MSC-Derived Small Extracellular Vesicles Exert Cardioprotective Effect Through Reducing VLCFAs and Apoptosis in Human Cardiac Organoid IRI Model.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 31% of all deaths globally. Myocardial ischemia-reperfusion injury (IRI), a common complication of CVDs, is a major cause of mortality and morbidity. Studies have shown efficacious use of mesenchymal stem cells-derived small extracellular vesicles (MSCs-EVs) to mitigate IRI in animals, but few research has been done on human-related models. In this study, human embryonic stem cell-derived chambered cardiac organoid (CCO) was used as a model system to study the effects of MSC-EVs on myocardial IRI. The results revealed that MSC-EVs treatment reduced apoptosis and improved contraction resumption of the CCOs. Metabolomics analysis showed that this effect could be attributed to EVs' ability to prevent the accumulation of unsaturated very long-chain fatty acids (VLCFAs). This was corroborated when inhibition of fatty acid synthase, which was reported to reduce VLCFAs, produced a similar protective effect to EVs. Overall, this study uncovered the mechanistic role of MSC-EVs in mitigating IRI that involves preventing the accumulation of unsaturated VLCFA, decreasing cell death, and improving contraction resumption in CCOs.

JAK2 as a surface marker for enrichment of human pluripotent stem cells-derived ventricular cardiomyocytes.

BACKGROUND: Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for cardiac disease modelling, drug discovery and regenerative medicine. Despite the advancement in various differentiation protocols, the heterogeneity of the generated population composed of diverse cardiac subtypes poses a significant challenge to their practical applications. Mixed populations of cardiac subtypes can compromise disease modelling and drug discovery, while transplanting them may lead to undesired arrhythmias as they may not integrate and synchronize with the host tissue's contractility. It is therefore crucial to identify cell surface markers that could enable high purity of ventricular CMs for subsequent applications. METHODS: By exploiting the fact that immature CMs expressing myosin light chain 2A (MLC2A) will gradually express myosin light chain 2 V (MLC2V) protein as they mature towards ventricular fate, we isolated signal regulatory protein alpha (SIRPA)-positive CMs expressing intracellular MLC2A or MLC2V using MARIS (method for analysing RNA following intracellular sorting). Subsequently, RNA sequencing analysis was performed to examine the gene expression profile of MLC2A + and MLC2V + sorted CMs. We identified genes that were significantly up-regulated in MLC2V + samples to be potential surface marker candidates for ventricular specification. To validate these surface markers, we performed immunostaining and western blot analysis to measure MLC2A and MLC2V protein expressions in SIRPA + CMs that were either positive or negative for the putative surface markers, JAK2 (Janus kinase 2) or CD200. We then characterized the electrophysiological properties of surface marker-sorted CMs, using fluo-4 AM, a green-fluorescent calcium indicator, to measure the cellular calcium transient at the single cell level. For functional validation, we investigated the response of the surface marker-sorted CMs to vernakalant, an atrial-selective anti-arrhythmic agent. RESULTS: In this study, while JAK2 and CD200 were identified as potential surface markers for the purification of ventricular-like CMs, the SIRPA+/JAK2+ population showed a higher percentage of MLC2V-expressing cells (~ 90%) compared to SIRPA+/CD200+ population (~ 75%). SIRPA+/JAK2+ sorted CMs exhibited ventricular-like electrophysiological properties, including slower beating rate, slower calcium depolarization and longer calcium repolarization duration. Importantly, vernakalant had limited to no significant effect on the calcium repolarization duration of SIRPA+/JAK2+ population, indicating their enrichment for ventricular-like CMs. CONCLUSION: Our study lays the groundwork for the identification of cardiac subtype surface markers that allow purification of cardiomyocyte sub-populations. Our findings suggest that JAK2 can be employed as a cell surface marker for enrichment of hPSC-derived ventricular-like CMs.

Upregulation of the JAK-STAT pathway promotes maturation of human embryonic stem cell-derived cardiomyocytes.

The immature characteristics and metabolic phenotypes of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) restrict their applications for disease modeling, drug discovery, and cell-based therapy. Leveraging on the metabolic shifts from glycolysis to fatty acid oxidation as CMs mature, a human hexokinase1-GFP metabolic reporter cell line (H7 HK1-GFP) was generated to facilitate the isolation of fetal or more matured hPSC-CMs. RNA sequencing of fetal versus more matured CMs uncovered a potential role of interferon-signaling pathway in regulating CM maturation. Indeed, IFN-γ-treated CMs resulted in an upregulation of the JAK-STAT pathway, which was found to be associated with increased expression of CM maturation genes, shift from MYH6 to MYH7 expression, and improved sarcomeric structure. Functionally, IFN-γ-treated CMs exhibited a more matured electrophysiological profile, such as increased calcium dynamics and action potential upstroke velocity, demonstrated through calcium imaging and MEA. Expectedly, the functional improvements were nullified with a JAK-STAT inhibitor, ruxolitinib.

