Human Adult Stem Cells Maintain a Constant Phenotype Profile Irrespective of Their Origin, Basal Media, and Long Term Cultures

The study aims to identify the phenotypic marker expressions of different human adult stem cells derived from, namely, bone marrow, subcutaneous fat, and omentum fat, cultured in different media, namely, DMEM-Low Glucose, Alpha-MEM, DMEM-F12 and DMEM-KO and under long term culture conditions (>P20). We characterized immunophenotype by using various hematopoietic, mesenchymal, endothelial markers, and cell adhesion molecules in the long term cultures (Passages-P1, P3, P5, P9, P12, P15, and P20.) Interestingly, data revealed similar marker expression profiles irrespective of source, basal media, and extensive culturing. This demonstrates that all adult stem cell sources mentioned in this study share similar phenotypic marker and all media seem appropriate for culturing these sources. However, a disparity was observed in the markers such as CD49d, CD54, CD117, CD29, and CD106, thereby warranting further research on these markers. Besides the aforesaid objective, it is understood from the study that immunophenotyping acts as a valuable tool to identify inherent property of each cell, thereby leading to a valuable cell based therapy

Enhanced Cardiac Regenerative Ability of Stem Cells After Ischemia-Reperfusion Injury Role of Human CD34+ Cells Deficient in MicroRNA-377

AbstractBackgroundMicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress.ObjectivesThe aim of this study was to explore the role of miR in human CD34+ cell (hCD34+) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions.MethodsIn response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction–based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34+ cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice.ResultsThe miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34+ cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control–transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34+ cells in immunodeficient mice, promoting neovascularization (at 28 days, post–I-R) and lower interstitial fibrosis, leading to improved left ventricular function.ConclusionsThese findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34+ cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis

Spaceflight-Associated Changes of snoRNAs in Peripheral Blood Mononuclear Cells and Plasma Exosomes—A Pilot Study

During spaceflight, astronauts are exposed to various physiological and psychological stressors that have been associated with adverse health effects. Therefore, there is an unmet need to develop novel diagnostic tools to predict early alterations in astronauts’ health. Small nucleolar RNA (snoRNA) is a type of short non-coding RNA (60–300 nucleotides) known to guide 2′-O-methylation (Nm) or pseudouridine (ψ) of ribosomal RNA (rRNA), small nuclear RNA (snRNA), or messenger RNA (mRNA). Emerging evidence suggests that dysregulated snoRNAs may be key players in regulating fundamental cellular mechanisms and in the pathogenesis of cancer, heart, and neurological disease. Therefore, we sought to determine whether the spaceflight-induced snoRNA changes in astronaut’s peripheral blood (PB) plasma extracellular vesicles (PB-EV) and peripheral blood mononuclear cells (PBMCs). Using unbiased small RNA sequencing (sRNAseq), we evaluated changes in PB-EV snoRNA content isolated from astronauts (n = 5/group) who underwent median 12-day long Shuttle missions between 1998 and 2001. Using stringent cutoff (fold change > 2 or log(2)-fold change >1, FDR < 0.05), we detected 21 down-and 9—up-regulated snoRNAs in PB-EVs 3 days after return (R + 3) compared to 10 days before launch (L-10). qPCR validation revealed that SNORA74A was significantly down-regulated at R + 3 compared to L-10. We next determined snoRNA expression levels in astronauts’ PBMCs at R + 3 and L-10 (n = 6/group). qPCR analysis further confirmed a significant increase in SNORA19 and SNORA47 in astronauts’ PBMCs at R + 3 compared to L-10. Notably, many downregulated snoRNA-guided rRNA modifications, including four Nms and five ψs. Our findings revealed that spaceflight induced changes in PB-EV and PBMCs snoRNA expression, thus suggesting snoRNAs may serve as potential novel biomarkers for monitoring astronauts’ health

Transcriptome wide changes in long noncoding RNAs in diabetic ischemic heart disease

