Biology subjects

Tewary, M.

Publications and source records attributed to Tewary, M..

7 recordsLinked to original sources

bioRxiv2022

Symmetry-breaking in adherent pluripotent stem cell-derived developmental patterns

The emergence of the anterior-posterior body axis during early gastrulation constitutes a symmetry-breaking event, which is key to the development of bilateral organisms, and its mechanism remains poorly understood. Two-dimensional gastruloids constitute a simple and robust framework to study early developmental events in vitro. Although spontaneous symmetry breaking has been observed in three dimensional (3D) gastruloids, the mechanisms behind this phenomenon are poorly understood. We thus set out to explore whether a controllable 2D system could be used to reveal the mechanisms behind the emergence of asymmetry in patterned cellular structures. We first computationally simulated the emergence of organization in micro-patterned mouse pluripotent stem cell (mPSC) colonies using a Turing-like activator-repressor model with activator-concentration-dependent flux boundary condition at the colony edge. This approach allows the self-organization of the boundary conditions, which results in a larger variety of patterns than previously observed. We found that this model recapitulated previous results of centro-symmetric patterns in large colonies, and also that in simulated small colony sizes, patterns with spontaneous asymmetries emerged. Model analysis revealed reciprocal effects between diffusion and size of the colony, with model-predicted asymmetries in small pattern sizes being dominated by diffusion, and centro-symmetric patterns being size-dominated. To test these predictions, we performed experiments on micro-patterned mPSC colonies of different sizes stimulated with Bone Morphogenetic Protein 4 (BMP4), and used Brachyury (BRA)-GFP expressing cells as pattern readout. We found that while large colonies showed centro-symmetric BRA patterns, the probability of colony polarization increased with decreasing sizes, with a maximum polarization frequency of 35% at [~]200m. These results indicate that a simple molecular activator-repressor system can provide cells with collective features capable of initiating a body-axes plan, and constitute a theoretical foundation for the engineering of asymmetry in developmental systems.

developmental biology↗

bioRxiv2020

Gene regulatory network (GRN) embedded agents connect cellular decision making to human pluripotent stem cell derived germ layer-like pattern formation

The emergence of germ layers in embryos during gastrulation is a key developmental milestone. How morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. To understand this, we developed a gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into cellular agents that respond to a dynamic signalling microenvironment. We found that cellular pattern order, composition, and dynamics were predictably manipulable based on the GRN wiring. We showed that feedback between OCT4, and BMP and WNT pathways created a dynamic OCT4 front that mediates the spatiotemporal evolution of developmental patterns. Translocation of this radial front can be predictively disrupted in vitro to control germ-layer pattern composition. This work links the emergence of multicellular patterns to regulatory network activity in individual hPSCs. We anticipate our approach will help to understand how GRN structure regulates organogenesis in different contexts.

developmental biology↗

bioRxiv2020

Endogenous suppression of WNT signalling in human embryonic stem cells leads to low differentiation propensity towards definitive endoderm

SummaryLow differentiation propensity towards a targeted lineage can significantly hamper the utility of individual human pluripotent stem cell (hPSC) lines in biomedical applications. Here, we use monolayer and micropatterned cell cultures, as well as transcriptomic profiling, to investigate how variability in signalling pathway activity between human embryonic stem cell lines affects their differentiation efficiency towards definitive endoderm (DE). We show that endogenous suppression of WNT signalling in hPSCs at the onset of differentiation prevents the switch from self-renewal to DE specification. Gene expression profiling reveals that this inefficient switch is reflected in NANOG expression dynamics. Importantly, we demonstrate that higher WNT stimulation or inhibition of the PI3K/AKT signalling can overcome the DE commitment blockage. Our findings highlight that redirection of the activity of Activin/NODAL pathway by WNT signalling towards mediating DE fate specification is a vulnerable spot, as disruption of this process can result in poor hPSC specification towards DE.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2018