Mending a broken heart: current strategies and limitations of cell-based therapy.

The versatility of pluripotent stem cells, attributable to their unlimited self-renewal capacity and plasticity, has sparked a considerable interest for potential application in regenerative medicine. Over the past decade, the concept of replenishing the lost cardiomyocytes, the crux of the matter in ischemic heart disease, with pluripotent stem cell-derived cardiomyocytes (PSC-CM) has been validated with promising pre-clinical results. Nevertheless, clinical translation was hemmed in by limitations such as immature cardiac properties, long-term engraftment, graft-associated arrhythmias, immunogenicity, and risk of tumorigenicity. The continuous progress of stem cell-based cardiac therapy, incorporated with tissue engineering strategies and delivery of cardio-protective exosomes, provides an optimistic outlook on the development of curative treatment for heart failure. This review provides an overview and current status of stem cell-based therapy for heart regeneration, with particular focus on the use of PSC-CM. In addition, we also highlight the associated challenges in clinical application and discuss the potential strategies in developing successful cardiac-regenerative therapy.

MicroRNA-493-5p-mediated repression of the MYCN oncogene inhibits hepatic cancer cell growth and invasion.

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer-related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR-493-5p, a major tumor suppressor miRNA that inactivates miR-483-3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR-493-5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR-493-5p given its significant inhibition through 3'-UTR targeting in miR-493-5p-rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR-493-5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR-493-5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR-493-5p activity. In summary, miR-493-5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.

MicroRNA-124a inhibits endoderm lineage commitment by targeting Sox17 and Gata6 in mouse embryonic stem cells.

The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, via miRNA profiling during endoderm differentiation, we discovered that miR-124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). To further investigate the functional role of miR-124a in early stages of differentiation, transfection of embryoid bodies with miR-124a mimic was performed. We showed that overexpression of miR-124a inhibits endoderm differentiation in vitro through targeting the 3'-untranslated region (UTR) of Sox17 and Gata6, revealing the existence of interplay between miR-124a and the Sox17/Gata6 transcription factors in hepato-specific gene regulation. In addition, we presented a feasible in vivo system that utilizes teratoma and gene expression profiling from microarray to quantitatively evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR-124a in teratomas by intratumor delivery of miR-124a mimic and Atelocollagen, significantly suppressed endoderm and mesoderm lineage differentiation while augmenting the differentiation into ectoderm lineage. Collectively, our findings suggest that miR-124a plays a significant role in mESCs lineage commitment.

MEG3-derived miR-493-5p overcomes the oncogenic feature of IGF2-miR-483 loss of imprinting in hepatic cancer cells.

Numerous studies have described the critical role played by microRNAs (miRNAs) in cancer progression and the potential of these small non-coding RNAs for diagnostic or therapeutic applications. However, the mechanisms responsible for the altered expression of miRNAs in malignant cells remain poorly understood. Herein, via epigenetic unmasking, we identified a group of miRNAs located in the imprinted delta like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3 (MEG3) locus that were repressed in hepatic tumor cells. Notably, miR-493-5p epigenetic silencing was correlated with hypermethylation of the MEG3 differentially regulated region (DMR) in liver cancer cell lines and tumor tissues from patients. Experimental rescue of miR-493-5p promoted an anti-cancer response by hindering hepatocellular carcinoma (HCC) cell growth in vitro and tumor progression in vivo. We found that miR-493-5p mediated part of its tumor-suppressor activity by abrogating overexpression of insulin-like growth factor 2 (IGF2) and the IGF2-derived intronic oncomir miR-483-3p in HCC cells characterized by IGF2 loss of imprinting (LOI). In summary, this study describes an unknown miRNA-dependent regulatory mechanism between two distinct imprinted loci and a possible therapeutic window for liver cancer patients exhibiting IGF2-miR-483 LOI and amplification.

Differentiation Therapy by Epigenetic Reconditioning Exerts Antitumor Effects on Liver Cancer Cells.

Primary liver tumors are mainly represented by hepatocellular carcinoma (HCC), one of the most aggressive and resistant forms of cancer. Liver tumorigenesis is characterized by an accumulation of epigenetic abnormalities, leading to gene extinction and loss of hepatocyte differentiation. The aim of this work was to investigate the feasibility of converting liver cancer cells toward a less aggressive and differentiated phenotype using a process called epigenetic reconditioning. Here, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine (5-AZA) promoted an anti-cancer response by inhibiting HCC cell tumorigenicity. Furthermore, epigenetic reconditioning improved sorafenib response. Remarkably, epigenetic treatment was associated with a significant restoration of differentiation, as attested by the increased expression of characteristic hepatocyte markers in reconditioned cells. In particular, we showed that reexpression of these epigenetically silenced liver genes following 5-AZA treatment or after knockdown of DNA methyltransferase 1 (DNMT1) was the result of regional CpG demethylation. Lastly, we confirmed the efficacy of HCC differentiation therapy by epigenetic reconditioning using an in vivo tumor growth model. In summary, this work demonstrates that epigenetic reconditioning using the demethylating compound 5-AZA shows therapeutic significance for liver cancer and is potentially attractive for the treatment of solid tumors.

