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Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells
Abstract
While encapsulation of cells within protective nanoporous gel cocoons increases cell retention and pro-survival integrin signaling, the influence of cocoon size and intra-capsular cell-cell interactions on therapeutic repair are unknown. Here, we employ a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells. Deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells and enhanced stimulation of endogenous repair. The latter being attributable to cell-cell stimulation of cytokine and extracellular vesicle production while also broadening of the miRNA cargo within extracellular vesicles. Thus, by tuning cocoon size and cell occupancy, the paracrine signature and retention of transplanted cells can be enhanced to promote paracrine stimulation of endogenous tissue repair.
... Negative immune responses against the injected cells can trigger their removal from the site of injury, therefore reducing retention and engraftment [6]. Commonly, vasculature and blood flow also inhibit the retention of injected cells, as free cells are very quickly cleared from the injection site from the constant flow of fluids throughout tissues [14,15]. Low engraftment and low retention both result in reduced therapeutic efficacy since the number of injected cells that remain localized and active are linked to positive therapeutic effects [14,15]. ...
... Commonly, vasculature and blood flow also inhibit the retention of injected cells, as free cells are very quickly cleared from the injection site from the constant flow of fluids throughout tissues [14,15]. Low engraftment and low retention both result in reduced therapeutic efficacy since the number of injected cells that remain localized and active are linked to positive therapeutic effects [14,15]. ...
... The hydrogel creates a barrier surrounding the encapsulated cells against undesired immune responses [16]. One previous study by Kanda et al showed that cell retention was enhanced with microencapsulated cells [15], presumably due to the larger volume which is more difficult for vasculature to clear. In vivo studies using encapsulated therapeutic cells have shown significant improvements in therapeutic outcomes over free floating cells. ...
Current cell-based strategies for repairing damaged tissue often show limited efficacy due to low cell retention at the site of injury. Encapsulation of cells within hydrogel microcapsules demonstrably increases cell retention but benefits can be limited due to premature cell escape from the hydrogel microcapsules and subsequent clearance from the targeted tissue. We propose a method of encapsulating cells in agarose microcapsules that have been modified to increase cell retention by providing cell attachment domains within the agarose hydrogel allowing cells to adhere to the microcapsules. We covalently modified agarose with the addition of the cell adhesion peptide, RGD (arginine, glycine, aspartic acid). We then used a microfluidic platform to encapsulate single cells within 50 µm agarose microcapsules. We tracked encapsulated cells for cell viability, egress from microcapsules and attachment to microcapsules at 2 h, 24 h, and 48 h after encapsulation. Many encapsulated cells eventually egress their microcapsule. Those that were encapsulated using RGD-modified agarose adhered to the outer surface of the microcapsule following egress. NIH 3T3 cells showed nearly 45% of egressed cells attached to the outside of RGD modified agarose microcapsules, while minimal cellular adhesion was observed when using unmodified agarose. Similarly, HUVECs had up to 33% of egressed cells attached and EDCs (explant-derived cardiac stem cells) showed up to 20% attachment with the presence of RGD binding domains within the agarose microcapsules.
... EVs were encapsulated using a novel microfluidic device designed for cell and small particle encapsulation, in a similar manner as described for cell encapsulation (Benavente-Babace et al., 2019; Kanda et al., 2020). A known quantity of EV protein (10-20 μg) was mixed with 1% ultra-low gelling temperature agarose (Sigma Aldrich, Oakville, ON, Canada), 1% gelatin (Sigma Aldrich, Oakville, ON, Canada) and kept at 37 • C during encapsulation. ...
... Nanoporous hydrogels have been previously used to encapsulate single cells forming a 'microgel' niche to protect cells for in vivo delivery (Kanda et al., 2018). A microfluidic approach (Benavente-Babace et al., 2019; Kanda et al., 2020) was employed to encapsulate EVs labelled with pkh26 in 1% agarose-1% gelatin producing microgels of uniform size (47 ± 0.4 μm) (Figure 2ac). Using confocal imaging z-stacks, labelled EVs were observed to be distributed throughout the spherical microgel, and appeared to be spatially co-localised with the labelled gelatin ( Figure 2d). ...
... In this study, we demonstrated that encapsulation of MSC-EVs in nano-porous microgels significantly improved lung targeted EV retention and enhanced EV uptake by resident lung cells. Microencapsulation has been previously employed for the delivery of therapeutic stem and progenitor cells (Kanda et al., 2018(Kanda et al., , 2020Karoubi et al., 2009). We sought to apply this technology for the targeted delivery of EVs to the lung. ...
Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell‐free ‘cellular’ therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano‐porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size‐based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose‐gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel‐loaded EVs demonstrated greater uptake by lung cells, in particular CD45⁺ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.
... To generate conditioned media for experimentation and to reflect the hypoxic lung milieu, cells were grown in their respective basal media (BM-MSC/HDCs: Nutristem basal media; UC-MSC: DMEM alone) at 1% oxygen for 48 h. EVs were isolated using ultracentrifugation (10,000×g 30 min and 100,000×g 3 h [31,32]. EV content, size, and surface marker expression were analyzed using nanoparticle tracking analysis (NanoSight LM10) and the Exo-Check Exosome Antibody Array (EXO-RAY200B, Systems Biosciences), respectively. ...
... The salutary effects of transplanted adult cells are largely mediated by paracrine transfer of bioactive molecules, such as cytokines or EVs. As such, we explored the cytokines and EVs produced by all 3 cell types in hopes of identifying the most effective therapy for COVID-19 lung injury [26,31,32]. As shown in Fig. 2A and Additional file 2: Fig. S2, 57 of the 105 proteins assayed in the membrane-based sandwich immunoassay were secreted above baseline by at least one of the 3 cell types. ...
... All cytokines in the conditioned media of all 3 cell types are implicated in proliferation, wound healing, or immunomodulation, highlighting the potential therapeutic utility of these cells in COVID-19 ARDS. EVs were isolated from conditioned media using differential ultracentrifugation [32]. As shown in Fig. 3A, all 3 isolates expressed surface markers commonly found in EVs (ICAM, ALIX, CD81, CD63, EPCAM, ANXAS, TSG101, FLOT-1) without markers indicative of cellular contamination (GM130). ...
Background
Although 90% of infections with the novel coronavirus 2 (COVID-19) are mild, many patients progress to acute respiratory distress syndrome (ARDS) which carries a high risk of mortality. Given that this dysregulated immune response plays a key role in the pathology of COVID-19, several clinical trials are underway to evaluate the effect of immunomodulatory cell therapy on disease progression. However, little is known about the effect of ARDS associated pro-inflammatory mediators on transplanted stem cell function and survival, and any deleterious effects could undermine therapeutic efficacy. As such, we assessed the impact of inflammatory cytokines on the viability, and paracrine profile (extracellular vesicles) of bone marrow-derived mesenchymal stromal cells, heart-derived cells, and umbilical cord-derived mesenchymal stromal cells.
Methods
All cell products were manufactured and characterized to established clinical release standards by an accredited clinical cell manufacturing facility. Cytokines and Extracellular vesicles in the cell conditioned media were profiled using proteomic array and nanoparticle tracking analysis. Using a survey of the clinical literature, 6 cytotoxic cytokines implicated in the progression of COVID-19 ARDS. Flow cytometry was employed to determine receptor expression of these 6 cytokines in three cell products. Based on clinical survey and flow cytometry data, a cytokine cocktail that mimics cytokine storm seen in COVID-19 ARDS patients was designed and the impact on cytokine cocktail on viability and paracrine secretory ability of cell products were assessed using cell viability and nanoparticle tracking analysis.
Results
Flow cytometry revealed the presence of receptors for all cytokines but IL-6, which was subsequently excluded from further experimentation. Despite this widespread expression, exposure of each cell type to individual cytokines at doses tenfold greater than observed clinically or in combination at doses associated with severe ARDS did not alter cell viability or extracellular vesicle character/production in any of the 3 cell products.
Conclusions
The paracrine production and viability of the three leading cell products under clinical evaluation for the treatment of severe COVID-19 ARDS are not altered by inflammatory mediators implicated in disease progression.
... Extracellular vesicles were encapsulated using a novel microfluidic device designed for cell and small particle encapsulation, in a similar manner as described for cell encapsulation 27,28 . In brief, a known quantity of EV protein was mixed with 1% ultra-low gelling temperature agarose (Sigma Aldrich, Oakville, ON, Canada), 1% gelatin (Sigma Aldrich, Oakville, ON, Canada) and kept . ...
... Microencapsulation has been previously employed for the delivery of therapeutic stem and progenitor cells. 22,23,27 We sought to apply this technology for the targeted delivery of EVs to the lung. We demonstrated reduced uptake by macrophages and ECs of EVs from microgels in vitro over 24 hours suggesting a more sustained release system compared to free EVs which were readily taken up. ...
Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free 'cellular' therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72 hours, while free EVs were rapidly cleared by the liver. Furthermore, microgel loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Therefore, microencapsulation of EVs is a novel tool for enhancing targeted delivery of EVs for future therapeutic applications.
... 3B ECM ve hidrojel sistemleri, mikroakışkan sistemlerinde bu amaç için, örneğin damlacık metodu kullanılarak uygulanmaktadır [66]. Bu yöntemde hücreler çeşitli hidrojel yapıları içerisine mikroakışkan platform tarafından kontrollü şekilde yüklenir [67]. Bu metot ile yalnızca hücrelerin değil; tek bir sferoidin bir damlacık içerisine yüklenebildiği sistemler de geliştirmek mümkündür [68]. ...
Microfluidic systems, which can be easily modified and integrated into many studies, have been the focus of attention of researchers in recent years. Thanks to microfluidic chips, controlled and optimized cell culture studies can be performed with less solution and continuous perfusion. In recent years, culturing the stem cells which has attracted the attention of regenerative medicine, alone or together with other cells and differentiating these cells in the desired direction, are frequently studied in chip systems. Adding hydrogels or decellularized organ matrices to these systems that will mimic the intercellular environment conditions gives results closer to in vivo. There are many studies showing that all the behaviors of the stem cells, including the differentiation, change considerably due to the material from which the chips are produced, the surface modifications, the flow rate, the medium content, the mechano-chemical properties of the hydrogels used, and electrical, chemical or mechanical stimulations. It is predicted that microfluidic chip systems will add a new dimension to personalized medicine, drug toxicity experiments, point-of-care rapid diagnostic kits, and many basic science researches in the future, and will be one of the more reliable and inexpensive potential methods, especially by replacing animal experiments. All these reasons make chip systems the focus of research. In this study; The fabrication, advantages, disadvantages, and applications of microfluidic chip systems in tissue engineering are discussed.
