María Eugenia Fernández-Santos

Cardiovascular diseases (CVDs) remain a leading source of morbidity and mortality worldwide. Although heart transplantation is an established treatment for selected patients with end-stage heart failure, the difficulty in obtaining enough donors and the problems associated with compatibility force the search for new strategies to mitigate these effects. Cardiac patches, a combination of therapeutic elements, such as cells and drugs deposited on a biomimetic scaffold, are particularly promising for repairing the long-term cardiac damage from a perspective based on tissue engineering. However, building these patches entails the compliance with extensive and exhaustive conditions and regulations. Key issues identified in the building process of cardiac patches are the cell–scaffold interaction, including the intensity and duration of this interaction. The present study is intended to develop a robust procedure that allows quantifying the adhesion between decellularized matrices and cells through single-cell force spectroscopy (SCFS) measurements. Determining this adhesion force is essential to ensure cell retention and therapeutic action in the damaged area and, therefore, to be able to design an advanced therapy medicinal product. The whole procedure is validated by measuring the adhesion of allogeneic adipose-derived mesenchymal stromal cells to a decellularized porcine myocardium patch.

Background: To report the 1‐year clinical outcomes of decellularised human corneal stromal lamina implantation, with or without autologous adipose‐derived adult stem cells (ADASCs), for advanced keratoconus in a European cohort. Methods: In this prospective, randomised Phase I/IIa trial, 10 eyes of 10 patients were allocated to decellularised stromal lamina implantation alone (G‐1; n = 5) or lamina recellularised with 1.0 × 10 ⁶ autologous ADASCs plus 2.0 × 10 ⁶ additional ADASCs injected into the stromal pocket (G‐2; n = 5). All procedures used femtosecond laser‐assisted 9.5‐mm intrastromal dissection. Results: No major intraoperative complications occurred except one G‐2 micro‐tear that resolved. No clinically relevant inflammation, rejection, or graft failure was observed; transparency was restored in all eyes by 3–6 months. At 12 months, central corneal thickness increased by 118.4 μm in G‐1 and 121.2 μm in G‐2. Decimal UDVA improved by 2.14 ± 0.71 lines in G‐1 and 1.30 ± 0.84 lines in G‐2; decimal CDVA improved by 2.48 ± 0.66 and 1.43 ± 0.53 lines, respectively. Manifest sphere improved to −1.10 D in G‐1 and −0.58 D in G‐2, whereas manifest cylinder changed minimally in both groups. Keratometric indices remained largely unchanged. In vivo confocal microscopy demonstrated progressive cellular repopulation of the implanted lamina during follow‐up in both groups. Conclusions: Decellularised stromal lamina implantation, with or without autologous ADASCs, was safe and feasible, maintaining corneal transparency and producing sustained stromal thickening with clinically meaningful visual and refractive improvement at 12 months. Larger adequately powered studies with longer follow‐up are required to assess clinical efficacy.

Engineered heart tissues (EHTs) have shown promise in partially restoring ejection fraction after myocardial infarction (MI); however, their potential to introduce electrophysiological heterogeneities and promote arrhythmias remains underexplored. This study assessed the arrhythmogenic risk following immature EHT engraftment in infarcted ventricles using computational simulations that replicate preclinical protocols. EHT computational models were developed and integrated into nine validated porcine-specific biventricular models from pigs with left circumflex (LCx, n = 4) or left anterior descending (LAD, n = 5) MIs. Ventricular tachycardia (VT) susceptibility was evaluated using an S1–S2 stimulation protocol across varying pacing sites and coupling intervals, accounting for infarct characteristics, implantation site, conductivity, and the ventricular conduction system (CS). VT burden was quantified with a 0–1 inducibility score (IS). In silico reentrant activity qualitatively reproduced the arrhythmic patterns observed experimentally in porcine MI models. VT vulnerability was greater in LAD than in LCx infarcts, consistent with a larger infarct size. Inclusion of the CS modified VT burden by providing conduction shortcuts that either facilitated or suppressed reentry. Remuscularization directly on the MI region (IS = 0.49) heightened VT inducibility in dense, transmural scars (IS = 0.16), whereas lateral EHT implantation (IS = 0.35) reduced this risk with respect to direct implantation. In non-transmural scars, VT inducibility varied with the implantation site. Matching EHT conductivity to host myocardium lowered or contained arrhythmogenicity (LCx-IS: from 0.5 to 0.25; LAD-IS: stable at 0.57). These results highlight the latent arrhythmic risk of EHT-mediated remuscularization after MI, identifying infarct substrate, EHT conductivity, and implantation site as critical determinants, and emphasize the importance of incorporating the CS for accurate risk assessment.

