emphasizes the value of using electroporated ELVs as vehicles for delivery of select miR cargo for cardiac repair.

Knockdown of deleterious miRNA in progenitor cell-derived small extracellular vesicles enhances tissue repair in myocardial infarction.

Small extracellular vesicles (sEVs) play a critical role in cardiac cell therapy by delivering molecular cargo and mediating cellular signaling. Among sEV cargo molecule types, microRNA (miRNA) is particularly potent and highly heterogeneous. However, not all miRNAs in sEV are beneficial. Two previous studies using computational modeling identified miR-192-5p and miR-432-5p as potentially deleterious in cardiac function and repair. Here, we show that knocking down miR-192-5p and miR-432-5p in cardiac c-kit+ cell (CPC)-derived sEVs enhances the therapeutic capabilities of sEVs in vitro and in a rat in vivo model of cardiac ischemia reperfusion. miR-192-5p- and miR-432-5p-depleted CPC-sEVs enhance cardiac function by reducing fibrosis and necrotic inflammatory responses. miR-192-5p-depleted CPC-sEVs also enhance mesenchymal stromal cell-like cell mobilization. Knocking down deleterious miRNAs from sEV could be a promising therapeutic strategy for treatment of chronic myocardial infarction.

Statistical modeling of extracellular vesicle cargo to predict clinical trial outcomes for hypoplastic left heart syndrome.

Cardiac-derived c-kit+ progenitor cells (CPCs) are under investigation in the CHILD phase I clinical trial (NCT03406884) for the treatment of hypoplastic left heart syndrome (HLHS). The therapeutic efficacy of CPCs can be attributed to the release of extracellular vesicles (EVs). To understand sources of cell therapy variability we took a machine learning approach: combining bulk CPC-derived EV (CPC-EV) RNA sequencing and cardiac-relevant in vitro experiments to build a predictive model. We isolated CPCs from cardiac biopsies of patients with congenital heart disease (n = 29) and the lead-in patients with HLHS in the CHILD trial (n = 5). We sequenced CPC-EVs, and measured EV inflammatory, fibrotic, angiogeneic, and migratory responses. Overall, CPC-EV RNAs involved in pro-reparative outcomes had a significant fit to cardiac development and signaling pathways. Using a model trained on previously collected CPC-EVs, we predicted in vitro outcomes for the CHILD clinical samples. Finally, CPC-EV angiogenic performance correlated to clinical improvements in right ventricle performance.

Single-Cell RNA Sequencing Reveals Distinct Cardiac-Derived Stromal Cell Subpopulations.

Human cardiac-derived c-kit+ stromal cells (CSCs) have demonstrated efficacy in preclinical trials for the treatment of heart failure and myocardial dysfunction. Unfortunately, large variability in patient outcomes and cell populations remains a problem. Previous research has demonstrated that the reparative capacity of CSCs may be linked to the age of the cells: CSCs derived from neonate patients increase cardiac function and reduce fibrosis. However, age-dependent differences between CSC populations have primarily been explored with bulk sequencing methods. In this work, we hypothesized that differences in CSC populations and subsequent cell therapy outcomes may arise from differing cell subtypes within donor CSC samples. We performed single-cell RNA sequencing on four neonatal CSC (nCSC) and five child CSC (cCSC) samples. Subcluster analysis revealed cCSC-enriched clusters upregulated in several fibrosis- and immune response-related genes. Module-based analysis identified upregulation of chemotaxis and ribosomal activity-related genes in nCSCs and upregulation of immune response and fiber synthesis genes in cCSCs. Further, we identified versican and integrin alpha 2 as potential markers for a fibrotic cell subtype. By investigating differences in patient-derived CSC populations at the single-cell level, this research aims to identify and characterize CSC subtypes to better optimize CSC-based therapy and improve patient outcomes.

Engineering Cardiac Small Extracellular Vesicle-Derived Vehicles with Thin-Film Hydration for Customized microRNA Loading.