... 3B ECM ve hidrojel sistemleri, mikroakışkan sistemlerinde bu amaç için, örneğin damlacık metodu kullanılarak uygulanmaktadır [66]. Bu yöntemde hücreler çeşitli hidrojel yapıları içerisine mikroakışkan platform tarafından kontrollü şekilde yüklenir [67]. Bu metot ile yalnızca hücrelerin değil; tek bir sferoidin bir damlacık içerisine yüklenebildiği sistemler de geliştirmek mümkündür [68]. ...
Kolayca modifiye edilebilir ve pek çok çalışmaya entegre edilebilir özellikleriyle mikroakışkan sistemler son yıllarda araştırmacıların ilgi odağındadır. Mikroakışkan çipler sayesinde daha az solüsyon ve sürekli perfüzyon ile kontrollü ve optimize hücre kültürü çalışmaları yapılabilmektedir. Son yıllarda özellikle rejeneratif tıbbın ilgisini çeken kök hücrelerin tek başına veya diğer hücrelerle birlikte kültürlenmesi ve kullanılan kök hücrelerin istenilen yönde farklılaştırılması çip sistemlerinde sıklıkla çalışılmaktadır. Bu sistemlere hücreler arası ortam koşullarını taklit edecek hidrojellerin veya hücrelerinden arındırılmış organ matrislerinin de ilave edilmesi in vivo'ya daha yakın sonuçlar vermektedir. Çiplerin üretildiği malzeme, yüzey modifikasyonları, akış hızı, besi yeri içeriği, kullanılan hidrojellerin mekano-kimyasal özellikleri, elektriksel, kimyasal ya da mekanik uyarımlar neticesinde kök hücrelerin farklılaşmaları da dahil tüm davranışlarının oldukça değiştiğini gösteren birçok çalışma mevcuttur. Mikroakışkan çip sistemlerinin ilerleyen zamanlarda kişiselleştirilmiş tıp, ilaç toksisite deneyleri, hasta-yanı hızlı tanı kitleri ve birçok temel bilim araştırmasına yeni bir boyut kazandıracağı, özellikle hayvan deneylerinin yerini alarak daha güvenilir ve ucuz potansiyel yöntemlerin başında geleceği öngörülmektedir. Tüm bu sebepler çip sistemlerini araştırma odağı yapmaktadır. Bu çalışmada; mikroakışkan çip sistemlerinin üretimi, avantajları, dezavantajları ve doku mühendisliği alanındaki uygulamaları tartışılmıştır.
... Cell encapsulation in hydrogels enhances their survival in transplanted tissue and makes regeneration easier, and also aids in the production of prohealing cytokines and extracellular vesicles. Microfluidic cocooning has several advantages over vortex cocooning, such as limited consumption of biomaterials, precise cocoon size and cell number, extremely limited shear stresses on cells during cocooning, high individual unit throughput, etc. [182]. Another key benefit of the microfluidic system is the ability to pattern cell and extracellular matrix (ECM) at the cell length scale. ...
Myocardium Infarction (MI) is one of the foremost cardiovascular diseases (CVDs) causing death worldwide, and its case numbers are expected to continuously increase in the coming years. Pharmacological interventions have not been at the forefront in ameliorating MI-related morbidity and mortality. Stem cell-based tissue engineering approaches have been extensively explored for their regenerative potential in the infarcted myocardium. Recent studies on microfluidic devices employing stem cells under laboratory set-up have revealed meticulous events pertaining to the pathophysiology of MI occurring at the infarcted site. This discovery also underpins the appropriate conditions in the niche for differentiating stem cells into mature cardiomyocyte-like cells and leads to engineering of the scaffold via mimicking of native cardiac physiological conditions. However, the mode of stem cell-loaded engineered scaffolds delivered to the site of infarction is still a challenging mission, and yet to be translated to the clinical setting. In this review, we have elucidated the various strategies developed using a hydrogel-based system both as encapsulated stem cells and as biocompatible patches loaded with cells and applied at the site of infarction.
... Previously, we have shown that encapsulation of heart-derived cells within protective hydrogel cocoons prior to intramyocardial injection increases acute and long-term engraftment. [91][92][93] Akin to many biomaterial studies, increased cell retention was associated with salutary effects on myocardial function and scar size. Dogma would traditionally interpret this result as proof that increased cell retention leads to better heart function. ...
Cardiovascular disease is the primary cause of death around the world. For almost two decades, cell therapy has been proposed as a solution for heart disease. In this article, we report on the "state-of-play" of cellular therapies for cardiac repair and regeneration. We outline the progression of new ideas from the preclinical literature to ongoing clinical trials. Recent data supporting the mechanics and mechanisms of myogenic and paracrine therapies are evaluated in the context of long-term cardiac engraftment. This discussion informs on promising new approaches to indicate future avenues for the field.
... Kanda et al also used a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells in the mouse model of ischemic cardiomyopathy. 125 They found that deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells, and enhanced stimulation of endogenous repair. ...
In the past decades, numerous preclinical studies and several clinical trials have evidenced the feasibility of cell transplantation in treating heart diseases. Over the years, different delivery routes of cell therapy have emerged and broadened the width of the field. However, a common hurdle is shared by all current delivery routes: low cell retention. A myriad of studies confirm that cell retention plays a crucial role in the success of cell-mediated cardiac repair. It is important for any delivery route to maintain donor cells in the recipient heart for enough time to not only proliferate by themselves, but also to send paracrine signals to surrounding damaged heart cells and repair them. In this review, we first undertake an in-depth study of primary theories of cell loss, including low efficiency in cell injection, "washout" effects, and cell death, and then organize the literature from the past decade that focuses on cell transplantation to the heart using various cell delivery routes, including intracoronary injection, systemic intravenous injection, retrograde coronary venous injection, and intramyocardial injection. In addition to a recapitulation of these approaches, we also clearly evaluate their strengths and weaknesses. Furthermore, we conduct comparative research on the cell retention rate and functional outcomes of these delivery routes. Finally, we extend our discussion to state-of-the-art bioengineering techniques that enhance cell retention, as well as alternative delivery routes, such as intrapericardial delivery. A combination of these novel strategies and more accurate assessment methods will help to address the hurdle of low cell retention and boost the efficacy of cell transplantation to the heart.
... Regarding the capsule porosity, we used a protocol described by our team, where samples were coated with nanogold prior to SEM imaging. 5,26 Under our experimental conditions, the 0% collagen microcapsules had a pore size of 0.23 ± 0.13 μm ( Figure 1C). Adding 25% collagen led to a decrease in the porosity to 0.16 ± 0.05 μm, while doubling the collagen to 50% led to an increase in the pore diameter to 0.39 ± 0.11 μm (see Figure S3 for representative images). ...
As cell therapies emerged, it was quickly realized that pro-regenerative cells directly injected into injured tissue struggled within the inflammatory microenvironment. By using microencapsulation, i.e. encapsulating cells within polymeric biomaterials, they are henceforth protected from the harmful extracellular cues, while still being able to receive oxygen, nutrients, and release secreted factors. Previous work showed that stem cells encapsulated within a biologically inert material (agarose) were able to significantly improve the function of the infarcted mouse heart. With the aim of using more bio-responsive microcapsules, we sought to develop an enzymatically degradable, type I collagen-based microcapsule for the intramyocardial delivery of bone marrow-derived mesenchymal stromal cells in a murine model of myocardial infarction.
Heart failure is an important cause of morbidity and mortality. More than 20 years ago, special interest was drawn to cell therapy as a means of restoring damaged hearts to working condition. But progress has not been straightforward as many of our initial assumptions turned out to be wrong. In this review, we critically examine the last 20 years of progress in cardiac cell therapy and focus on several of the popular beliefs surrounding cell therapy to illustrate the mechanisms involved in restoring heart function after cardiac injury. Are they true or false?
The cell origin-specific payloads within extracellular vesicles (EVs) mediate therapeutic bioactivity for a wide variety of stem cell types. In this study, we profiled the microRNA (miRNA) and protein cargos found within EVs produced by three clinical-grade stem cell products of different ontogenies being considered for clinical application, namely bone marrow-derived mesenchymal stromal cells (BM-MSCs), heart-derived cells (HDCs), and umbilical cord-derived MSCs (UC-MSCs). Although several miRNAs (757) and proteins (420) were found in common, each producer cell type expressed unique miRNA profiles when the most highly expressed transcripts were compared. Differential expression analysis revealed that BM-MSCs and HDCs were quite similar, while UC-MSCs had the greatest number of unique miRNAs and proteins. Despite these differences, all three EVs promoted cell adhesion/migration, immune response, platelet aggregation, protein translation/stabilization, and RNA processing. EVs from BM-MSCs were implicated in apoptosis, cell-cycle progression, collagen formation, heme pigment synthesis, and smooth muscle differentiation, while HDC and UC-MSC EVs were found to regulate complement activation, endopeptidase activity, and matrix metallopeptidases. Overall, miRNA and protein profiling reveal functional differences between three leading stem cell products. These findings provide a framework for mechanistic exploration of candidate therapeutic molecules driving the salutary effects of EVs.