The efficient design of biohybrid materials requires controlling the interaction between the cell and the material for a wide range of possible combinations. Single cell force spectroscopy (SCFS), an atomic force microscopy (AFM) experimental procedure based on the binding of an individual cell to an AFM cantilever and the assessment of the adhesion force between the cell and a target substrate, represents one of the most promising alternatives to characterize the interaction between cell and material. However, SCFS relies on the efficient binding of the cell to the AFM in order to avoid drawbacks, such as the detachment of the cell. In this work, three different versatile and robust procedures are presented that allow for the binding of either non-adherent (CD4⁺ T-lymphocytes) or adherent (mesenchymal stem cells, MSC) cells to the AFM probe. The three crosslinking strategies comprise (1) the streptavidin/biotin system, (2) sulfhydryl group-based crosslinkers, and (3) “click” (bioorthogonal) chemistry. Additionally, three decoration schemes of the functionalized AFM probes are explored: a specific antibody, concanavalin A, and direct binding of the cell through azide-derivatized membrane proteins. Differences are observed between these alternatives and it is found that the strength of the interaction (in decreasing order) is as follows: specific antibody, concanavalin A, and binding through azide-derivatized proteins.

Engineered heart tissues (EHTs) have shown promise in partially restoring ejection fraction after myocardial infarction (MI); however, their potential to introduce electrophysiological heterogeneities and promote arrhythmias remains underexplored. This study assessed the arrhythmogenic risk following immature EHT engraftment in infarcted ventricles using computational simulations that replicate preclinical protocols. EHT computational models were developed and integrated into nine validated porcine-specific biventricular models from pigs with left circumflex (LCx, n=4) or left anterior descending (LAD, n=5) MIs. Ventricular tachycardia (VT) susceptibility was evaluated using an S1-S2 stimulation protocol across varying pacing sites and coupling intervals, accounting for infarct characteristics, implantation site, conductivity, and the ventricular conduction system (CS). VT burden was quantified with a 0-1 inducibility score (IS). In silico reentrant activity reproduced the arrhythmic patterns observed experimentally in porcine MI models. VT vulnerability was greater in LAD than in LCx infarcts, consistent with a larger infarct size. Inclusion of the CS modified VT burden by providing conduction shortcuts that either facilitated or suppressed reentry. Remuscularization directly on the MI region (IS = 0.49) heightened VT inducibility in dense, transmural scars (IS = 0.16), whereas lateral EHT implantation (IS = 0.35) reduced this risk with respect to direct implantation. In non-transmural scars, VT inducibility varied with the implantation site. Matching EHT conductivity to host myocardium lowered or contained arrhythmogenicity (LCx-IS: from 0.5 to 0.25; LAD-IS: stable at 0.57). These results highlight the latent arrhythmic risk of EHT-mediated remuscularization after MI, identifying infarct substrate, EHT conductivity, and implantation site as critical determinants, and emphasize the importance of incorporating the CS for accurate risk assessment. Author summary: Remuscularization with engineered heart tissue patches has shown preclinical potential to restore cardiac ejection fraction after myocardial infarction. However, the introduction of electrophysiological heterogeneities from these patches remains underexplored. To address this, we performed in silico investigations of post-infarction arrhythmogenesis and its progression after patch engraftment in porcine models with vessel-specific infarcts, applying a comprehensive arrhythmia inducibility protocol. We first computationally reproduced the experimentally observed arrhythmic patterns in pigs. Before patch engraftment, we found that larger infarcts increased ventricular tachycardia vulnerability. The inclusion of the ventricular conduction system modified arrhythmic outcomes, as electrical shortcuts could either facilitate or suppress reentry. Following remuscularization, highly transmural scars exhibited higher arrhythmicity, with lateral patch implantation mitigating it compared to direct placement. In contrast, for poorly transmural scars, the relationship between arrhythmia inducibility and patch location was unclear, highlighting digital twins as a tool for personalized risk prediction. When host-like patch conductivity was used, arrhythmia inducibility after engraftment was reduced or contained. Overall, our results: i) reveal the latent arrhythmogenicity associated with patch-mediated remuscularization of infarcted hearts; ii) identify infarction substrate, patch conductivity, and implantation site as key arrhythmogenic determinants; and iii) emphasize the importance of accurately modeling the cardiac conduction system.

