Biology subjects

Amin, G.

Publications and source records attributed to Amin, 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

Nicotinamide riboside: A promising therapy for MI-induced acute kidney injury by upregulating nicotinamide phosphoribosyltransferase-mediated NAD levels

BackgroundCardiorenal syndrome (CRS) type 1 is characterized by the development of acute kidney injury (AKI) following acute cardiac illness and notably acute myocardial infarction (MI). AKI is considered an independent risk factor increasing mortality rate substantially. Nicotinamide dinucleotide (NAD) is an important coenzyme in energy metabolism and oxidative phosphorylation and in its oxidized form, a substrate for multiple NAD+-dependent enzymes such as Sirtuins and poly-ADP ribose polymerases. Decreased cardiac NAD levels along with a down-regulation of the nicotinamide phosphoribosyl transferase (NAMPT) have been reported following MI. A compensatory upregulation in nicotinamide riboside kinase (NMRK) 2, an NAD+ biosynthetic enzyme that uses nicotinamide riboside (NR) to generate NAD+ takes place in the heart after MI but the impact on kidney NAD metabolism and function has not been addressed before. MethodsMI was induced by ligating the left anterior descending coronary artery in 2 months old C57BL6/J mice, followed by the administration of NR (IP injection, 400mg/kg/day) for four and seven days. We hypothesized that NR treatment could be a potential promising therapy for MI-induced AKI. ResultsOur findings showed no significant improvement in cardiac ejection fraction following NR treatment at days 4 and 7 post-MI, whereas kidney functions were enhanced and morphological alterations and cell death decreased. The observed renal protection seems to be mediated by an up-regulation of NAMPT-mediated increase in renal NAD levels, notably in distal tubules. ConclusionOur findings indicate that NR could be a potential promising therapy for AKI following an early stage of MI.

pharmacology and toxicology↗

bioRxiv2023

The transcription factor OCT6 promotes the dissolution of the naive pluripotent state by repressing Nanog and activating a formative state gene regulatory network.

Animal development relies on complex gene regulatory networks (GRNs) that govern the nearly irreversible changes that occur during cell differentiation. In this work we aimed to determine key transcription factors (TFs) associated with the dissolution of the naive pluripotent state and the acquisition of a formative identity. We identified OCT6 as one of the earliest TFs induced during the onset of mouse embryonic stem cell (mESCs) differentiation. To investigate its role, we generated an Oct6 knockout mESC line, which failed to acquire the characteristic cell morphology associated with the formative state. Transcriptome analysis of differentiating cells revealed nearly 300 differentially expressed genes compared to wild-type cells, including pluripotency TFs Nanog, Klf2, Nr5a2, Prdm14, and Esrrb, that failed to correctly downregulate. Notably, premature expression of OCT6 in naive cells triggered a rapid morphological transformation mirroring differentiation, accompanied by self-induction of Oct6 and expression of TFs such as Sox3, Zic2/3, Foxp1, as well as the formative genes Dnmt3A and FGF5. Strikingly, the majority of OCT6 expressing cells did not express NANOG. Gene expression and single molecule RNA-FISH analysis confirmed that this regulation was at the transcriptional level. Collectively, our results establish OCT6 as a key TF in the dissolution of the naive pluripotent state and support a model where Oct6 and Nanog form a double negative feedback loop which could act as a toggle switch important for the transition to the formative state. HighlightsO_LIOct6 is rapidly induced as mESCs exit ground state pluripotency. C_LIO_LILoss of OCT6 negatively affects the transition to formative pluripotency. C_LIO_LIPremature expression of OCT6 in mESCs is sufficient to induce a formative-like phenotype. C_LIO_LIOCT6 and NANOG repress each other forming a double negative feedback loop. C_LI

developmental biology↗

bioRxiv2021

HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.

cancer biology↗

bioRxiv2019

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted PGCs

The CRISPR/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rates during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cells (PGCs) transplantation to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. First, we optimized the procedure for gRNA targeted PGCs transplantation (PGCT), by increasing the efficiencies of genome mutation in PGCs and induction of PGCs fates in donor embryos for PGCT. Second, the combined CRISPR/Cas9 with PGCT was utilized for generation of maternal zygotic (MZ) mutants of tcf7l1a (essential gene for head development), pou5f3 (essential gene for zygotic genome activation) and chd (essential gene for dorsal development) at F1 generation with high efficiency. Finally, we revealed some novel phenotypes in the maternal zygotic mutant of tcf7l1a and chd, while MZtcf7l1a showed elevated neural crest development, and MZchd have stronger ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish.

developmental biology↗