Cardiovascular diseases have been the leading cause of death worldwide during the past several decades. Cell loss is the main problem that resulted in cardiac dysfunction and further mortality. Cell therapy aiming to replenish the lost cells is proposed to treat cardiovascular diseases especially ischemic heart diseases which lead to a big portion of cell loss. Due to the direct injection's low cell retention and survival ratio, cell therapy using biomaterials as cell carriers attracts more and more attention because of their promotion of cell delivery and maintenance at the aiming sites. In this review, we systematically summarized the three main factors involved in cell therapy for myocardial tissue regeneration: cell sources (somatic cells, stem cells and engineered cells), chemical components of cell carriers (natural materials, synthetic materials and electroactive materials), and categories of cell delivery materials (patches, microspheres, injectable hydrogels, nanofiber and microneedles, etc.). An introduction of the methods including magnetic resonance/radionuclide/photoacoustic and fluorescence imaging for tracking the behavior of transplanted cells in vivo is also included. Current challenges of biomaterials‐based cell therapy and their future directions are provided to give both beginners and professionals a clear view of the development and future trends in this area. This article is protected by copyright. All rights reserved
Micrometer‐sized hydrogels are crosslinked three‐dimensional network matrices with high‐water contents and dimensions ranging from several to hundreds of micrometers. Due to their excellent biocompatibility and capability to mimic physiological microenvironments in vivo, micrometer‐sized hydrogels have attracted much attention in the biomedical engineering field. Their biological properties and applications are primarily influenced by their chemical compositions and geometries. However, inhomogeneous morphologies and uncontrollable geometries limit traditional micrometer‐sized hydrogels obtained by bulk mixing. In contrast, microfluidic technology holds great potential for the fabrication of micrometer‐sized hydrogels since their geometries, sizes, structures, compositions, and physicochemical properties can be precisely manipulated on demand based on the excellent control over fluids. Therefore, micrometer‐sized hydrogels fabricated by microfluidic technology have been applied in the biomedical field, including drug encapsulation, cell encapsulation, and tissue engineering. This review introduces micrometer‐sized hydrogels with various geometries synthesized by different microfluidic devices, highlighting their advantages in various biomedical applications over those from traditional approaches. Overall, emerging microfluidic technologies enrich the geometries and morphologies of hydrogels and accelerate translation for industrial production and clinical applications. This article is protected by copyright. All rights reserved
In vitro models have been used as a complementary tool to animal studies in understanding the nervous system's physiological mechanisms and pathological disorders, while also serving as platforms to evaluate the safety and efficiency of therapeutic candidates. Following recent advances in materials science, micro- and nanofabrication techniques and cell culture systems, in vitro technologies have been rapidly gaining the potential to bridge the gap between animal and clinical studies by providing more sophisticated models that recapitulate key aspects of the structure, biochemistry, biomechanics, and functions of human tissues. This was made possible, in large part, by the development of biomaterials that provide cells with physicochemical features that closely mimic the cellular microenvironment of native tissues. Due to the well-known material-driven cellular response and the importance of mimicking the environment of the target tissue, the selection of optimal biomaterials represents an important early step in the design of biomimetic systems to investigate brain structures and functions. This review provides a comprehensive compendium of commonly used biomaterials as well as the different fabrication techniques employed for the design of neural tissue models. Furthermore, the authors discuss the main parameters that need to be considered to develop functional platforms not only for the study of brain physiological functions and pathological processes but also for drug discovery/development and the optimization of biomaterials for neural tissue engineering.
Herein, we developed a flexible and cost-effective manual droplet operation system (MDOS) for performing miniaturized cell assays as well as single cell analysis. The MDOS consists of a manual x-y-z translation stage for liquid transferring and switching, a high-precision syringe pump for liquid driving and metering, a tapered capillary probe for droplet manipulation, a droplet array chip for droplet loading and reaction, sample/reagent reservoirs for storage, and a microscope for droplet observation, with a total expense of only $4,000. By using the flexible combination of three elementary operations of the x-y-z stage's moving and the pump's aspirating and depositing, the MDOS can manually achieve multiple droplet handling operations in the nanoliter to picoliter range, including droplet generation, assembling, fusion, diluting, and splitting. On this basis, multiple cell-related operations could be performed, such as nanoliter-scale in-droplet cell culture, cell coculture, drug stimulation, cell washing, and cell staining, as well as formation of picoliter single-cell droplets. The feasibility and flexibility of the MDOS was demonstrated in multi-mode miniaturized cell assays, including cell-based drug test, first-pass effect assay, and single-cell enzyme assay. The MDOS with the features of low cost, easy to build and flexible to use, could provide a promising alternative for performing miniaturized assays in routine laboratories, in addition to conventional microfluidic chip-based systems and automated robot systems.
Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition¹, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue². Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.
Droplet microfluidics offers exquisite control over the flows of multiple fluids in microscale, enabling fabrication of advanced microparticles with precisely tunable structures and compositions in a high throughput manner. The combination of these remarkable features with proper materials and fabrication methods has enabled high efficiency, direct encapsulation of actives in microparticles whose features and functionalities can be well controlled. These microparticles have great potential in a wide range of bio-related applications including drug delivery, cell-laden matrices, biosensors and even as artificial cells. In this review, we briefly summarize the materials, fabrication methods, and microparticle structures produced with droplet microfluidics. We also provide a comprehensive overview of their recent uses in biomedical applications. Finally, we discuss the existing challenges and perspectives to promote the future development of these engineered microparticles.
Background:
Aging is one of the key factors that regulate the function of human bone marrow mesenchymal stem cells (hBM-MSCs) and related changes in microRNA (miRNA) expression. However, data reported on aging-related miRNA changes in hBM-MSCs are limited.
Methods:
We demonstrated previously that miR-10a is significantly decreased in aged hBM-MSCs and restoration of the miR-10a level attenuated cell senescence and increased the differentiation capacity of aged hBM-MSCs by repressing Krüpple-like factor 4 (KLF4). In the present study, miR-10a was overexpressed or KLF4 was downregulated in old hBM-MSCs by lentiviral transduction. The hypoxia-induced apoptosis, cell survival, and cell paracrine function of aged hBM-MSCs were investigated in vitro. In vivo, miR-10a-overexpressed or KLF4-downregulated old hBM-MSCs were implanted into infarcted mouse hearts after myocardial infarction (MI). The mouse cardiac function of cardiac angiogenesis was measured and cell survival of aged hBM-MSCs was investigated.
Results:
Through lentivirus-mediated upregulation of miR-10a and downregulation of KLF4 in aged hBM-MSCs in vitro, we revealed that miR-10a decreased hypoxia-induced cell apoptosis and increased cell survival of aged hBM-MSCs by repressing the KLF4-BAX/BCL2 pathway. In vivo, transplantation of miR-10a-overexpressed aged hBM-MSCs promoted implanted stem cell survival and improved cardiac function after MI. Mechanistic studies revealed that overexpression of miR-10a in aged hBM-MSCs activated Akt and stimulated the expression of angiogenic factors, thus increasing angiogenesis in ischemic mouse hearts.
Conclusions:
miR-10a rejuvenated aged hBM-MSCs which improved angiogenesis and cardiac function in injured mouse hearts.
Natriuretic Peptide Receptor 3 (NPR3), the clearance receptor for extracellular bio-active natriuretic peptides (NPs), plays important roles in the homeostasis of body fluid volume and vascular tone. Using luciferase reporter and antagomir-based silencing assays, we demonstrated that the expression of NPR3 could be modulated by microRNA-143 (miR-143-3p), a microRNA species with up-regulated circulating concentrations in clinical heart failure. The regulatory effect of miR-143 on NPR3 expression was further evidenced by the reciprocal relationship between miR-143 and NPR3 levels observed in hypoxia-treated human cardiac cells and in left ventricular tissue from rats undergoing experimental myocardial infarction. Further analysis indicated elevation of miR-143 in response to hypoxic challenge reflects transcriptional activation of the miR-143 host gene (MIR143HG). This was corroborated by demonstration of the induction of host gene promoter activity upon hypoxic challenge. Moreover, miR-143 was shown to enhance its own expression by increasing MIR143HG promoter activity, as well as targeting the expressions of NPPA, NPPC, NR3C2, and CRHR2 in cardiac cells. Taken together, these findings suggest that the elevation of miR-143 upon hypoxic insult may be part of a microRNA-based feed forward loop that results in fine tuning the levels of NPs and neurohormonal receptors in cardiac cell lineages.
MiRNA is a class of small non-coding RNA which has an important effect on posttranscriptional gene regulation. It can regulate the expression of the target gene at the mRNA level and further influence the protein level of the target gene. We found that ULK1 may be the target gene of miR-26a-5p, and ULK1 (unc-51 like autophagy activating kinase 1) is a key component in autophagy pathway. In this study, we overexpressed miR-26a-5p by transfecting miR-26a-5p mimic into cells and simultaneously inhibited miR-26a-5p by transfecting miR-26a-5p inhibitor into cells. We demonstrated that overexpression of miR-26a-5p can reduce the expression of ULK1 and collagen I, and decrease the activation of LC3-I to LC3-II. In contrast, inhibition of miR-26a-5p can increase the expression of ULK1 and collagen I, and increase the activation of LC3-I to LC3-II. The Dual-luciferase reporter assay showed that miR-26a-5p directly acted on the 3'UTR of ULK1 and thus affected the expression of ULK1. As such, our study demonstrated that miR-26a-5p might regulate the autophagy in cardiac fibroblasts by targeting ULK1, which may have an effect on cardiac fibrosis. To our knowledge, this is the first study that shows miR-26a-5p regulates the autophagic pathway in cardiac fibroblasts.
Cells can be microencapsulated in synthetic hydrogel microspheres (microgels) using droplet microfluidics, but microfluidic devices with a single droplet generating geometry have limited throughput, especially as microgel diameter decreases. Here we demonstrate microencapsulation of human mesenchymal stem cells (hMSCs) in small (<100 μm diameter) microgels utilizing parallel droplet generators on a two-layer elastomer device, which has 600% increased throughput vs. single-nozzle devices. Distribution of microgel diameters were compared between products of parallel vs. single-nozzle configurations for two square nozzle widths, 35 and 100 μm. Microgels produced on parallel nozzles were equivalent to those produced on single nozzles, with substantially the same polydispersity. Microencapsulation of hMSCs was compared for parallel nozzle devices of each width. Thirty five micrometer wide nozzle devices could be operated at twice the cell concentration of 100 μm wide nozzle devices but produced more empty microgels than predicted by a Poisson distribution. Hundred micrometer wide nozzle devices produced microgels as predicted by a Poisson distribution. Polydispersity of microgels did not increase with the addition of cells for either nozzle width. hMSCs encapsulated on 35 μm wide nozzle devices had reduced viability (~70%) and a corresponding decrease in vascular endothelial growth factor (VEGF) secretion compared to hMSCs cultured on tissue culture (TC) plastic. Encapsulating hMSCs using 100 μm wide nozzle devices mitigated loss of viability and function, as measured by VEGF secretion.