More than 10% of adults in the United States have type 2 diabetes mellitus (DM) with a 2-4 times higher prevalence of ischemic heart disease than the non-diabetics. Despite extensive research approaches to limit this life-threatening condition have proven unsuccessful, highlighting the need for understanding underlying molecular mechanisms. Long noncoding RNAs (lncRNAs), which regulate gene expression by acting as signals, decoys, guides, or scaffolds have been implicated in diverse cardiovascular conditions. However, their role in ischemic heart disease in DM remains poorly understood. We provide new insights into the lncRNA expression profile after ischemic heart disease in DM mice. We performed unbiased RNA sequencing of well-characterized type 2 DM model db/db mice or its control db/+ subjected to sham or MI surgery. Computational analysis of the RNA sequencing of these LV tissues identified several differentially expressed lncRNAs between (db/db sham vs. db/db MI) including Gm19522 and Gm8075. lncRNA Gm-19522 may regulate DNA replication via DNA protein kinases, while lncRNA Gm-8075 is associated with cancer gene dysregulation and PI3K/Akt pathways. Thus, the downregulation of lncRNAs Gm19522 and Gm8075 post-MI may serve as potential biomarkers or novel therapeutic targets to improve cardiac repair/recovery in diabetic ischemic heart disease.</p

Astronauts Plasma-Derived Exosomes Induced Aberrant EZH2-Mediated H3K27me3 Epigenetic Regulation of the Vitamin D Receptor

There are unique stressors in the spaceflight environment. Exposure to such stressors may be associated with adverse effects on astronauts\u27 health, including increased cancer and cardiovascular disease risks. Small extracellular vesicles (sEVs, i.e., exosomes) play a vital role in intercellular communication and regulate various biological processes contributing to their role in disease pathogenesis. To assess whether spaceflight alters sEVs transcriptome profile, sEVs were isolated from the blood plasma of 3 astronauts at two different time points: 10 days before launch (L-10) and 3 days after return (R+3) from the Shuttle mission. AC16 cells (human cardiomyocyte cell line) were treated with L-10 and R+3 astronauts-derived exosomes for 24 h. Total RNA was isolated and analyzed for gene expression profiling using Affymetrix microarrays. Enrichment analysis was performed using Enrichr. Transcription factor (TF) enrichment analysis using the ENCODE/ChEA Consensus TF database identified gene sets related to the polycomb repressive complex 2 (PRC2) and Vitamin D receptor (VDR) in AC16 cells treated with R+3 compared to cells treated with L-10 astronauts-derived exosomes. Further analysis of the histone modifications using datasets from the Roadmap Epigenomics Project confirmed enrichment in gene sets related to the H3K27me3 repressive mark. Interestingly, analysis of previously published H3K27me3-chromatin immunoprecipitation sequencing (ChIP-Seq) ENCODE datasets showed enrichment of H3K27me3 in the VDR promoter. Collectively, our results suggest that astronaut-derived sEVs may epigenetically repress the expression of the VDR in human adult cardiomyocytes by promoting the activation of the PRC2 complex and H3K27me3 levels

Enhanced Cardiac Regenerative Ability of Stem Cells After Ischemia-Reperfusion Injury Role of Human CD34+ Cells Deficient in MicroRNA-377

AbstractBackgroundMicroRNA (miR) dysregulation in the myocardium has been implicated in cardiac remodeling after injury or stress.ObjectivesThe aim of this study was to explore the role of miR in human CD34+ cell (hCD34+) dysfunction in vivo after transplantation into the myocardium under ischemia-reperfusion (I-R) conditions.MethodsIn response to inflammatory stimuli, the miR array profile of endothelial progenitor cells was analyzed using a polymerase chain reaction–based miR microarray. miR-377 expression was assessed in myocardial tissue from human patients with heart failure (HF). We investigated the effect of miR-377 inhibition on an hCD34+ cell angiogenic proteome profile in vitro and on cardiac repair and function after I-R injury in immunodeficient mice.ResultsThe miR array data from endothelial progenitor cells in response to inflammatory stimuli indicated changes in numerous miR, with a robust decrease in the levels of miR-377. Human cardiac biopsies from patients with HF showed significant increases in miR-377 expression compared with nonfailing control hearts. The proteome profile of hCD34+ cells transfected with miR-377 mimics showed significant decrease in the levels of proangiogenic proteins versus nonspecific control–transfected cells. We also validated that serine/threonine kinase 35 is a target of miR-377 using a dual luciferase reporter assay. In a mouse model of myocardial I-R, intramyocardial transplantation of miR-377 silenced hCD34+ cells in immunodeficient mice, promoting neovascularization (at 28 days, post–I-R) and lower interstitial fibrosis, leading to improved left ventricular function.ConclusionsThese findings indicate that HF increased miR-377 expression in the myocardium, which is detrimental to stem cell function, and transplantation of miR-377 knockdown hCD34+ cells into ischemic myocardium promoted their angiogenic ability, attenuating left ventricular remodeling and cardiac fibrosis

