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MSCs products as well as their derived extracellular vesicles, are currently being explored as advanced biologics in cell-based therapies with high expectations for their clinical use in the next few years. In recent years, various strategies designed for improving the therapeutic potential of mesenchymal stromal cells (MSCs), including pre-conditioning for enhanced cytokine production, improved cell homing and strengthening of immunomodulatory properties, have been developed but the manufacture and handling of these cells for their use as advanced therapy medicinal products (ATMPs) remains insufficiently studied, and available data are mainly related to non-industrial processes. In the present article, we will review this topic, analyzing current information on the specific regulations, the selection of living donors as well as MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.), in-process quality controls for ensuring cell efficiency and safety during all stages of the manual and automatic (bioreactors) manufacturing process, including cryopreservation, the use of cell banks, handling medicines, transport systems of ATMPs, among other related aspects, according to European and US legislation. Our aim is to provide a guide for a better, homogeneous manufacturing of therapeutic cellular products with special reference to MSCs.

Stem cell therapy has emerged as a promising new area in regenerative medicine allowing the recovery of viable tissues. Among the many sources of adult stem cells, bone marrow-derived are easy to expand in culture via plastic adherence and their multipotentiality for differentiation make them ideal for clinical applications. Interestingly, several studies have indicated that MSCs expansion in vitro may be limited mainly due to cell aging related to the number of cell divisions in culture. We have determined that MSCs exhibit a progressive decline across successive passages in the expression of stem cell markers, in plasticity and in the inflammatory response, presenting low immunogenicity. We have exposed human MSCs after several passages to TLRs ligands and analyzed their inflammatory response. These cells responded to pro-inflammatory stimuli (i.e., NOS-2 expression) and to anti-inflammatory cytokines (i.e., HO1 and Arg1) until two expansions, rapidly declining upon subculture. Moreover, in the first passages, MSCs were capable to release IL1β, IL6 and IL8, as well as to produce active MMPs allowing them to migrate. Interestingly enough, after two passages, anaerobic glycolysis was enhanced releasing high levels of lactate to the extracellular medium. All these results may have important implications for the safety and efficacy of MSCs-based cell therapies.

Abstract Background Chronic lower limb ischemia develops earlier and more frequently in patients with type 2 diabetes mellitus. Diabetes remains the main cause of lower-extremity non-traumatic amputations. Current medical treatment, based on antiplatelet therapy and statins, has demonstrated deficient improvement of the disease. In recent years, research has shown that it is possible to improve tissue perfusion through therapeutic angiogenesis. Both in animal models and humans, it has been shown that cell therapy can induce therapeutic angiogenesis, making mesenchymal stromal cell-based therapy one of the most promising therapeutic alternatives. The aim of this study is to evaluate the feasibility, safety, and efficacy of cell therapy based on mesenchymal stromal cells derived from adipose tissue intramuscular administration to patients with type 2 diabetes mellitus with critical limb ischemia and without possibility of revascularization. Methods A multicenter, randomized double-blind, placebo-controlled trial has been designed. Ninety eligible patients will be randomly assigned at a ratio 1:1:1 to one of the following: control group (n = 30), low-cell dose treatment group (n = 30), and high-cell dose treatment group (n = 30). Treatment will be administered in a single-dose way and patients will be followed for 12 months. Primary outcome (safety) will be evaluated by measuring the rate of adverse events within the study period. Secondary outcomes (efficacy) will be measured by assessing clinical, analytical, and imaging-test parameters. Tertiary outcome (quality of life) will be evaluated with SF-12 and VascuQol-6 scales. Discussion Chronic lower limb ischemia has limited therapeutic options and constitutes a public health problem in both developed and underdeveloped countries. Given that the current treatment is not established in daily clinical practice, it is essential to provide evidence-based data that allow taking a step forward in its clinical development. Also, the multidisciplinary coordination exercise needed to develop this clinical trial protocol will undoubtfully be useful to conduct academic clinical trials in the field of cell therapy in the near future. Trial registration ClinicalTrials.gov NCT04466007 . Registered on January 07, 2020. All items from the World Health Organization Trial Registration Data Set are included within the body of the protocol.

Due to their suppressive capacity, the adoptive transfer of regulatory T cells (Treg) has acquired a growing interest in controlling exacerbated inflammatory responses. Limited Treg recovery and reduced quality remain the main obstacles in most current protocols where differentiated Treg are obtained from adult peripheral blood. An alternate Treg source is umbilical cord blood, a promising source of Treg cells due to the higher frequency of naïve Treg and lower frequency of memory T cells present in the fetus’ blood. However, the Treg number isolated from cord blood remains limiting. Human thymuses routinely discarded during pediatric cardiac surgeries to access the retrosternal operative field has been recently proposed as a novel source of Treg for cellular therapy. This strategy overcomes the main limitations of current Treg sources, allowing the obtention of very high numbers of undifferentiated Treg. We have developed a novel good manufacturing practice (GMP) protocol to obtain large Treg amounts, with very high purity and suppressive capacity, from the pediatric thymus (named hereafter thyTreg). The total amount of thyTreg obtained at the end of the procedure, after a short-term culture of 7 days, reach an average of 1,757 x106 (range 50 x 106 – 13,649 x 106) cells from a single thymus. The thyTreg product obtained with our protocol shows very high viability (mean 93.25%; range 83.35% – 97.97%), very high purity (mean 92.89%; range 70.10% – 98.41% of CD25+FOXP3+ cells), stability under proinflammatory conditions and a very high suppressive capacity (inhibiting in more than 75% the proliferation of activated CD4+ and CD8+ T cells in vitro at a thyTreg:responder cells ratio of 1:1). Our thyTreg product has been approved by the Spanish Drug Agency (AEMPS) to be administered as cell therapy. We are recruiting patients in the first-in-human phase I/II clinical trial worldwide that evaluates the safety, feasibility, and efficacy of autologous thyTreg administration in children undergoing heart transplantation (NCT04924491). The high quality and amount of thyTreg and the differential features of the final product obtained with our protocol allow preparing hundreds of doses from a single thymus with improved therapeutic properties, which can be cryopreserved and could open the possibility of an “off-the-shelf” allogeneic use in another individual.