A Phase I/II clinical trial was conducted in pediatric patients with high-risk malignancies to assess the safety and efficacy of NK cell infusion post-haplo-HSCT. Patients received either alloreactive NK cells from KIR/HLA-mismatched donors or IL-15-stimulated NK cells from KIR/HLA-matched donors, across three dose-escalating cohorts. The IL15-NK cell infusion product demonstrated enhanced activating receptor expression and functional capacity. Increased NKG2D expression was observed in NK cells from patients receiving IL15-NK cells. One-year follow-up showed accelerated immune reconstitution in both the allo-NK and IL15-NK cell arms. IL15-NK recipients exhibited a pro-inflammatory cytokine profile (IL-2, IL-4, IL-6, IFN-γ). Elevated CD69 expression was associated with a higher risk of transplant-related complications.

Purpose: Cryopreserved homografts for valve replacement surgeries face a major problem regarding their durability after implantation and decellularized pulmonary heart valves have raised as potential new generation substitute for these surgeries. The present study aims to document the work performed for the safe implementation in public tissue banks of a new decellularization method for human pulmonary heart valves, based on previous risk evaluation. Methods: After assessing new preparation method associated risks, using EuroGTP-II methodologies, an extensive array of in vitro studies were defined to validate the new technique, mitigate the risks and provide quality and safety data. Results: Initial evaluation of risks using EuroGTP II tool, showed Final Risk Score of 23 (high risk), and four studies were devised to mitigate identified risks: (i) tissue structure integrity; (ii) cell content; (iii) microbiological safety; and (iv) cytotoxicity evaluation in final tissue preparation. Protein quantification, mechanical properties, and histological evaluation indicated no tissue damage, reducing implant failure probability, while cellular content removal demonstrated a 99% DNA removal and microbiological control ensured contamination absence. Moreover, in vitro results showed no cytotoxicity. Risk re-evaluation indicated a risk reduction to moderate risk (Final Risk Score = 10), suggesting that further evidence for safe clinical use would be needed at pre-clinical in vivo evaluation to mitigate remaining risks. Conclusions: The studies performed and reviewed bibliography were able to significantly reduce the original level of risk associated with the clinical application of this homograft’s preparation. However, additional in vivo studies and tissue stability tests are still necessary to address the remaining risks associated with reagents’ effect on extracellular matrix and storage conditions, which could influence implant failure, before the clinical evaluation procedures can be implemented to determine the efficacy and safety of the new decellularized heart valves.

Background: Despite tremendous therapeutic advancements, a significant proportion of coronary artery disease patients suffer from refractory angina pectoris, that is, quality-of-life-compromising angina that is non-manageable with established pharmacological and interventional treatment options. Adenoviral vascular endothelial growth factor-D^ΔNΔC (AdVEGF-D)-encoding gene therapy (GT) holds promise for the treatment of refractory angina. Methods: ReGenHeart is an investigator-initiated, multicentre, randomised, placebo-controlled and double-blinded phase 2 clinical trial that aims to study the safety and efficacy of intramyocardially administered angiogenic AdVEGF-D GT for refractory angina. Patients will be randomised in a 2:1 ratio and blocks of six to receive either AdVEGF-D or placebo. Primary endpoints are improvements in functional capacity assessed with the 6 min walking test and angina symptoms with Canadian Cardiovascular Society class after 6 month follow-up. Secondary endpoints are improvements in myocardial perfusion assessed with either positron emission tomography or single-photon emission CT after 6 month follow-up and functional capacity and angina symptoms after 12 months. In addition, changes in the quality of life, the use of angina medication and the incidence of major adverse cardiac and cerebrovascular events will be evaluated. Conclusions: The phase 2 ReGenHeart trial will provide knowledge of the safety and efficacy of AdVEGF-D GT to ameliorate symptoms in refractory angina patients, extending and further testing positive results from the preceding phase 1/2a trial.

Due to their suppressive capacity, regulatory T cells (Tregs) have attracted growing interest as an adoptive cellular therapy for the prevention of allograft rejection, but limited Treg recovery and lower quality of adult-derived Tregs could represent an obstacle to success. To address this challenge, we developed a new approach that provides large quantities of Tregs with high purity and excellent features, sourced from thymic tissue routinely removed during pediatric cardiac surgeries (thyTregs). We report on a 2-year follow-up of the first patient treated worldwide with thyTregs, included in a phase I/II clinical trial evaluating the administration of autologous thyTreg in infants undergoing heart transplantation. In addition to observing no adverse effects that could be attributed to thyTreg administration, we report that the Treg frequency in the periphery was preserved during the 2-year follow-up period. These initial results are consistent with the trial objective, which is to confirm safety of the autologous thyTreg administration and its capacity to restore the Treg pool.