Epigenetic reprogramming using 5-azacytidine promotes an anti-cancer response in pancreatic adenocarcinoma cells.

Curative management of pancreatic adenocarcinoma is limited because this malignancy remains resistant to most chemotherapeutic drugs. Strategies that reverse epigenetic alterations offer a unique opportunity for cancer cell reprogramming, which is valuable for development of new treatments. The aim of this work was to reprogram pancreatic ductal adenocarcinoma (PDAC) cells toward a less aggressive and drug-responsive phenotype. The process applied is called "epigenetic reprogramming". To evaluate the efficiency of PDAC epigenetic reprogramming, we assessed tumor growth and drug response in PANC-1 cells after exposure to non-cytotoxic doses of the demethylating agent 5-azacytidine (5-AZA). Here, we showed that an epigenetic regimen using 5-AZA promoted an anti-cancer response by inhibiting PDAC tumor growth in vivo after the engraftment of treated cells. Remarkably, the subsequent addition of gemcitabine (GEM) to the 5-AZA-mediated reprogramming resulted in a marked growth inhibition effect in GEM-resistant pancreatic cancer cells. We observed that various characteristic peptides expressed in the pancreas, which included the antiproliferative hormone somatostatin (SST) and the SST receptor 2 (SSTR2), were significantly upregulated in the epigenetically reprogrammed PDAC cells. The inhibitory effect of octreotide (OCT), an SST analog, was tested on PDAC cells and found to be improved after cell reprogramming. Furthermore, we found that SST gene expression restoration following 5-AZA treatment or following knockdown of the DNA methyltransferase (DNMT) 1 enzyme was associated with the reversion of SST epigenetic silencing through regional CpG demethylation. Lastly, we confirmed the efficacy of 5-AZA-based epigenetic reprogramming in vivo using a PDAC tumor growth model. In conclusion, this study demonstrates that epigenetic reprogramming using the demethylating compound 5-AZA shows anti-cancer effects in PANC-1 cells and is potentially attractive for the treatment of solid tumors.

Mesenchymal stem cell-derived extracellular vesicles: a glimmer of hope in treating Alzheimer's disease.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular plaques resulting from the accumulation of beta-amyloid peptide (Aß). To date, a definitive cure for this disease is still lacking as the currently approved drugs used are mainly symptomatic treatments. The revolutionary discovery of extracellular vesicles (EVs) has shed new light on the development of disease-modifying treatments for AD, owing to their potential in delivering the therapeutic agents to the brain. The feasibility of harnessing EVs for clinical applications is highly dependent on the donor cell, which determines the intrinsic properties of EVs. The merit of mesenchymal stem cells (MSCs) as therapeutic delivery vehicles, and the proven therapeutic effects of the EVs derived from these cells, make researchers esteem MSCs as ideal producers of EVs. Therefore, MSC-derived EVs (MSC-EVs) emerge to be an appealing therapeutic delivery approach for the treatment of AD. Here, we discuss perspectives on the therapeutic strategies using MSC-EVs to treat AD and the associated challenges in clinical application.

Preliminary study on overproduction of reactive oxygen species by neutrophils in diabetes mellitus.

AIM: To assess the amount and pattern of reactive oxygen species (ROS) production in diabetic patient-derived neutrophils. METHODS: Blood samples from type 2 diabetes mellitus (DM) patients and volunteers (controls) were subjected to neutrophil isolation and the assessment of neutrophil oxidative burst using chemiluminescence assay. Neutrophils were activated by using phorbol myristate acetate (PMA) and neutrophils without activation were kept as a negative control. The chemiluminescence readings were obtained by transferring cell suspension into a 1.5 mL Eppendorf tube, with PMA and luminol. Reaction mixtures were gently vortexed and placed inside luminometer for a duration of 5 min. RESULTS: Our results showed that in the resting condition, the secretion of ROS in normal non-diabetic individuals was relatively low compared to diabetic patients. However, the time scale observation revealed that the secreted ROS declined accordingly with time in non-diabetic individuals, yet such a reduction was not detected in diabetic patients where at all the time points, the secretion of ROS was maintained at similar magnitudes. This preliminary study demonstrated that ROS production was significantly higher in patients with DM compared to non-diabetic subjects in both resting and activated conditions. CONCLUSION: The respiratory burst activity of neutrophils could be affected by DM and the elevation of ROS production might be an aggravating factor in diabetic-related complications.