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5GFP reporter cardiac fibroblasts. We first established a new NKX2.5GFP reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5GFP hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5GFP cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.

Translational science has been introduced as the nexus among the scientific and the clinical field, which allows researchers to provide and demonstrate that the evidence-based research can connect the gaps present between basic and clinical levels. This type of research has played a major role in the field of cardiovascular diseases, where the main objective has been to identify and transfer potential treatments identified at preclinical stages into clinical practice. This transfer has been enhanced by the intromission of digital health solutions into both basic research and clinical scenarios. This review aimed to identify and summarize the most important translational advances in the last years in the cardiovascular field together with the potential challenges that still remain in basic research, clinical scenarios, and regulatory agencies.

BackgroundMechanical stretch increases Na+ inflow into myocytes, related to mechanisms including stretch-activated channels or Na+/H+ exchanger activation, involving Ca2+ increase that leads to changes in electrophysiological properties favoring arrhythmia induction. Ranolazine is an antianginal drug with confirmed beneficial effects against cardiac arrhythmias associated with the augmentation of INaL current and Ca2+ overload.ObjectiveThis study investigates the effects of mechanical stretch on activation patterns in atrial cell monolayers and its pharmacological response to ranolazine.MethodsConfluent HL-1 cells were cultured in silicone membrane plates and were stretched to 110% of original length. The characteristics of in vitro fibrillation (dominant frequency, regularity index, density of phase singularities, rotor meandering, and rotor curvature) were analyzed using optical mapping in order to study the mechanoelectric response to stretch under control conditions and ranolazine action.ResultsHL-1 cell stretch increased fibrillatory dominant frequency (3.65 ± 0.69 vs. 4.35 ± 0.74 Hz, p < 0.01) and activation complexity (1.97 ± 0.45 vs. 2.66 ± 0.58 PS/cm2, p < 0.01) under control conditions. These effects were related to stretch-induced changes affecting the reentrant patterns, comprising a decrease in rotor meandering (0.72 ± 0.12 vs. 0.62 ± 0.12 cm/s, p < 0.001) and an increase in wavefront curvature (4.90 ± 0.42 vs. 5.68 ± 0.40 rad/cm, p < 0.001). Ranolazine reduced stretch-induced effects, attenuating the activation rate increment (12.8% vs. 19.7%, p < 0.01) and maintaining activation complexity—both parameters being lower during stretch than under control conditions. Moreover, under baseline conditions, ranolazine slowed and regularized the activation patterns (3.04 ± 0.61 vs. 3.65 ± 0.69 Hz, p < 0.01).ConclusionRanolazine attenuates the modifications of activation patterns induced by mechanical stretch in atrial myocyte monolayers.

Background. Paralysis of one vocal fold leads to glottal gap and vocal fold insufficiency that has significant impact upon a patient’s quality of life. Fillers have been tested to perform intracordal injections, but they do not provide perdurable results. Early data suggest that enriching fat grafts with adipose-derived regenerative cells (ADRCs) promote angiogenesis and modulate the immune response, improving graft survival. The aim of this study is to propose ADRC-enriched adipose tissue grafts as effective filler for the paralyzed vocal fold to use it for functional reconstruction of the glottal gap. Method. This is the first phase I-IIA clinical trial (phase I/IIA clinical trial, unicentric, randomized, controlled, and two parallel groups), to evaluate the safety of a new therapy with ADRC-enriched fat grafting (ADRC: group I) for laryngoplasty after unilateral vocal fold paralysis. Control group patients received centrifuged autologous fat (CAF: group II) grafts. Overall mean age is 52.49 ± 16.60 years. Group I (ADRC): 7 patients (3 males and 4 females), 52.28 ± 20.95 year. Group II (CAF): 7 patients (3 males and 4 females), 52.71 ± 12.59 year. Results. VHI-10 test showed that preoperative mean score was 24.21 ± 8.28. Postoperative mean score was 6.71 ± 6.75. Preoperative result in group I was 21.14 ± 3.58 and postoperative result was 3.14 ± 3.53. Preoperative result for group II was 27.29 ± 10.66. Postoperative score in group II was 10.29 ± 7.52. Wilcoxon and the Student t-tests showed that the patient’s self-perception of posttreatment improvement is larger when ADRCs are used. Comparing pre- and posttreatment voice quality analysis, group I showed a p=0.053. Group II showed a p=0.007. There would be no significant differentiation between pre- and posttreatment results. This is true for group II and limited for group I. Conclusions. This prospective trial demonstrates the safety and efficacy of the treatment of glottal gap defects utilizing ADRC-enriched fat grafts. This trial is registered with NCT02904824.