Statement of significance:
Stem cell based-therapies represent a possible solution to repair damaged myocardial tissue by promoting cardioprotection, angiogenesis, and reduced fibrosis. However, recent evidence indicates that most of the positive outcomes are likely due to the release of paracrine factors (cytokines, growth factors, and exosomes) from the cells and not because of the local engraftment of stem cells. This cocktail of essential growth factors and paracrine signals is known as secretome can be isolated in vitro, and the biomolecule composition can be controlled by varying stem-cell culture conditions. Here, we propose a straightforward strategy to deliver secretome produced from hASCs by using a nanocomposite injectable hydrogel made of gelatin and Laponite®. The designed secretome-loaded hydrogel represents a promising alternative to traditional stem cell therapy for the treatment of acute myocardial infarction.
Stem cell encapsulation technology demonstrates much promise for the replacement of damaged tissue in several diseases, including spinal cord injury (SCI). The use of biocompatible microcapsules permits the control of stem cell fate in situ to facilitate the replacement of damaged/lost tissue. In this work, a novel customized microfluidic device was developed for the reproducible encapsulation of Neural Stem Cells (NSCs) and Dental Pulp Stem Cells (DPSCs) within monodisperse, alginate-collagen microcapsules. Both cell types survived within the microcapsules for up to 21 days in culture. Stem cells demonstrated retention of their multipotency and neuronal differentiation properties upon selective release from the microcapsules, as demonstrated by high proliferation rates and the production of stem cell and neuronal lineage markers. When cell laden microcapsules were transplanted into an organotypic SCI model, the microcapsules effectively retained the transplanted stem cells at the site of implantation. Implanted cells survived over a 10 day period in culture after transplantation and demonstrated commitment to a neural lineage. These results demonstrate the great benefits of using microfluidics for stem cell encapsulation in a quick, effortless, aseptic manner. Our device provides a quick, effective, and aseptic method for the encapsulation of two different stem cell types (DPSCs and NSCs) within alginate-collagen microcapsules. Since stem cells were able to retain their viability and neural differentiation capacity within such microcapsules, this method provides a useful technique to study stem cell behavior within three dimensional (3D) environments.
Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, economical and widely used methods for evaluating gene expression. However, the utility of this method continues to be undermined by a number of challenges including normalization using appropriate reference genes. The need to develop tailored and effective strategies is further underscored by the burgeoning field of extracellular vesicle (EV) biology. EVs contain unique signatures of small RNAs including microRNAs (miRs). In this study we develop and validate a comprehensive strategy for identifying highly stable reference genes in a therapeutically relevant cell type, cardiosphere-derived cells. Data were analysed using the four major approaches for reference gene evaluation: NormFinder, GeNorm, BestKeeper and the Delta Ct method. The weighted geometric mean of all of these methods was obtained for the final ranking. Analysis of RNA sequencing identified miR-101-3p, miR-23a-3p and a previously identified EV reference gene, miR-26a-5p. Analysis of a chip-based method (NanoString) identified miR-23a, miR-217 and miR-379 as stable candidates. RT-qPCR validation revealed that the mean of miR-23a-3p, miR-101-3p and miR-26a-5p was the most stable normalization strategy. Here, we demonstrate that a comprehensive approach of a diverse data set of conditions using multiple algorithms reliably identifies stable reference genes which will increase the utility of gene expression evaluation of therapeutically relevant EVs.
Heart failure is the number one killer worldwide with ~50% of patients dying within 5 years of prognosis. The discovery of stem cells, which are capable of repairing the damaged portion of the heart, has created a field of cardiac regenerative medicine, which explores various types of stem cells, either autologous or endogenous, in the hope of finding the “holy grail” stem cell candidate to slow down and reverse the disease progression. However, there are many challenges that need to be overcome in the search of such a cell candidate. The ideal cells have to survive the harsh infarcted environment, retain their phenotype upon administration, and engraft and be activated to initiate repair and regeneration in vivo. Early bench and bedside experiments mostly focused on bone marrow-derived cells; however, heart regeneration requires multiple coordinations and interactions between various cell types and the extracellular matrix to form new cardiomyocytes and vasculature. There is an observed trend that when more than one cell is coadministered and cotransplanted into infarcted animal models the degree of regeneration is enhanced, when compared to single-cell administration. This review focuses on stem cell candidates, which have also been tested in human trials, and summarizes findings that explore the interactions between various stem cells in heart regenerative therapy.
Purpose of review:
In this review, we highlight the most important cellular and molecular mechanisms that contribute to cardiac inflammation and fibrosis. We also discuss the interplay between inflammation and fibrosis in various precursors of heart failure (HF) and how such mechanisms can contribute to myocardial tissue remodelling and development of HF.
Recent findings:
Recently, many research articles attempt to elucidate different aspects of the interplay between inflammation and fibrosis. Cardiac inflammation and fibrosis are major pathophysiological mechanisms operating in the failing heart, regardless of HF aetiology. Currently, novel therapeutic options are available or are being developed to treat HF and these are discussed in this review. A progressive disease needs an aggressive management; however, existing therapies against HF are insufficient. There is a dynamic interplay between inflammation and fibrosis in various precursors of HF such as myocardial infarction (MI), myocarditis and hypertension, and also in HF itself. There is an urgent need to identify novel therapeutic targets and develop advanced therapeutic strategies to combat the syndrome of HF. Understanding and describing the elements of the inflammatory and fibrotic pathways are essential, and specific drugs that target these pathways need to be evaluated.
Background
Cardiomyocyte apoptosis is a common pathological manifestation that occurs in several heart diseases. This study aimed to explore the mechanism of microRNA-486 (miR-486) in cardiomyocyte apoptosis by interfering with the p53-activated BCL-2 associated mitochondrial pathway.
Methods
miR-486 mimics and inhibitors were transfected into the primary cardiomyocytes of suckling Sprague-Dawley rat pups, and H2O2 was used to induce apoptosis. Flow cytometry and TUNEL were both used to detect cardiomyocyte apoptosis, while the relative mRNA transcript and protein levels of miR-486, p53, Bbc3, BCL-2, and cleaved caspase-3 were detected using RT-PCR and western blot analysis, respectively.
Results
miR-486 overexpression significantly decreased the expressions of p53, Bbc3 and cleaved caspase-3 (P < 0.05), and BCL-2 expression was significantly increased (P < 0.05), which in turn caused a significant decrease in the rate of cardiomyocyte apoptosis (P < 0.05). In contrast, miR-486 silencing resulted in an elevated rate of cardiomyocyte apoptosis (P < 0.05).
Conclusion
miR-486 may regulate cardiomyocyte apoptosis via p53-mediated BCL-2 associated mitochondrial apoptotic pathway. Therefore, up-regulating miR-486 expression in cardiomyocytes can effectively reduce the activation of the BCL-2 associated mitochondrial apoptotic pathway, consequently protecting cardiomyocytes.
Recent evidence suggests that hypoxia caused by acute myocardial infarction can induce cardiomyocyte apoptosis. Exosomes are signalling mediators that contribute to intercellular communication by transporting cytosolic components including miRNAs, mRNAs, and proteins. However, the systemic regulation and function of exosomal miRNAs in hypoxic cardiomyocytes are currently not well understood. Here, we used small RNA sequencing to investigate the effects of hypoxia stress on miRNAome of rat cardiomyoblast cells (H9c2) and corresponding exosomes. We identified 92 and 62 miRNAs in cells and exosomes, respectively, that were differentially expressed between hypoxia and normoxia. Hypoxia strongly modulated expression of hypoxia-associated miRNAs in H9c2 cells, and altered the miRNAome of H9c2 cells-derived exosomes. Functional enrichment analysis revealed extensive roles of differentially expressed exosomal miRNAs in the HIF-1 signalling pathway and in apoptosis-related pathways including the TNF, MAPK, and mTOR pathways. Furthermore, gain- and loss-of-function analysis demonstrated potential anti-apoptotic effects of the hypoxia-induced exosomal miRNAs, including miR-21-5p, miR-378-3p, miR-152-3p, and let-7i-5p; luciferase reporter assay confirmed that Atg12 and Faslg are targets of miR-152-3p and let-7i-5p, respectively. To summarize, this study revealed that hypoxia-induced exosomes derived from H9c2 cells loaded cardioprotective miRNAs, which mitigate hypoxia-induced H9c2 cells apoptosis.
Background
MicroRNA (miRNA) is a type of noncoding RNA that can repress the expression of target genes through posttranscriptional regulation. In addition to numerous physiologic roles for miRNAs, they play an important role in pathophysiologic processes affecting cardiovascular health. Previously, we reported that nuclear encoded microRNA (miR‐181c) is present in heart mitochondria, and importantly, its overexpression affects mitochondrial function by regulating mitochondrial gene expression.
Methods and Results
To investigate further how the miR‐181 family affects the heart, we suppressed miR‐181 using a miR‐181‐sponge containing 10 repeated complementary miR‐181 “seed” sequences and generated a set of H9c2 cells, a cell line derived from rat myoblast, by stably expressing either a scrambled or miR‐181‐sponge sequence. Sponge‐H9c2 cells showed a decrease in reactive oxygen species production and reduced basal mitochondrial respiration and protection against doxorubicin‐induced oxidative stress. We also found that miR‐181a/b targets phosphatase and tensin homolog (PTEN), and the sponge‐expressing stable cells had increased PTEN activity and decreased PI3K signaling. In addition, we have used miR‐181a/b−/− and miR‐181c/d−/− knockout mice and subjected them to ischemia‐reperfusion injury. Our results suggest divergent effects of different miR‐181 family members: miR‐181a/b targets PTEN in the cytosol, resulting in an increase in infarct size in miR‐181a/b−/− mice due to increased PTEN signaling, whereas miR‐181c targets mt‐COX1 in the mitochondria, resulting in decreased infarct size in miR‐181c/d−/− mice.
Conclusions
The miR‐181 family alters the myocardial response to oxidative stress, notably with detrimental effects by targeting mt‐COX1 (miR‐181c) or with protection by targeting PTEN (miR‐181a/b).
