RESUMO
Cardiac biological pacing (BP) is one of the future directions for bradyarrhythmias intervention. Currently, cardiac pacemaker cells (PCs) used for cardiac BP are mainly derived from pluripotent stem cells (PSCs). However, the production of high-quality cardiac PCs from PSCs remains a challenge. Here, we developed a cardiac PC differentiation strategy by adopting dual PC markers and simulating the developmental route of PCs. First, two PC markers, Shox2 and Hcn4, were selected to establish Shox2:EGFP; Hcn4:mCherry mouse PSC reporter line. Then, by stepwise guiding naïve PSCs to cardiac PCs following naïve to formative pluripotency transition and manipulating signaling pathways during cardiac PCs differentiation, we designed the FSK method that increased the yield of SHOX2+; HCN4+ cells with typical PC characteristics, which was 12 and 42 folds higher than that of the embryoid body (EB) and the monolayer M10 methods respectively. In addition, the in vitro cardiac PCs differentiation trajectory was mapped by single-cell RNA sequencing (scRNA-seq), which resembled in vivo PCs development, and ZFP503 was verified as a key regulator of cardiac PCs differentiation. These PSC-derived cardiac PCs have the potential to drive advances in cardiac BP technology, help with the understanding of PCs (patho)physiology, and benefit drug discovery for PC-related diseases as well.
Assuntos
Diferenciação Celular , Miócitos Cardíacos , Células-Tronco Pluripotentes , Animais , Camundongos , Diferenciação Celular/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Corpos Embrioides/citologia , Corpos Embrioides/metabolismoRESUMO
The spalt (Sal) gene family has four members (Sall1-4) in vertebrates, all of which play pivotal roles in various biological processes and diseases. However, the expression and function of SALL2 in development are still less clear. Here, we first charted SALL2 protein expression pattern during mouse embryo development by immunofluorescence, which revealed its dominant expression in the developing nervous system. With the establishment of Sall2 deficient mouse embryonic stem cells (ESCs), the in vitro neural differentiation system was leveraged to interrogate the function of SALL2, which showed impaired neural differentiation of Sall2 knockout (KO) ESCs. Furthermore, neural stem cells (NSCs) could not be derived from Sall2 KO ESCs and the generation of neural tube organoids (NTOs) was greatly inhibited in the absence of SALL2. Meanwhile, transgenic expression of E1 isoform of SALL2 restored the defects of neural differentiation in Sall2 KO ESCs. By chromatin immunoprecipitation sequencing (ChIP-seq), Tuba1a was identified as downstream target of SALL2, whose function in neural differentiation was confirmed by rescuing neural phenotypes of Sall2 KO ESCs when overexpressed. In sum, by elucidating SALL2 expression dynamics during early mouse development and mechanistically characterizing its indispensable role in neural differentiation, this study offers insights into SALL2's function in human nervous system development, associated pathologies stemming from its mutations and relevant therapeutic strategy.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas , Fatores de Transcrição , Animais , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Neurogênese , Camundongos Knockout , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
BACKGROUND: Cardiovascular diseases (CVDs) have the highest mortality worldwide. Human pluripotent stem cells (hPSCs) and their cardiomyocyte derivatives (hPSC-CMs) offer a valuable resource for disease modeling, pharmacological screening, and regenerative therapy. While most CVDs are linked to significant over-production of reactive oxygen species (ROS), the effects of current antioxidants targeting excessive ROS are limited. Nanotechnology is a powerful tool to develop antioxidants with improved selectivity, solubility, and bioavailability to prevent or treat various diseases related to oxidative stress. Cerium oxide nanozymes (CeONZs) can effectively scavenge excessive ROS by mimicking the activity of endogenous antioxidant enzymes. This study aimed to assess the nanotoxicity of CeONZs and their potential antioxidant benefits in stressed human embryonic stem cells (hESCs) and their derived cardiomyocytes (hESC-CMs). RESULTS: CeONZs demonstrated reliable nanosafety and biocompatibility in hESCs and hESC-CMs within a broad range of concentrations. CeONZs exhibited protective effects on the cell viability of hESCs and hESC-CMs by alleviating excessive ROS-induced oxidative stress. Moreover, CeONZs protected hESC-CMs from doxorubicin (DOX)-induced cardiotoxicity and partially ameliorated the insults from DOX in neonatal rat cardiomyocytes (NRCMs). Furthermore, during hESCs culture, CeONZs were found to reduce ROS, decrease apoptosis, and enhance cell survival without affecting their self-renewal and differentiation potential. CONCLUSIONS: CeONZs displayed good safety and biocompatibility, as well as enhanced the cell viability of hESCs and hESC-CMs by shielding them from oxidative damage. These promising results suggest that CeONZs may be crucial, as a safe nanoantioxidant, to potentially improve the therapeutic efficacy of CVDs and be incorporated into regenerative medicine.
Assuntos
Cério , Miócitos Cardíacos , Células-Tronco Pluripotentes , Humanos , Ratos , Animais , Espécies Reativas de Oxigênio/metabolismo , Estresse Oxidativo , Diferenciação Celular , Antioxidantes/farmacologia , Doxorrubicina/farmacologiaRESUMO
BACKGROUND: Transcription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear. METHODS: With NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data. RESULTS: Either HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs. CONCLUSIONS: HAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células-Tronco Embrionárias Humanas , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/genética , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células-Tronco Embrionárias Humanas/metabolismoRESUMO
Anthracyclines including doxorubicin (DOX) are still the most widely used and efficacious antitumor drugs, although their cardiotoxicity is a significant cause of heart failure. Despite considerable efforts being made to minimize anthracycline-induced cardiac adverse effects, little progress has been achieved. In this study, we aimed to explore the role and underlying mechanism of SNX17 in DOX-induced cardiotoxicity. We found that SNX17 was downregulated in cardiomyocytes treated with DOX both in vitro and in vivo. DOX treatment combined with SNX17 interference worsened the damage to neonatal rat ventricular myocytes (NRVMs). Furthermore, the rats with SNX17 deficiency manifested increased susceptibility to DOX-induced cardiotoxicity (myocardial damage and fibrosis, impaired contractility and cardiac death). Mechanistic investigation revealed that SNX17 interacted with leiomodin-2 (LMOD2), a key regulator of the thin filament length in muscles, via its C-TERM domain and SNX17 deficiency exacerbated DOX-induced cardiac systolic dysfunction by promoting aberrant LMOD2 degradation through lysosomal pathway. In conclusion, these findings highlight that SNX17 plays a protective role in DOX-induced cardiotoxicity, which provides an attractive target for the prevention and treatment of anthracycline induced cardiotoxicity.