High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning

In vitro models of post-implantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon BMP4 treatment of geometrically-confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line associated heterogeneity was found to be attributable to endogenous nodal expression, with upregulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal downregulation correlated with a neurulation-associated gene profile expression. We harness this knowledge to establish a platform of pre-neurulation-like fate patterning in geometrically confined hPSC colonies that arises due to a stepwise activation of reaction-diffusion and positional-information. Our work identifies a Nodal signalling dependent switch in peri-gastrulation versus pre-neurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of self-organized fate specification in differentiating epiblast and ectodermal tissues.

developmental biology↗

bioRxiv2018

Context-explorer: Analysis of spatially organized protein expression in high-throughput screens

A growing body of evidence highlights the importance of the cellular microenvironment as a regulator of phenotypic and functional cellular responses to perturbations. We have previously developed cell patterning techniques to control population context parameters, and here we demonstrate Context-explorer (CE), a software tool to improve investigation of microenvironmental variables through colony level analyses. We demonstrate the capabilities of CE in the analysis of human and mouse pluripotent stem cells (hPSCs, mPSCs) patterned in colonies of defined size and shape in multi-well plates.\n\nCE employs a density-based clustering algorithm to identify cell colonies within micropatterned wells. Using this automatic colony classification methodology, we obtain accuracies comparable to manual colony counts in a fraction of the time. Classifying cells according to their relative position within a colony enables statistical analysis of radial spatial trends in protein expression within multiple colonies in the same treatment group. When applied to colonies of hPSCs, our analysis reveals a radial gradient in the expression of the pluripotency inducing transcription factors SOX2 and OCT4, and a similar trend in the intra-colony location of different cellular phenotypes. We extend these analyses to colonies of different sizes and shapes and demonstrate how the metrics derived by CE can be used to asses the patterning fidelity of micropatterned plates.\n\nWe have incorporated a number of features to enhance the usability and utility of CE. To appeal to a broad scientific community, all of the softwares functionality is accessible from a graphical user interface, and convenience functions for several common data operations are included. CE is compatible with existing image analysis programs such as CellProfiler and extends the analytical capabilities already provided by these tools. Taken together, CE facilitates investigation of spatially heterogeneous cell populations in fundamental research and drug development validation programs.

cell biology↗

bioRxiv2018

Identifying the genetic basis of variation in cell behaviour in human iPS cell lines from healthy donors

Large cohorts of human iPSCs from healthy donors are potentially a powerful tool for investigating the relationship between genetic variants and cellular phenotypes. Here we integrate high content imaging, gene expression and DNA sequence datasets for over 100 human iPSC lines to identify the genetic basis of inter-individual variability in cell behaviour. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we identified genes that correlated in expression with intrinsic (genetic) and extrinsic (ECM) factors. However, variation in mRNA levels could not account for outlier cell behaviour. Instead, we identified rare, deleterious SNVs in the coding sequence of genes involved in ECM adhesion that occurred in cell lines that were outliers for one or more phenotypes such as cell spreading. These also correlated with altered germ layer differentiation on micropatterned surfaces. Our study thus establishes a strategy for integrating genetic and cell biological measurements for high-throughput analysis.

cell biology↗

bioRxiv2017

A defined platform of human peri-gastrulation-like biological fate patterning reveals coordination between Reaction-Diffusion and Positional-Information

How position dependent cell fate acquisition occurs during embryogenesis has been a central question in developmental biology. To study this process, we developed a defined, high-throughput assay using BMP4 to induce peri-gastrulation-like fate patterning in geometrically constrained human pluripotent stem cell colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient - an observation mechanistically compliant with a BMP4-NOGGIN Reaction-Diffusion (RD) model. Consequent fate acquisition occurred as a function of both the pSMAD1 signaling strength, and induction time - consistent with the Positional-Information (PI) paradigm. Our findings implicate coordination between RD and PI underlying the peri-gastrulation-like fate patterning. This model not only predicts experimental results of perturbing key parameters like colony size, and BMP4 dose, but also identifies experimental conditions that rescue patterning in colonies of sizes that have been reported to be patterning-reticent, and recapitulate RD-like periodic patterns in large colonies.

developmental biology↗