Cell therapies for myocardial infarction, including cardiac ckit+ progenitor cell (CPC) therapies, have been promising, with clinical trials underway. Recently, paracrine signaling, specifically through small extracellular vesicle (sEV) release, was implicated in cell-based cardiac repair. sEVs carry cardioprotective cargo, including microRNA (miRNA), within a complex membrane and improve cardiac outcomes similar to that of their parent cells. However, miRNA loading efficiency is low, and sEV yield and cargo composition vary with parent cell conditions, minimizing sEV potency. Synthetic mimics allow for cargo-loading control but consist of much simpler membranes, often suffering from high immunogenicity and poor stability. Here, we aim to combine the benefits of sEVs and synthetic mimics to develop sEV-like vesicles (ELVs) with customized cargo loading. We developed a modified thin-film hydration (TFH) mechanism to engineer ELVs from CPC-derived sEVs with pro-angiogenic miR-126 encapsulated. Characterization shows miR-126+ ELVs are similar in size and structure to sEVs. Upon administration to cardiac endothelial cells (CECs), ELV uptake is similar to sEVs too. Further, when functionally validated with a CEC tube formation assay, ELVs significantly improve tube formation parameters compared to sEVs. This study shows TFH-ELVs synthesized from sEVs allow for select miRNA loading and can improve in vitro cardiac outcomes.

Designing a 3D Printing Based Auxetic Cardiac Patch with hiPSC-CMs for Heart Repair.

Myocardial infarction is one of the largest contributors to cardiovascular disease and reduces the ability of the heart to pump blood. One promising therapeutic approach to address the diminished function is the use of cardiac patches composed of biomaterial substrates and cardiac cells. These patches can be enhanced with the application of an auxetic design, which has a negative Poisson's ratio and can be modified to suit the mechanics of the infarct and surrounding cardiac tissue. Here, we examined multiple auxetic models (orthogonal missing rib and re-entrant honeycomb in two orientations) with tunable mechanical properties as a cardiac patch substrate. Further, we demonstrated that 3D printing based auxetic cardiac patches of varying thicknesses (0.2, 0.4, and 0.6 mm) composed of polycaprolactone and gelatin methacrylate can support induced pluripotent stem cell-derived cardiomyocyte function for 14-day culture. Taken together, this work shows the potential of cellularized auxetic cardiac patches as a suitable tissue engineering approach to treating cardiovascular disease.

Biomimetic nanovesicle design for cardiac tissue repair.

Cardiovascular disease is a major cause of mortality and morbidity worldwide. Exosome therapies are promising for cardiac repair. Exosomes transfer cargo between cells, have high uptake by native cells and are ideal natural carriers for proteins and nucleic acids. Despite their proreparative potential, exosome production is dependent on parent cell state with typically low yields and cargo variability. Therefore, there is potential value in engineering exosomes to maximize their benefits by delivering customized, potent cargo for cardiovascular disease. Here, we outline several methods of exosome engineering focusing on three important aspects: optimizing cargo, homing to target tissue and minimizing clearance. Finally, we put these methods in context of the cardiac field and discuss the future potential of vesicle design.

Nanoparticle-Hydrogel System for Post-myocardial Infarction Delivery of MicroRNA.

Effective therapies for cardiac repair and regeneration after myocardial infarction (MI) are rather limited. Although microRNAs (miRs) are known to play an important role in improving cardiac function after MI at a cellular level, delivering and retaining miRs at the target site has been challenging. To address this dilemma, several miR carriers have been developed, but these face their own limitations such as immunogenicity and poor targeting to the infarct site. In this Perspective, we summarize different mechanisms for miR administration and localization to cardiac tissue, with a specific focus on the clinically relevant injectable hydrogel and nanoparticle system developed by Yang et al. and reported in this issue of ACS Nano. We also highlight future directions for this field and outline the remaining unanswered questions.

Predicting Functional Responses of Progenitor Cell Exosome Potential with Computational Modeling.

Congenital heart disease can lead to severe right ventricular heart failure (RVHF). We have shown that aggregated c-kit+ progenitor cells (CPCs) can improve RVHF repair, likely due to exosome-mediated effects. Here, we demonstrate that miRNA content from monolayer (2D) and aggregated (3D) CPC exosomes can be related to in vitro angiogenesis and antifibrosis responses using partial least squares regression (PLSR). PLSR reduced the dimensionality of the data set to the top 40 miRNAs with the highest weighted coefficients for the in vitro biological responses. Target pathway analysis of these top 40 miRNAs demonstrated significant fit to cardiac angiogenesis and fibrosis pathways. Although the model was trained on in vitro data, we demonstrate that the model can predict angiogenesis and fibrosis responses to exosome treatment in vivo with a strong correlation with published in vivo responses. These studies demonstrate that PLSR modeling of exosome miRNA content has the potential to inform preclinical trials and predict new promising CPC therapies. Stem Cells Translational Medicine 2019;8:1212-1221.