The tumor microenvironment (TME) has an impact on breast cancer progression
by creating a pro-inflammatory milieu within the tumor. However, little is known about
the roles of miRNAs in cells of the TME during this process. We identified six putative
oncomiRs in a breast cancer dataset, all strongly correlating with poor overall patient
survival. Out of the six candidates, miR-1246 was upregulated in aggressive breast
cancer subtypes and expressed at highest levels in mesenchymal stem/stroma cells
(MSCs). Functionally, miR-1246 led to a p65-dependent increase in transcription and
release of pro-inflammatory mediators IL-6, CCL2 and CCL5 in MSCs, and increased
NF-κB activity. The pro-inflammatory phenotype of miR-1246 in MSCs was independent
of TNFα stimulations and mediated by direct targeting of the tumor-suppressors
PRKAR1A and PPP2CB. In vitro recapitulation of the TME revealed increased Stat3
phosphorylation in breast epithelial (MCF10A) and cancer cells (SK-BR-3, MCF7,
T47D) upon incubation with conditioned medium (CM) of MSCs overexpressing miR-
1246. Additionally, this stimulation enhanced proliferation of MCF10A cells, increased
migration of MDA-MB-231 cells and induced attraction of THP-1 monocytic cells. Our
data shows that miR-1246 acts as both key-enhancer of pro-inflammatory responses
in MSCs and putative oncomiR in breast cancer, suggesting its influence on cancerrelated
inflammation and breast cancer progression.
Background/aims:
Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure, but the mechanisms undelying cardiac fibrosis remained unknown. microRNAs (miRNAs) are novel mechanisms leading to fibrotic diseases, including cardiac fibrosis. Previous studies revealed that miR-22 might be a potential target. However, the roles and mechanisms of miR-22 in cardiac fibrosis remained ill defined. The present study thus addressed the impact of miR-22 in cardiac fibrosis.
Methods:
After seven days following coronary artery occlusion in mice, tissues used for histology were collected and processed for Masson's Trichrome staining. In addition, cardiac fibroblasts were transfected with mimics and inhibitors of miR-22 using Lipofectamin 2000, and luciferase activity was measured in cell lysates using a luciferase assay kit. Western blotting was used to detect the expression of collagen1, α-SMA and TGFβRI proteins levels, and real time-PCR was employed to measure the Col1α1, Col3α1, miR-22 and TGFβRI mRNA levels.
Results:
In this study, we found that miR-22 was dynamically downregulated following MI induced by permanent ligation of the left anterior descending coronary artery for 7 days, an effect paralleled by significant collagen deposition. Inhibition of miR-22 with AMO-22 resulted in increased expression of Col1α1, Col3α1 and fibrogenesis in cultured cardiac fibroblasts. Conversely, overexpression of miR-22 in cultured cardiac fibroblasts significantly abrogated angiotensin II-induced collagen formation and fibrogenesis. Furthermore, we found that TGFβRI is a direct target for miR-22, and downregulation of TGFβR may have mediated the antifibrotic effect of miR-22.
Conclusion:
Our data clearly demonstrate that miR-22 acts as a novel negative regulator of angiotensin II-induced cardiac fibrosis by suppressing the expression of TGFβRI in the heart and may represent a new potential therapeutic target for treating cardiac fibrosis.
Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis.
The acute phase protein Pentraxin 3 (PTX3) plays a non-redundant role as a soluble pattern recognition receptor for selected pathogens and it represents a rapid biomarker for primary local activation of innate immunity and inflammation. Recent evidence indicates that PTX3 exerts an important role in modulating the cardiovascular system in humans and experimental models. In particular, there are conflicting points concerning the effects of PTX3 in cardiovascular diseases (CVD) since several observations indicate a cardiovascular protective effect of PTX3 while others speculate that the increased plasma levels of PTX3 in subjects with CVD correlate with disease severity and with poor prognosis in elderly patients.
In the present review, we discuss the multifaceted effects of PTX3 on the cardiovascular system focusing on its involvement in atherosclerosis, endothelial function, hypertension, myocardial infarction and angiogenesis. This may help to explain how the specific modulation of PTX3 such as the use of different dosing, time, and target organs could help to contain different vascular diseases. These opposite actions of PTX3 will be emphasized concerning the modulation of cardiovascular system where potential therapeutic implications of PTX3 in humans are discussed.
Background:
Insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) are among the most promising growth factors for promoting cardiorepair. Here, we evaluated the combination of cell- and gene-based therapy using mesenchymal stem cells (MSC) genetically modified to overexpress IGF-1 or HGF to treat acute myocardial infarction (AMI) in a porcine model.
Methods:
Pig MSC from adipose tissue (paMSC) were genetically modified for evaluation of different therapeutic strategies to improve AMI treatment. Three groups of infarcted Large White pigs were compared (I, control, non-transplanted; II, transplanted with paMSC-GFP (green fluorescent protein); III, transplanted with paMSC-IGF-1/HGF). Cardiac function was evaluated non-invasively using magnetic resonance imaging (MRI) for 1 month. After euthanasia and sampling of the animal, infarcted areas were studied by histology and immunohistochemistry.
Results:
Intramyocardial transplant in a porcine infarct model demonstrated the safety of paMSC in short-term treatments. Treatment with paMSC-IGF-1/HGF (1:1) compared with the other groups showed a clear reduction in inflammation in some sections analyzed and promoted angiogenic processes in ischemic tissue. Although cardiac function parameters were not significantly improved, cell retention and IGF-1 overexpression was confirmed within the myocardium.
Conclusions:
The simultaneous administration of IGF-1- and HGF-overexpressing paMSC appears not to promote a synergistic effect or effective repair. The combined enhancement of neovascularization and fibrosis in paMSC-IGF-1/HGF-treated animals nonetheless suggests that sustained exposure to high IGF-1 + HGF levels promotes beneficial as well as deleterious effects that do not improve overall cardiac regeneration.
MicroRNAs have been implicated in some biological and pathological processes, including the myocardial ischemia/reperfusion (I/R) injury. Recent findings demonstrated that miR-93 might provide a potential cardioprotective effect on ischemic heart disease. This study was to investigate the role of miR-93 in I/R-induced cardiomyocyte injury and the potential mechanism. In this study, we found that hypoxia/reoxygenation (H/R) dramatically increased LDH release, MDA contents, ROS generation, and endoplasmic reticulum stress (ERS)-mediated cardiomyocyte apoptosis, which were attenuated by co-transfection with miR-93 mimic. Phosphatase and tensin homolog (PTEN) was identified as the target gene of miR-93. Furthermore, miR-93 mimic significantly increased p-Akt levels under H/R, which was partially released by LY294002. In addtion, Ad-miR-93 also attenuated myocardial I/R injury in vivo, manifested by reduced LDH and CK levels, infarct area and cell apoptosis. Taken together, our findings indicates that miR-93 could protect against I/R-induced cardiomyocyte apoptosis by inhibiting PI3K/AKT/PTEN signaling.
Background
Although patient-sourced cardiac stem cells repair damaged myocardium, the extent to which medical co-morbidities influence cardiac-derived cell products is uncertain. Therefore, we investigated the influence of atherosclerotic risk factors on the regenerative performance of human cardiac explant-derived cells (EDCs). Methods
In this study, the Long Term Stratification for survivors of acute coronary syndromes model was used to quantify the burden of cardiovascular risk factors within a group of patients with established atherosclerosis. EDCs were cultured from human atrial appendages and injected into immunodeficient mice 7 days post-left coronary ligation. Cytokine arrays and enzyme linked immunoassays were used to determine the release of cytokines by EDCs in vitro, and echocardiography was used to determine regenerative capabilities in vivo. ResultsEDCs sourced from patients with more cardiovascular risk factors demonstrated a negative correlation with production of pro-healing cytokines (such as stromal cell derived factor 1α) and exosomes which had negative effects on the promotion of angiogenesis and chemotaxis. Reductions in exosomes and pro-healing cytokines with accumulating medical co-morbidities were associated with increases in production of the pro-inflammatory cytokine interleukin-6 (IL-6) by EDCs. Increased patient co-morbidities were also correlated with significant attenuation in improvements of left ventricular ejection fraction. Conclusions
The regenerative performance of the earliest precursor cell population cultured from human explant tissue declines with accumulating medical co-morbidities. This effect is associated with diminished production of pro-cardiogenic cytokines and exosomes while IL-6 is markedly increased. Predictors of cardiac events demonstrated a lower capacity to support angiogenesis and repair injured myocardium in a mouse model of myocardial infarction.
Myocardial ischemia-reperfusion (I-R) injury lacks effective treatments. The miR-17-92 cluster plays important roles in regulating proliferation, apoptosis, cell cycle and other pivotal processes. However, their roles in myocardial I-R injury is largely unknown. In this study, we found that miR-19b was the only member of the miR-17-92 cluster that was downregulated in infarct area of heart samples from a murine model of I-R injury. Meanwhile, downregulation of miR-19b was also detected in H2O2-treated H9C2 cells in vitro mimicking oxidative stress occurring during myocardial I-R injury. Using flow cytometry and Western blot analysis, we found that overexpression of miR-19b decreased H2O2-induced apoptosis and improved cell survival, whiledownregulation of that had inverse effects. Furthermore, PTEN was negatively regulated by miR-19b at the protein level while silencing PTEN could completely block the aggravated impact of miR-19b inhibitor on H2O2-induced apoptosis in H9C2 cardiomyocytes, indicating PTEN as a downstream target of miR-19b controlling H2O2-induced apoptosis. These data indicate that miR-19b overexpression might be a novel therapy for myocardial I-R injury.
Myocardial infarction (MI) affects millions of people worldwide. MI causes massive cardiac cell death and heart function decrease. However, heart tissue cannot effectively regenerate by itself. While stem cell therapy has been considered an effective approach for regeneration, the efficacy of cardiac stem cell therapy remains low due to inferior cell engraftment in the infarcted region. This is mainly a result of low cell retention in the tissue and poor cell survival under ischemic, immune rejection and inflammatory conditions. Various approaches have been explored to improve cell engraftment: increase of cell retention using biomaterials as cell carriers; augmentation of cell survival under ischemic conditions by preconditioning cells, genetic modification of cells, and controlled release of growth factors and oxygen; and enhancement of cell survival by protecting cells from excessive inflammation and immune surveillance. In this paper, we review current progress, advantages, disadvantages, and potential solutions of these approaches.
Stem cell-based repair and regeneration for cardiac regeneration following myocardial injury remain unmet challenges largely due to low viability of cells transplanted in the recipient sites. Accumulating evidence has revealed that local existence of reactive oxygen species (ROS) causes transplanted cell death via both apoptosis and autophagy. Ham and colleagues have identified let-7b as one of the primary mediators for ROS-induced apoptosis and autophagy of mesenchymal stem cells (MSCs) through direct targeting of caspase-3. Importantly, intramyocardial injection of let-7b-modified MSCs significantly enhanced ventricular function and facilitated myocardial repair by protecting transplanted cells from apoptosis and autophagy in the rat cardiac ischemia-reperfusion model. These findings provide novel insights into the roles of microRNA underlying stem cell survival following in vivo delivery, and offer further evidence that microRNA-modified MSC transplantation might be an effective therapeutic approach for tissue repair and regeneration.