Integrated single-cell RNA-seq analysis reveals mitochondrial calcium signaling as a modulator of endothelial-to-mesenchymal transition

Endothelial cells (ECs) are highly plastic, capable of differentiating into various cell types. Endothelial-to-mesenchymal transition (EndMT) is crucial during embryonic development and contributes substantially to vascular dysfunction in many cardiovascular diseases (CVDs). While targeting EndMT holds therapeutic promise, understanding its mechanisms and modulating its pathways remain challenging. Using single-cell RNA sequencing on three in vitro EndMT models, we identified conserved gene signatures. We validated original regulators in vitro and in vivo during embryonic heart development and peripheral artery disease. EndMT induction led to global expression changes in all EC subtypes rather than in mesenchymal clusters. We identified mitochondrial calcium uptake as a key driver of EndMT; inhibiting mitochondrial calcium uniporter (MCU) prevented EndMT in vitro, and conditional Mcu deletion in ECs blocked mesenchymal activation in a hind limb ischemia model. Tissues from patients with critical limb ischemia with EndMT features exhibited significantly elevated endothelial MCU. These findings highlight MCU as a regulator of EndMT and a potential therapeutic target

Establishment of a rat model of myocardial infarction with a high survival rate: A suitable model for evaluation of efficacy of stem cell therapy

The most common rat model of myocardial infarction (MI) is by ligation of left anterior descending (LAD) coronary artery but it is associated with high mortality and large variations in the infarct size. We evolved certain innovations/modifications in the existing technique including immobilization of the heart without exteriorization, identification of the LAD by pressing it proximal to the site of ligation by an ear-bud, and subsequently its ligation 8 mm from its origin, no touch technique of the lungs during surgery, removal of air from the chest cavity prior to its closure using an in-house tubing, and deflation of the lungs before extubation. We induced MI in 24 Sprague- Dawley (SD) rats using these modifications and carried out post-MI evaluation of hemodynamic parameters, serum cardiac enzymes and histological studies upto 90 days using 13 sham operated and 3 healthy SD rats as controls. Three of the 24 rats (13%) died <24 hours of MI, but thereafter no mortality was observed till the follow-up period of 90 days. The infarct size was consistent in all the rats (21±4% of left ventricular area).? This model with low early and no long-term mortality may be suitable for studying efficacy of stem cell therapy in MI, where a follow-up of at least 13 weeks is required to assess myocardial regeneration

Elucidating the Functions of Non-Coding RNAs from the Perspective of RNA Modifications

It is now commonly accepted that most of the mammalian genome is transcribed as RNA, yet less than 2% of such RNA encode for proteins. A majority of transcribed RNA exists as non-protein-coding RNAs (ncRNAs) with various functions. Because of the lack of sequence homologies among most ncRNAs species, it is difficult to infer the potential functions of ncRNAs by examining sequence patterns, such as catalytic domains, as in the case of proteins. Added to the existing complexity of predicting the functions of the ever-growing number of ncRNAs, increasing evidence suggests that various enzymes modify ncRNAs (e.g., ADARs, METTL3, and METTL14), which has opened up a new field of study called epitranscriptomics. Here, we examine the current status of ncRNA research from the perspective of epitranscriptomics.</jats:p