Biology subjects

Sevlever, G.

Publications and source records attributed to Sevlever, G..

5 recordsLinked to original sources

bioRxiv2025

Metabolic Maturation Unveils Left Ventricular Identity in WNT ON/OFF Human Pluripotent Stem Cell-Derived Cardiomyocytes

Deriving high-purity mature left ventricular (LV) cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) is a priority for cardiovascular research and for future therapeutic applications. Small-molecule WNT modulation (WNT ON/OFF) is currently the predominant differentiation method; however, a critical discordance exists regarding its cardiac subtype outcome. While lineage tracing suggests a First Heart Field (FHF) bias, phenotypic characterizations report significant heterogeneity regarding definitive ventricular markers, leading to controversy about the cardiac subtypes generated by this method. Here, we demonstrate that this apparent heterogeneity is a result of CM immaturity. Using single-cell protein analysis, we first show that WNT ON/OFF generates an NKX2.5+ progenitor pool that robustly co-expresses HAND1, confirming uniform FHF specification regardless of differentiation efficiency. We then demonstrate that the CM population negative for the ventricular marker MYL2 observed at early differentiation timepoints mostly represents immature LV cardiomyocytes that have not yet acquired their definitive phenotype. By implementing a targeted metabolic maturation regime, we unlocked this identity, achieving 95% MYL2+/HAND1+/TBX5+ LV CMs by day 38, substantially earlier and with higher chamber-specific purity than previously reported. This phenotypic resolution was accompanied by advanced structural maturation, including sarcomeric protein isoform switching, multinucleation, and notably, the assembly of polarized XIRP2+ intercalated discs, a hallmark of postnatal CM maturation not previously described in 2D differentiations. Validated across three independent hPSC lines, these findings provide the field with a rapid, high-fidelity platform for generating pure mature LV cardiomyocytes for disease modeling and therapeutic research.

developmental biology↗

bioRxiv2024

SHEN-LONG, a novel cardiac regulatory locus, regulates cardiac master transcription factor NKX2-5 in pluripotent stem cell derived cardiomyocytes

Cardiac development is a finely regulated process, transforming undifferentiated cells into the specialized cell components of the heart. Super-Enhancers (SE), clusters of enhancers densely populated with transcription factors, play a central role in cell fate decisions, including cardiogenesis. In this work, we studied a cardiac SE in chr3q25.31. This locus also contains a cardiac specific long non-coding RNA: LINC00881 (LINC881). We termed this region of interest SHEN-LONG (Super Heart ENhancer and LONG non-coding LINC881), and selected it for further functional characterization. Our analysis revealed SHEN-LONG is a hotspot of cardiac kernel transcription factor binding sites. Cardiac differentiation of human iPSC with partial Knock Out of SHEN-LONG resulted in cardiomyocytes with reduced NKX2-5 expression, a master cardiac transcription factor, suggesting a significant role of SHEN-LONG in this process. Whole transcriptome sequencing of SHEN-LONG KO cardiomyocytes produced 134 differentially expressed genes located at great distances (>4Mb) emphasizing its long-range functional impact. A regulation mediated essentially by the SE and not by LINC881 was confirmed when LINC881 overexpression did not recover NKX2-5 expression. These findings provide insights into a novel NKX2-5 regulatory mechanism. The knowledge gained in this work may pave the way for advances in therapeutic interventions for cardiovascular disorders.

molecular biology↗

bioRxiv2021

Small extracellular vesicles from iPSC-MSC lose their regenerative potential upon UV-C irradiation

Mesenchymal Stem Cells derived from induced Pluripotent Stem cells (iPSC-MSC) have become a promising alternative to classical Mesenchymal Stem Cells in regenerative medicine. Their properties -as immunomodulatory and regenerative capacities-are in part due to the secretion of Extracellular Vesicles (EVs). Small EVs (sEVs) with sizes that range from 50 to 120 nm contain proteins, lipids, and nucleic acids that exert a role in cellular communication. Their content will depend on the cell of origin and its physiological state, thus the message they convey might change in response to changes in cellular conditions. In particular, the DNA damage response (DDR) has been reported to modulate sEVs secretion. In this work, we analyze how UV-C radiation upon iPSC-MSC alter sEVs secretion, cargo and bystander effect. Here, we confirm that UV-C radiation causes DDR in a dose dependent manner. In addition, we found that UV-C induced stress did not modulate the expression of genes that participate in sEVs biogenesis pathway. Consequently, we found that the amount of sEVs secreted by radiated and non-irradiated cells remained stable. However, sEVs from radiated cells were unable to promote cell migration in their target cells. Moreover, a label-free proteomic analysis revealed that UV-C induced DDR produces sEVs with an altered cargo, rich in migration-inhibiting proteins, and resulting in a less stromal-oriented repertoire.

cell biology↗

bioRxiv2020

celldeath: a tool for simple detection of cell death in transmitted light microscopy images by visual deep learning analysis

Cell death experiments are routinely done in many labs around the world, these experiments are the backbone of many assays for drug development. Cell death detection is usually performed in many ways, and requires time and reagents. However, cell death is preceded by slight morphological changes in cell shape and texture. In this paper, we trained a neural network to classify cells undergoing cell death. We found that the network was able to highly predict cell death after one hour of exposure to camptothecin. Moreover, this prediction largely outperforms human ability. Finally, we provide a simple python tool that can broadly be used to detect cell death.

cell biology↗

bioRxiv2019

Analysis of the expression of PIWI-interacting RNAs during cardiac differentiation of human pluripotent stem cells

PIWI-interacting RNAs (piRNAs) are a class of non-coding RNAs initially thought to be restricted almost exclusively to germ line cells. In recent years, accumulating evidence has demonstrated that piRNAs are actually expressed in somatic cells like pluripotent, neural, cardiac and even cancer cells. However, controversy still remains around the existence and function of somatic piRNAs. Using small RNA-seq samples from H9 pluripotent stem cells differentiated to mesoderm progenitors and cardiomyocytes we identified the expression of 447 piRNAs, of which 241 were detected in pluripotency, 218 in mesoderm and 171 in cardiac cells. The majority of them originated from the sense strand of protein coding and lncRNAs genes in all stages of differentiation, though no evidences for secondary piRNAs (ping-pong loop) were found. Genes hosting piRNAs in cardiac samples were related to critical biological processes in the heart, like contraction and cardiac muscle development. Our results indicate that somatic piRNAs might have a role in fine-tuning the expression of genes involved in the differentiation of pluripotent cells to cardiomyocytes.

molecular biology↗