MicroRNAs (miRs) regulate a number of physiological and pathological processes, including myocardial chronic hypoxia. Previous studies revealed that the expression of miR-146b is increased in vitro and in vivo following the induction of hypoxia. In the present study, the role of miR‑146b in hypoxic cardiomyocytes, and the mechanisms underlying its activity, were investigated. The expression of miR‑146b was measured in tissue samples from patients with congenital heart disease by reverse transcription‑quantitative polymerase chain reaction. The rat H9c2 cardiomyocyte cell line was transfected with an miR‑146b inhibitor or the experimental controls, and the cells were maintained under hypoxic conditions for 72 h. The expression of miR‑146b increased following the induction of hypoxia. Transfection with the miR‑146b inhibitor enhanced the release of lactate dehydrogenase and increased hypoxia‑induced apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP nick‑end labeling, Hoechst 33258 staining, JC‑1 assay (measuring mitochondrial membrane permeability) and annexin V/propidium iodide analysis. A decreased expression of Bcl‑2 was observed, whereas the expression levels of cleaved‑caspase 3 and Bax were increased. Western blot analysis and a dual luciferase reporter assay confirmed that ribonuclease L is a direct target of miR‑146b. Furthermore, inhibition of miR-146b increased the activation of nuclear factor-κB and signal transducer and activator of transcription 3. In conclusion, the inhibition of miR‑146b may increase hypoxia-induced cardiomyocyte apoptosis.
Background:
Insulin-like growth factor 1 (IGF-1) activates prosurvival pathways and improves postischemic cardiac function, but this key cytokine is not robustly expressed by cultured human cardiac stem cells. We explored the influence of an enhanced IGF-1 paracrine signature on explant-derived cardiac stem cell-mediated cardiac repair.
Methods and results:
Receptor profiling demonstrated that IGF-1 receptor expression was increased in the infarct border zones of experimentally infarcted mice by 1 week after myocardial infarction. Human explant-derived cells underwent somatic gene transfer to overexpress human IGF-1 or the green fluorescent protein reporter alone. After culture in hypoxic reduced-serum media, overexpression of IGF-1 enhanced proliferation and expression of prosurvival transcripts and prosurvival proteins and decreased expression of apoptotic markers in both explant-derived cells and cocultured neonatal rat ventricular cardiomyocytes. Transplant of explant-derived cells genetically engineered to overexpress IGF-1 into immunodeficient mice 1 week after infarction boosted IGF-1 content within infarcted tissue and long-term engraftment of transplanted cells while reducing apoptosis and long-term myocardial scarring.
Conclusions:
Paracrine engineering of explant-derived cells to overexpress IGF-1 provided a targeted means of improving cardiac stem cell-mediated repair by enhancing the long-term survival of transplanted cells and surrounding myocardium.
There is a recognized and growing need for rapid and efficient cell assays, where the size of microfluidic devices lend themselves to the manipulation of cellular populations down to the single cell level. An exceptional way to analyze cells independently is to encapsulate them within aqueous droplets surrounded by an immiscible fluid, so that reagents and reaction products are contained within a controlled microenvironment. Most cell encapsulation work has focused on the development and use of passive methods, where droplets are produced continuously at high rates by pumping fluids from external pressure-driven reservoirs through defined microfluidic geometries. With limited exceptions, the number of cells encapsulated per droplet in these systems is dictated by Poisson statistics, reducing the proportion of droplets that contain the desired number of cells and thus the effective rate at which single cells can be encapsulated. Nevertheless, a number of recently developed actively-controlled droplet production methods present an alternative route to the production of droplets at similar rates and with the potential to improve the efficiency of single-cell encapsulation. In this critical review, we examine both passive and active methods for droplet production and explore how these can be used to deterministically and non-deterministically encapsulate cells.
Cell therapy is one of the most promising areas within regenerative medicine. However, its full potential is limited by the rapid loss of introduced therapeutic cells before their full effects can be exploited, due in part to anoikis, and in part to the adverse environments often found within the pathologic tissues that the cells have been grafted into. Encapsulation of individual cells has been proposed as a means of increasing cell viability. In this study, we developed a facile, high throughput method for creating temperature responsive microcapsules comprising agarose, gelatin and fibrinogen for delivery and subsequent controlled release of cells. We verified the hypothesis that composite capsules combining agarose and gelatin, which possess different phase transition temperatures from solid to liquid, facilitated the destabilization of the capsules for cell release. Cell encapsulation and controlled release was demonstrated using human fibroblasts as model cells, as well as a therapeutically relevant cell line-human umbilical vein endothelial cells (HUVECs). While such temperature responsive cell microcapsules promise effective, controlled release of potential therapeutic cells at physiological temperatures, further work will be needed to augment the composition of the microcapsules and optimize the numbers of cells per capsule prior to clinical evaluation.
Epicardial injection of heart-derived cell products is safe and effective post-myocardial infarction (MI), but clinically-translatable transendocardial injection has never been evaluated. We sought to assess the feasibility, safety and efficacy of percutaneous transendocardial injection of heart-derived cells in porcine chronic ischemic cardiomyopathy.
We studied a total of 89 minipigs; 63 completed the specified protocols. After NOGA-guided transendocardial injection, we quantified engraftment of escalating doses of allogeneic cardiospheres or cardiosphere-derived cells in minipigs (n = 22) post-MI. Next, a dose-ranging, blinded, randomized, placebo-controlled ("dose optimization") study of transendocardial injection of the better-engrafting product was performed in infarcted minipigs (n = 16). Finally, the superior product and dose (150 million cardiospheres) were tested in a blinded, randomized, placebo-controlled ("pivotal") study (n = 22). Contrast-enhanced cardiac MRI revealed that all cardiosphere doses preserved systolic function and attenuated remodeling. The maximum feasible dose (150 million cells) was most effective in reducing scar size, increasing viable myocardium and improving ejection fraction. In the pivotal study, eight weeks post-injection, histopathology demonstrated no excess inflammation, and no myocyte hypertrophy, in treated minipigs versus controls. No alloreactive donor-specific antibodies developed over time. MRI showed reduced scar size, increased viable mass, and attenuation of cardiac dilatation with no effect on ejection fraction in the treated group compared to placebo.
Dose-optimized injection of allogeneic cardiospheres is safe, decreases scar size, increases viable myocardium, and attenuates cardiac dilatation in porcine chronic ischemic cardiomyopathy. The decreases in scar size, mirrored by increases in viable myocardium, are consistent with therapeutic regeneration.
Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell-derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non-coding RNA, microRNA-24 (miR-24), as a pro-survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR-24 via viral transduction or chemical transfection, where the observed protective role of miR-24 in cardiomyocytes might have partially resulted from its effect on non-cardiomyocyte cells. To elucidate the cardiomyocyte-specific effects of miR-24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR-24 expression is driven by the cardiac-specific Myh6 promoter. The Myh6-miR-24 transgenic mice did not exhibit apparent difference from their wild-type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post-MI compared to their wild-type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non-genetic approach, raising the possibility that non-genetic approaches of introducing miR-24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte-specific role of miR-24 in the adult heart, and may provide insight to strategy design when considering miRNA-based therapies for cardiovascular diseases.
Self-assembling heart-derived stem cell clusters named cardiospheres (CSps) improve function and attenuate remodeling in rodent models of acute myocardial infarction. The effects of CSps in chronically remodeled myocardium post-MI, and the underlying mechanisms, remain unknown. One month after permanent coronary ligation, rats were randomly assigned to injection of vehicle (controls) or CSps in the peri-infarct area. One month post-injection, CSps increased left ventricular function, reduced scar mass and collagen density, and enhanced vascularity within the infarct zone compared to controls. Immunoblots revealed Tgfβ-1/smad cascade downregulation and an increase in soluble endoglin post-CSp injection. Six months post-transplantation, left ventricular function further improved and cardiomyocyte hypertrophy was attenuated in the CSp-treated group. In vitro, co-culture of CSps with fibroblasts recapitulated the suppression of the Tgf-β1/smad pathway changes, responses which were blunted by neutralizing antibody against endoglin. Thus, cardiosphere transplantation enhances angiogenesis and reduces fibrosis in chronically infarcted myocardium, leading to partial reversal of cardiac dysfunction. The underlying mechanism involves inhibition of Tgf-β1/smad signaling by CSp-secreted soluble endoglin.
Endothelial progenitor cells (EPCs) and human mesenchymal stem cells (hMSCs) have shown great regenerative potential to repair damaged tissue; however, their injection in vivo results in low retention and poor cell survival. Early clinical research has focused on cell‐encapsulation, to improve viability and integration of delivered cells. However, this strategy has been limited by the inability to reproduce large volumes of standardized microcapsules and the lack of information on cell‐specific egress and timed release from hydrogel microcapsules. Here, we address both of these limitations. First, we use a droplet microfluidic platform to generate monodisperse, agarose microcapsules and second, we encapsulate and characterize egress of therapeutically relevant cells (human umbilical vein endothelial cells, EPCs, and hMSCs). With increased temporal resolution, we demonstrate distinct differences in egress between cell types. Importantly, therapeutic cells (hMSCs) egress quickly, in < 6 hours following encapsulation. Further, we examined potential escape mechanisms, and show that proliferation can be exploited by cells for microcapsule translocation. We also systematically characterized the egress of fibroblasts (as model cells) following alterations to the microcapsules. Specifically, we show that microcapsule size and hydrogel density impacts cell egress efficiency. Overall, our results demonstrate the need for characterization of cell‐specific egress and tuning of the cocoon microenvironment, prior to delivery, for timely release and successful engraftment.
