Biology subjects

Gonzalez, D. M.

Publications and source records attributed to Gonzalez, D. M..

6 recordsLinked to original sources

bioRxiv2024

Transient Notch Activation Converts Pluripotent Stem Cell-Derived Cardiomyocytes towards a Purkinje Fiber Fate

Cardiac Purkinje fibers form the most distal part of the ventricular conduction system. They coordinate contraction and play a key role in ventricular arrhythmias. While many cardiac cell types can be generated from human pluripotent stem cells, methods to generate Purkinje fiber cells remain limited, hampering our understanding of Purkinje fiber biology and conduction system defects. To identify signaling pathways involved in Purkinje fiber formation, we analyzed single cell data from murine embryonic hearts and compared Purkinje fiber cells to trabecular cardiomyocytes. This identified several genes, processes, and signaling pathways putatively involved in cardiac conduction, including Notch signaling. We next tested whether Notch activation could convert human pluripotent stem cell-derived cardiomyocytes to Purkinje fiber cells. Following Notch activation, cardiomyocytes adopted an elongated morphology and displayed altered electrophysiological properties including increases in conduction velocity, spike slope, and action potential duration, all characteristic features of Purkinje fiber cells. RNA-sequencing demonstrated that Notch-activated cardiomyocytes undergo a sequential transcriptome shift, which included upregulation of key Purkinje fiber marker genes involved in fast conduction such as SCN5A, HCN4 and ID2, and downregulation of genes involved in contractile maturation. Correspondingly, we demonstrate that Notch-induced cardiomyocytes have decreased contractile force in bioengineered tissues compared to control cardiomyocytes. We next modified existing in silico models of human pluripotent stem cell-derived cardiomyocytes using our transcriptomic data and modeled the effect of several anti-arrhythmogenic drugs on action potential and calcium transient waveforms. Our models predicted that Purkinje fiber cells respond more strongly to dofetilide and amiodarone, while cardiomyocytes are more sensitive to treatment with nifedipine. We validated these findings in vitro, demonstrating that our new cell-specific in vitro model can be utilized to better understand human Purkinje fiber physiology and its relevance to disease.

developmental biology↗

bioRxiv2023

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole genome sequencing (WGS), but still generate hundreds of artefacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolkit (PTATO), to accurately detect single base substitutions, small insertions and deletions (indels) and structural variants in PTA-based WGS data. PTATO includes a machine learning approach to distinguish PTA-artefacts from true mutations with high sensitivity (up to 90% for base substitution and 95% for indels), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that many hematopoietic stem and progenitor cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS technologies, have normal somatic single base substitution burdens, but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

genomics↗

bioRxiv2022

RNF43 mutations facilitate mucinous colorectal cancer metastasis via formation of a tumour-intrinsic niche

Colorectal cancer (CRC) progression is characterised by a remarkable increase in plasticity and cellular heterogeneity. The ability of cells to shift states and adopt mixed lineage potential exacerbates during metastasis and associates with poor patient survival. How these atypical differentiated states emerge and drive cancer progression however has remained unclear. Here, we investigate this issue for BRAF-mutant CRC that commonly arise via the serrated pathway and are linked to poor prognosis via incompletely understood progression steps. Using patient-derived organoids, gene editing and murine tumour models, we show that mutations in RNF43, a regulator of WNT receptor abundance, endow BRAF-mutant CRC with metastatic capacity by driving a non-dividing tumour-intrinsic niche cell (TINC) state that provides growth factor self-sufficiency to the cancer tissue. TINCs are enriched in RNF43-mutant, mucinous and metastatic samples of human CRC, and ablation of TINCs lead to re-acquired external growth factor dependency during CRC organoid outgrowth. Together, our findings uncover a mechanism by which tumours leverage cellular plasticity to mediate self-organised stem- and niche-cell interactions. Our results argue that formation of a tumour-intrinsic niche is a prerequisite for BRAF-mutant CRC seeding to distant organs and that interference with niche formation may help avoid metastatic relapse.

cancer biology↗

bioRxiv2021

Understanding Mechanisms of Chamber-Specific Differentiation Through Combination of Lineage Tracing and Single Cell Transcriptomics

The specification and differentiation of atrial and ventricular myocardial cell types during development is incompletely understood. We have previously shown that Foxa2 expression during gastrulation identifies a population of ventricular fated progenitors, allowing for labeling of these cells prior to the morphogenetic events that lead to chamber formation and acquisition of bona fide atrial or ventricular identity. In this study, we performed single cell RNA sequencing of Foxa2Cre;mTmG embryos at the cardiac crescent (E8.25), primitive heart tube (E8.75) and heart tube (E9.25) stage in order to understand the transcriptional mechanisms underlying formation of atrial and ventricular cell types at the earliest stages of cardiac development. We find that progression towards differentiated myocardial cell types occurs primarily based on heart field progenitor identity, and that different progenitor populations contribute to ventricular or atrial identity through separate differentiation mechanisms. We identified a number of candidate markers that define such differentiation processes, as well as differential regulation of metabolic processes that distinguish atrial and ventricular fated cells at the earliest stages of development. We further show that exogenous injection with retinoic acid during formation of the cardiac primordia causes defects in ventricular chamber size and is associated with dysregulation in FGF signaling in anterior second heart field cells and a shunt in differentiation towards orthogonal lineages. Retinoic acid also causes defects in cell-cycle exit in myocardial committed progenitors that result in formation of hypomorphic ventricles with decreased expression of important metabolic processes and sarcomere assembly. Collectively, our data identify, at a single cell level, distinct lineage trajectories during cardiac progenitor cell specification and differentiation, and the precise effects of manipulating cardiac progenitor field patterning via exogenous retinoic acid signaling.

developmental biology↗

bioRxiv2021

PPARdelta signaling activation improves metabolic and contractile maturation of human pluripotent stem cell-derived cardiomyocytes.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) provide an unprecedented opportunity to study human heart development and disease. A major caveat however is that they remain functionally and structurally immature in culture, limiting their potential for disease modeling and regenerative approaches. Here, we address the question of how different metabolic pathways can be modulated in order to induce efficient hPSC-CM maturation. We show that PPAR signaling acts in an isoform-specific manner to balance glycolysis and fatty acid oxidation (FAO). PPARD activation or inhibition results in efficient respective up- or down-regulation of the gene regulatory networks underlying FAO in hPSC-CMs. PPARD induction further increases mitochondrial and peroxisome content, enhances mitochondrial cristae formation and augments FAO flux. Lastly PPARD activation results in enhanced myofibril organization and improved contractility. Transient lactate exposure, commonly used in hPSC-CM purification protocols, induces an independent program of cardiac maturation, but when combined with PPARD activation equally results in a metabolic switch to FAO. In summary, we identify multiple axes of metabolic modifications of hPSC-CMs and a role for PPARD signaling in inducing the metabolic switch to FAO in hPSC-CMs. Our findings provide new and easily implemented opportunities to generate mature hPSC-CMs for disease modeling and regenerative therapy.

cell biology↗

bioRxiv2020

A Library Of Induced Pluripotent Stem Cells From Clinically Well-Characterized, Diverse Healthy Human Individuals

A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22-61. The hiPSCs match the karyotype and short tandem repeat identity of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of Mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-seq derived cell clusters and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.

cell biology↗