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Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture

ABSTRACT: Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture

PROVIDER: PRJNA565015 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10099900_1.fastq.gz	Fastqsanger.gz
	SRR10099900_2.fastq.gz	Fastqsanger.gz
	SRR10099901_1.fastq.gz	Fastqsanger.gz
	SRR10099901_2.fastq.gz	Fastqsanger.gz
	SRR10099902_1.fastq.gz	Fastqsanger.gz

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Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture.

Project description:In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/β-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition and TGFβ signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish a hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.

| S-EPMC10399980 | biostudies-literature

Optogenetic control of Wnt signaling models cell-intrinsic embryogenic patterning using 2D human pluripotent stem cell culture

Project description:Purpose: To establish a molecular fingerprint for optoWnt-induced differentiation, we measured global transcriptional changes using bulk-population RNA-seq of optoWnt and WT hESCs after 48 hrs of light stimulation. Illuminated WT cells served as a phototoxicity control, and unilluminated optoWnt cells controlled for potential CRISPR/Cas9 knock-in effects, cell perturbation due to optoWnt expression, and dark-state Wnt pathway activation. We also measured feedback between optoWnt/WT populations during co-culture (1:1) by FACS sorting for each population after 48 hrs of growth. Methods: Bulk RNA-seq of hESCs using Illumina TruSeq Stranded mRNA Library Prep Kit and sequenced on HiSeq 4000 Results: Principal component analysis (PCA) showed clustering of biological triplicates for each condition and strong transcriptional changes upon optoWnt stimulation that account for 95% gene variance among samples. Dark and illuminated monoculture WT cells clustered together, and differential analysis of monocultures showed minimal gene expression differences, demonstrating minimal phototoxicity effects after 48 hrs of continuous 0.8 µW/mm-2 blue light stimulation. In contrast, optoWnt stimulation induced a broad transcriptional effect, with ~5,500 differentially expressed genes between the dark and illuminated conditions. Direct B-catenin target genes, such as CDX1, DKK1, T, and TBX3, are among the most differentially expressed genes, all with a log fold change (LFC) of ~ 9 – 13. Lastly, WT cells grown in illuminated co-culture with optoWnt cells showed differential gene expression and upregulation of TGF-B target genes when compared with WT cells grown in monoculture or in dark co-cultures, indicating signaling feedback between the optoWnt and WT populations.

2023-07-01 | GSE137289 | GEO

Optogenetic manipulation of BMP signalling to drive chondrogenic differentiation of hPSCs

Project description:Optogenetics is a rapidly advancing technology that combines photochemical, optical and synthetic biology techniques to control cellular behaviour and physiology. Combining sensitive light responsive optogenetic intervention tools with human pluripotent stem cell differentiation models has the potential to refine differentiation and unpick the processes by which morphogenetic signals direct tissue patterning, organisation and cell specification. Here, we utilised an optogenetic bone morphogenetic protein (BMP) signalling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We initially engineered light-sensitive hESCs through CRISPR/Cas9-mediated integration of the optoBMP system into the AAVS1 locus. Activation of optoBMP with blue light in lieu of BMP family growth factors during differentiation resulted in activation of BMP signalling pathway mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells left in the dark. Furthermore, cells differentiated with light were capable of forming chondrogenic pellets consisting of a hyaline-like cartilaginous matrix.

2023-11-09 | E-MTAB-13377 | biostudies-arrayexpress

Endogenous FGF gradients are required for cell fate patterning in 2D human gastruloids

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Project description:Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4, known as a 2D gastruloid, are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h.

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Sensing Cardiac Electrical Activity with a Cardiomyocyte Targeted Optogenetic Voltage Indicator

Project description:Rationale: Monitoring and controlling cardiomyocyte activity with optogenetic tools offers exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. Objective: To test the hypothesis that cardiomyocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electrical activity in isolated cardiomyocytes and the whole heart as well as function and maturity in induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Methods and Results: We first generated mice with cardiomyocyte-restricted expression of VSFP2.3 and demonstrated distinct sarcolemmal localization of VSFP2.3 without any signs for associated pathologies (assessed by echocardiography). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch-clamping. The utility of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiomyocytes. Optical cardiograms (OCGs) could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in iPSC-derived cardiomyocytes. Conclusions: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof-of-concept for its utility in optogenetic sensing of physiological and pathological excitation in mature and immature cardiomyocytes in vitro and in vivo. Determination of transgene (VSFP2.3) cardiotoxicity

2015-09-02 | E-GEOD-69190 | biostudies-arrayexpress

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