Tissue and stem cell encapsulation andtransplantation were considered as promising tools in the treatment of patients with diabetes mellitus. The aim of this study was to evaluate the effect of microfluidic encapsulation on the differentiation of trabecular meshwork mesenchymal stem cells (TM‐MSC), into insulin‐producing cells (IPCs) both in vitro and in vivo. The presence of differentiated cells in microfibers (three dimensional [3D]) and tissue culture plates (TCPS; two dimensional [2D]) culture was evaluated by detecting mRNA and protein expression of pancreatic islet‐specific markers as well as measuring insulin release of cells in response to glucose challenges. Finally, semi‐differentiated cells in microfibers (3D) and 2D cultures were used to control the glucose level in diabetic rats. The results of this study showed that MSCs differentiated in alginate microfibers (fabricated by microfluidic device) express more Pdx‐1 mRNA (1.938‐fold, p‐value: 0.0425) and Insulin mRNA (2.841‐fold, p‐value: 0.0001) compared with those cultured on TCPS. Furthermore, cell encapsulation in microfluidic derived microfibers decreased the level of blood glucose in diabetic rats. The approach used in this study showed the possibility of alginate microfibers as a matrix for differentiation of TM‐MSCs (as a new source) into IPCs. In addition, it could minimize different steps in stem cell differentiation, handling, and encapsulation, which lead to loss of an unlimited number of cells.
Fibroblast growth factor 19 (FGF19) has emerged as a crucial cytoprotective regulator that antagonizes cell apoptosis and oxidative stress under adverse conditions. However, whether FGF19 plays a cytoprotective role in preventing myocardial damage during myocardial ischemia/reperfusion injury remains unknown. In this study, we aimed to investigate the potential role of FGF19 in regulating hypoxia/reoxygenation (H/R)-induced injury of cardiomyocytes in vitro. We found that that FGF19 expression was upregulated in response to H/R treatment in cardiomyocytes. Silencing of FGF19 significantly inhibited viability and increased apoptosis and reactive oxygen species (ROS) generation in cardiomyocytes with H/R treatment. In contrast, overexpression of FGF19 improved viability and inhibited apoptosis and ROS generation induced by H/R treatment, showing a cardioprotective effect. Moreover, we found that FGF19 regulated the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2). In addition, FGF19 promoted the activation of Nrf2-mediated antioxidant response element (ARE) antioxidant signaling. Notably, treatment with a GSK-3β inhibitor significantly abrogated the adverse effects of FGF19 silencing on H/R-induced injury, whereas silencing of Nrf2 partially blocked the FGF19-mediated cardioprotective effect against H/R-induced injury in cardiomyocytes. Taken together, our findings demonstrate that FGF19 alleviates H/R-induced apoptosis and oxidative stress in cardiomyocytes by inhibiting GSK-3β activity and promoting the activation of Nrf2/ARE signaling, providing a potential therapeutic target for prevention of myocardial injury.
Ischaemic heart disease is a leading cause of death worldwide. Injury to the heart is followed by loss of the damaged cardiomyocytes, which are replaced with fibrotic scar tissue. Depletion of cardiomyocytes results in decreased cardiac contraction, which leads to pathological cardiac dilatation, additional cardiomyocyte loss, and mechanical dysfunction, culminating in heart failure. This sequential reaction is defined as cardiac remodelling. Many therapies have focused on preventing the progressive process of cardiac remodelling to heart failure. However, after patients have developed end-stage heart failure, intervention is limited to heart transplantation. One of the main reasons for the dramatic injurious effect of cardiomyocyte loss is that the adult human heart has minimal regenerative capacity. In the past 2 decades, several strategies to repair the injured heart and improve heart function have been pursued, including cellular and noncellular therapies. In this Review, we discuss current therapeutic approaches for cardiac repair and regeneration, describing outcomes, limitations, and future prospects of preclinical and clinical trials of heart regeneration. Substantial progress has been made towards understanding the cellular and molecular mechanisms regulating heart regeneration, offering the potential to control cardiac remodelling and redirect the adult heart to a regenerative state.
Although cocooning explant-derived cardiac stem cells in protective nanogels prior to intramyocardial injection boosts long-term cell retention, the number of EDCs that finally engraft is trivial and unlikely to account for salutary effects on myocardial function and scar size. As such, we investigated the effect of varying the nanogel content within micro-capsules to alter the physical properties of cocoons without influencing cocoon dimensions. Increasing nanogel concentration enhanced cell migration and viability while improving cell-mediated repair of injured myocardium. Given the latter occurred with nanogel content having no detectable effect on the long-term engraftment of transplanted cells, we found that changing the physical properties of cocoons prompted explant-derived cardiac stem cells to produce greater amounts of cytokines, microparticles and microRNAs that boosted the generation of new blood vessels and new cardiomyocytes. Thus, by altering the physical properties of cocoons by varying nanogel content, the paracrine signature of encapsulated cells can be enhanced to promote greater endogenous repair of injured myocardium.
After a myocardial infarction, heart tissue becomes irreversibly damaged, leading to scar formation and inevitably ischemic heart failure. Of the many available interventions after a myocardial infarction, such as percutaneous intervention or pharmacological optimization, none can reverse the ischemic insult on the heart and restore cardiac function. Thus, the only available cure for patients with scarred myocardium is allogeneic heart transplantation, which comes with extensive costs, risks, and complications. However, multiple studies have shown that the heart is, in fact, not an end-stage organ and that there are endogenous mechanisms in place that have the potential to spark regeneration. Stem cell therapy has emerged as a potential tool to tap into and activate this endogenous framework. Particularly promising are stem cells derived from cardiac tissue itself, referred to as cardiosphere-derived cells (CDCs). CDCs can be extracted and isolated from the patient's myocardium and then administered by intramyocardial injection or intracoronary infusion. After early success in the animal model, multiple clinical trials have demonstrated the safety and efficacy of autologous CDC therapy in humans. Clinical trials with allogeneic CDCs showed early promising results and pose a potential "off-the-shelf" therapy for patients in the acute setting after a myocardial infarction. The mechanism responsible for CDC-induced cardiac regeneration seems to be a combination of triggering native cardiomyocyte proliferation and recruitment of endogenous progenitor cells, which most prominently occurs via paracrine effects. A further understanding of the mediators involved in paracrine signaling can help with the development of a stem cell-free therapy, with all the benefits and none of the associated complications.
Pressure overload causes cardiac fibroblast activation and transdifferentiation, leading to increased interstitial fibrosis formation and subsequently myocardial stiffness, diastolic and systolic dysfunction, and eventually heart failure. A better understanding of the molecular mechanisms underlying pressure overload-induced cardiac remodeling and fibrosis will have implications for heart failure treatment strategies. The microRNA (miRNA)-221/222 family, consisting of miR-221-3p and miR-222-3p, is differentially regulated in mouse and human cardiac pathology and inversely associated with kidney and liver fibrosis. We investigated the role of this miRNA family during pressure overload-induced cardiac remodeling. In myocardial biopsies of patients with severe fibrosis and dilated cardiomyopathy or aortic stenosis, we found significantly lower miRNA-221/222 levels as compared to matched patients with nonsevere fibrosis. In addition, miRNA-221/222 levels in aortic stenosis patients correlated negatively with the extent of myocardial fibrosis and with left ventricular stiffness. Inhibition of both miRNAs during AngII (angiotensin II)-mediated pressure overload in mice led to increased fibrosis and aggravated left ventricular dilation and dysfunction. In rat cardiac fibroblasts, inhibition of miRNA-221/222 derepressed TGF-β (transforming growth factor-β)-mediated profibrotic SMAD2 (mothers against decapentaplegic homolog 2) signaling and downstream gene expression, whereas overexpression of both miRNAs blunted TGF-β-induced profibrotic signaling. We found that the miRNA-221/222 family may target several genes involved in TGF-β signaling, including JNK1 (c-Jun N-terminal kinase 1), TGF-β receptor 1 and TGF-β receptor 2, and ETS-1 (ETS proto-oncogene 1). Our findings show that heart failure-associated downregulation of the miRNA-221/222 family enables profibrotic signaling in the pressure-overloaded heart.
Introduction: Over the past decade, it has become clear that long-term engraftment of any ex vivo expanded cell product transplanted into injured myocardium is modest and all therapeutic regeneration is mediated by stimulation of endogenous repair rather than differentiation of transplanted cells into working myocardium. Given that increasing the retention of transplanted cells boosts myocardial function, focus on the fundamental mechanisms limiting retention and survival of transplanted cells may enable strategies to help to restore normal cardiac function.
Areas covered: This review outlines the challenges confronting cardiac engraftment of ex vivo expanded cells and explores means of enhancing cell-mediated repair of injured myocardium.
Expert opinion: Stem cell therapy has already come a long way in terms of regenerating damaged hearts though the poor retention of transplanted cells limits the full potential of truly cardiotrophic cell products. Multifaceted strategies directed towards fundamental mechanisms limiting the long-term survival of transplanted cells will be needed to enhance transplanted cell retention and cell-mediated repair of damaged myocardium for cardiac cell therapy to reach its full potential.
Mesenchymal stromal cells (MSCs) secrete paracrine factors that play crucial roles during tissue regeneration. Whether this paracrine function is influenced by the properties of biomaterials in general, and those used for cell delivery in particular, largely remains unexplored. Here, we investigated if three-dimensional culture in distinct microenvironments - nanoporous hydrogels (mean pore size ∼5 nm) and macroporous scaffolds (mean pore size ∼120 μm) - affects the secretion pattern of MSCs, and consequently leads to differential paracrine effects on target progenitor cells such as myoblasts. We report that compared to MSCs encapsulated in hydrogel, scaffold seeded MSCs show an enhanced secretion profile and exert beneficial paracrine effects on various myoblast functions including migration and proliferation. Additionally, we show that the heightened paracrine effects of scaffold seeded cells can in part be attributed to N-cadherin mediated cell-cell interactions during culture. In hydrogels, this physical interaction between cells is prevented by the encapsulating matrix. Functionally blocking N-cadherin negatively affected the secretion profile and paracrine effects of MSCs on myoblasts, with stronger effects observed for scaffold seeded compared to hydrogel encapsulated cells. Together, these findings demonstrate that the therapeutic potency of MSCs can be enhanced by biomaterials that promote cell-cell interactions.
The self-diffusion of neat water, dimethylsulfoxide (DMSO), octanol and the molecular components in a water-DMSO solution were measured by 1H and 2H NMR diffusion experiments for those fluids imbibed into Controlled Pore Glasses (CPG). Their highly interconnected structure is scaled by pore size and shows invariant pore topology independently of the size. The nominal pore diameter of the explored CPGs varied from 7.5 nm to 72.9 nm. Hence, the ∼μm mean-square diffusional displacement during the explored diffusion times was much larger than the individual pore size and the experiment yielded the average diffusion coefficient. Great care was taken to establish the actual pore volumes of the CPGs. Transverse relaxation experiments processed by Inverse Laplace Transformation were performed to verify that the liquids explored filled exactly the available pore volume. Relative to the respective diffusion coefficients obtained in bulk phases, we observe a reduction in the diffusion coefficient that is independent of pore size for the larger pores and becomes stronger towards the smaller pores. Geometric tortuosity governs the behavior at larger pore sizes while the interaction with pore walls becomes the dominant factor at our smallest pore diameter. Deviation from the trends predicted by the Renkin equation indicates that the interaction with the pore wall is not just simple steric one but is in part dependent on the specific features of the molecules explored here.
Statement of significance:
Tracking the dynamic behaviour of transplanted bone-marrow mononuclear cells (BM-MNCs) is a long-standing research goal. Conventional methods involving contrast and tracer agents interfere with cellular function while also yielding false signals. The use of bioluminescence addresses these shortcomings while allowing for real-time non-invasive tracking in vivo. Given the failures of transplanted BM-MNCs to engraft into injured tissue, biomaterial scaffolds capable of attracting and enhancing BM-MNC engraftment at sites of injury are highly sought in numerous tissue engineering applications. To this end, the results from this study demonstrate a new longitudinal tracking model that can non-invasively determine exogenous BM-MNC homing and engraftment to biomaterials, providing a valuable tool to inform the design of scaffolds with implications for countless tissue engineering applications.
The mechanism of cardiac hypertrophy involving microRNAs (miRNAs) is attracting increasing attention. Our study aimed to investigate the role of miR-10a in cardiac hypertrophy development and the underlying regulatory mechanism.
Transverse abdominal aortic constriction (TAAC) surgery was performed to establish a cardiac hypertrophy rat model, and angiotensin II (AngII) was used to induce cardiac hypertrophy in cultured neonatal rat cardiomyocytes. Expression of T-box 5 (TBX5) and miR-10a was altered by cell transfection of siRNA or miRNA mimic/inhibitor. Leucine incorporation assay, histological and cytological examination, quantitative real-time PCR (qRT-PCR), and Western blot were performed to detect the effects of miR-10a and TBX5 on cardiac hypertrophy. Dual-luciferase reporter assay was conducted to verify the regulation of TBX5 by miR-10a.
miR-10a was down-regulated, and TBX5 was up-regulated in the rat model and AngII-stimulated cardiomyocytes. miR-10a inhibited TBX5 expression by directly targeting the binding site in Tbx5 3’UTR. Overexpression of miR-10a in AngII-treated cardiomyocytes decreased relative cell area, and significantly reduced the mRNA levels of natriuretic peptide A (Nppa), myosin heavy chain 7 cardiac muscle beta (Myh7), and leucine incorporation (P < 0.01 or P < 0.001). Knockdown of Tbx5 had similar effects on AngII-induced cardiomyocytes.
Our findings indicate that miR-10a may inhibit cardiac hypertrophy via targeting Tbx5. Thus, miR-10a provides promising therapeutic strategies for the treatment of cardiac hypertrophy.
Existing techniques to encapsulate cells into microscale hydrogels generally yield high polymer-to-cell ratios and lack control over the hydrogel's mechanical properties. Here, we report a microfluidic-based method for encapsulating single cells in an approximately six-micrometre layer of alginate that increases the proportion of cell-containing microgels by a factor of ten, with encapsulation efficiencies over 90%. We show that in vitro cell viability was maintained over a three-day period, that the microgels are mechanically tractable, and that, for microscale cell assemblages of encapsulated marrow stromal cells cultured in microwells, osteogenic differentiation of encapsulated cells depends on gel stiffness and cell density. We also show that intravenous injection of singly encapsulated marrow stromal cells into mice delays clearance kinetics and sustains donor-derived soluble factors in vivo. The encapsulation of single cells in tunable hydrogels should find use in a variety of tissue engineering and regenerative medicine applications.
Regeneration of diseased tissue is one of the foremost concerns for millions of patients who suffer from tissue damage each year. Local delivery of cell-laden hydrogels offers an attractive approach for tissue repair. However, due to the typical macroscopic size of these cell constructs, the encapsulated cells often suffer from poor nutrient exchange. These issues can be mitigated by incorporating cells into microscopic hydrogels, or microgels, whose large surface-to-volume ratio promotes efficient mass transport and enhanced cell-matrix interactions. Using microfluidic technology, monodisperse cell-laden microgels with tunable sizes can be generated in a high-throughput manner, making them useful building blocks that can be assembled into tissue constructs with spatially controlled physicochemical properties. In this review, we examine microfluidics-generated cell-laden microgels for tissue regeneration applications. We provide a brief overview of the common biomaterials, gelation mechanisms, and microfluidic device designs that are used to generate these microgels, and summarize the most recent works on how they are applied to tissue regeneration. Finally, we discuss future applications of microfluidic cell-laden microgels as well as existing challenges that should be resolved to stimulate their clinical application.
In the past decade, substantial evidence supports the paradigm that stem cells exert their reparative and regenerative effects, in large part, through the release of biologically active molecules acting in a paracrine fashion on resident cells. The data suggest the existence of a tissue microenvironment where stem cell factors influence cell survival, inflammation, angiogenesis, repair, and regeneration in a temporal and spatial manner.
Rationale:
The let-7 family of microRNAs (miRs) regulates critical cell functions, including survival signaling, differentiation, metabolic control and glucose utilization. These functions may be important during myocardial ischemia. MiR-let-7 expression is under tight temporal and spatial control through multiple redundant mechanisms that may be stage-, isoform- and tissue-specific.
Objective:
To determine the mechanisms and functional consequences of miR-let-7 regulation by hypoxia in the heart.
Methods and results:
MiR-let-7a, -7c and -7g were downregulated in the adult mouse heart early after coronary occlusion, and in neonatal rat ventricular myocytes subjected to hypoxia. Let-7 repression did not require glucose depletion, and occurred at a post-transcriptional level. Hypoxia also induced the RNA binding protein Lin28, a negative regulator of let-7. Hypoxia induced neither Lin28 induction nor miR-let-7 repression in cardiac fibroblasts. Both changes were abrogated by treatment with the histone deacetylase inhibitor trichostatin A. Restoration of let-7g to hypoxic myocytes and to ischemia-reperfused mouse hearts in vivo via lentiviral transduction potentiated the hypoxia-induced phosphorylation and activation of Akt, and prevented hypoxia-dependent caspase activation and death. Mechanistically, phosphotidyl inositol 3'kinase interacting protein 1 (PIK3IP1), a negative regulator of PI3K, was identified as a novel target of miR-let-7 by a crosslinking technique showing that miR-let-7g specifically targets PI3KIP1 to the cardiac myocyte Argonaute complex RISC. Finally, in non-failing and failing human myocardium, we found specific inverse relationships between Lin28 and miR-let-7g, and between miR-let-7g and PIK3IP1.
Conclusion:
A conserved hypoxia-responsive Lin28-miR-let-7-PIK3IP1 regulatory axis is specific to cardiac myocytes and promotes apoptosis during myocardial ischemic injury.
Key points
Wnt signalling is activated in arrhythmogenic heart diseases, but its role in the regulation of cardiac ion channel expression is unknown.
Exposure of neonatal rat ventricular myocytes to Wnt3a, an activator of canonical Wnt signalling, decreases Scn5a mRNA, Na v 1.5 protein and Na ⁺ current density.
Wnt3a does not affect the inward rectifier K ⁺ current or L‐type Ca ²⁺ channels.
The Wnt pathway is a negative regulator of cardiac Na ⁺ channel expression and may play a role in altered ion channel expression in heart disease.
Abstract
Wnt signalling plays crucial roles in heart development, but is normally suppressed postnatally. In arrhythmogenic conditions, such as cardiac hypertrophy and heart failure, Wnt signalling is reactivated. To explore the potential role of Wnt signalling in arrhythmogenic electrical remodelling, we examined voltage‐dependent ion channels in cardiomyocytes. Treatment of neonatal rat ventricular myocytes with either recombinant Wnt3a protein or CHIR‐99021 (CHIR, a glycogen synthase kinase‐3β inhibitor) caused a dose‐dependent increase in Wnt target gene expression ( Axin2 and Lef1 ), indicating activation of the Wnt/β‐catenin pathway. Cardiac Na ⁺ current ( I Na ) density was reduced by Wnt3a (−20 ± 4 vs . control −59 ± 7 pA pF ⁻¹ , at −30 mV) or CHIR (−22 ± 5 pA pF ⁻¹ ), without changes in steady‐state activation, inactivation or repriming kinetics. Wnt3a and CHIR also produced dose‐dependent reductions in the mRNA level of Scn5a (the cardiac Na ⁺ channel α subunit gene), as well as a 56% reduction (by Wnt3a) in the Na v 1.5 protein level. Consistent with I Na reduction, action potentials in Wnt3a‐treated neonatal rat ventricular myocytes had a lower upstroke amplitude (91 ± 3 vs . control 137 ± 2 mV) and decreased maximum upstroke velocity (70 ± 10 vs . control 163 ± 15 V s ⁻¹ ). In contrast, inward rectifier K ⁺ current and L‐type Ca ²⁺ channels were not affected by Wnt3a treatment. Taken together, our data indicate that the Wnt/β‐catenin pathway suppresses I Na in postnatal cardiomyocytes and may contribute to ion channel remodelling in heart disease.
Over the past 2 decades, there have been numerous stem cell studies focused on cardiac diseases, ranging from proof-of-concept to phase 2 trials. This series of papers focuses on the legacy of these studies and the outlook for future treatment of cardiac diseases with stem cell therapies. The first section by Drs. Rosen and Myerburg is an independent review that analyzes the basic science and translational strategies supporting the rapid advance of stem cell technology to the clinic, the philosophies behind them, trial designs, and means for going forward that may impact favorably on progress. The second and third sections were collected as responses to the initial section of this review. The commentary by Drs. Francis and Cole discusses the review by Drs. Rosen and Myerburg and details how trial outcomes can be affected by noise, poor trial design (particularly the absence of blinding), and normal human tendencies toward optimism and denial. The final, independent paper by Dr. Marbán takes a different perspective concerning the potential for positive impact of stem cell research applied to heart disease and future prospects for its clinical application. (Compiled by the JACC editors)