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

Low 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

Controlled Organization of Cell Fates in Spatially Confined Stem Cell Populations

During embryonic development, cells divide and differentiate over space and time as instructed by their environment to create complex, functionally diverse tissues. Our understanding of the underlying orchestrating rules is incomplete, but recent studies have started to reveal how to instruct cultured pluripotent cell populations to undergo similar developmental-like organizational events. Here, we induce spatially polarized cell fate organization in micropatterned colonies, previously only reported for non-adherent or locally induced cell populations. Underlying this discovery was the augmentation of our micropatterning high-throughput platform through the development of analytical frameworks to automate the quantification of heterogeneous cell responses. These developments enabled rapid hypotheses generation and testing, which enabled new insights into the underlying biology of how and when cell fate organization occurs. Specifically, we developed analytical frameworks to accurately identify colonies of cells within an image and localization of positively expressing regions within these, which enabled automatic quantification of spatial fate organization. We show that when mouse pluripotent stem cells are differentiated on micropatterns towards gastrulation-like fates, their normally symmetrical spatial organization of cell fates can be modified by changing the micropattern diameter and cell density. By differentiating cells at low to medium density on circular micropatterns of 200-300 um in diameter, we induce polarized organization of primitive streak-like and anterior epiblast-like cells, reminiscent of how these populations are localized during development. We study the emergence of this organization using live imaging and found that polarization occurs largely due to reorganization within the colony post-induction. Overall, our results show that system size, both in terms of colony geometry and cell number at the time of differentiation, is critical for polarized cell fate organization. We hypothesize that this could indicate a need for developmentally relevant system sizes in polarization of micropatterned colonies, and that it might be driven by initial heterogeneities in colony morphology or cell fate distribution, or minor fluctuation that are allowed to amplify and perturb a homogeneous state. These insights on how to control and quantify fate organization in cell populations can advance both our understanding of developmental processes and how to create complex tissues with regenerative engineering.Ph.D

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 cell fate acquisitions through community 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 geometries in multi-well plates. CE employs a density-based clustering algorithm to identify cell colonies. Using this automatic colony classification methodology, we reach accuracies comparable to manual colony counts in a fraction of the time, both in micropatterned and unpatterned wells. Classifying cells according to their relative position within a colony enables statistical analysis of spatial organization in protein expression within colonies. When applied to colonies of hPSCs, our analysis reveals a radial gradient in the expression of the transcription factors SOX2 and OCT4. 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. We have incorporated a number of features to enhance the usability and utility of CE. To appeal to a broad scientific community, all of the software's 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 for fundamental research and drug development validation programs

A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning

How position-dependent cell fate acquisition occurs during embryogenesis is a central question in developmental biology. To study this process, we developed a defined, high-throughput assay to induce peri-gastrulation-associated patterning in geometrically confined human pluripotent stem cell (hPSC) colonies. We observed that, upon BMP4 treatment, phosphorylated SMAD1 (pSMAD1) activity in the colonies organized into a radial gradient. We developed a reaction-diffusion (RD)-based computational model and observed that the self-organization of pSMAD1 signaling was consistent with the RD principle. Consequent fate acquisition occurred as a function of both pSMAD1 signaling strength and duration of induction, consistent with the positional-information (PI) paradigm. We propose that the self-organized peri-gastrulation-like fate patterning in BMP4-treated geometrically confined hPSC colonies arises via a stepwise model of RD followed by PI. This two-step model predicted experimental responses to perturbations of key parameters such as colony size and BMP4 dose. Furthermore, it also predicted experimental conditions that resulted in RD-like periodic patterning in large hPSC colonies, and rescued peri-gastrulation-like patterning in colony sizes previously thought to be reticent to this behavior.This work was funded by the Canadian Institutes for Health Research and Medicine by Design, a Canada First Research Excellence Program at the University of Toronto.M.T.received funding from CREATE Materials, Mimetics, and Manufacturing from the Natural Sciences and Engineering Research Council of Canada. P.W.Z. is the Canada Research Chair in Stem Cell Engineering

Virtual cells in a virtual microenvironment recapitulate early development-like patterns in human pluripotent stem cell colonies

The mechanism by which morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. Here, we developed a minimal gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into “cellular” agents that respond to a dynamic morphogenetic signaling microenvironment. Simulations demonstrated that GRN wiring had significant non-intuitive effects on tissue pattern order, composition, and dynamics. Experimental perturbation of GRN connectivities supported model predictions and demonstrated the role of OCT4 as a master regulator of peri-gastrulation fates. Our so-called GARMEN strategy provides a multiscale computational platform to understand how single-cell-based regulatory interactions scale to tissue domains. This foundation provides new opportunities to simulate the impact of network motifs on normal and aberrant tissue development. </p

High-throughput micropatterning platform reveals Nodal-dependent bisection of peri-gastrulation-associated versus preneurulation-associated fate patterning.

In vitro models of postimplantation 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 bone morphogenetic protein 4 (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 up-regulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal down-regulation correlated with a preneurulation-associated gene profile expression. We harness this knowledge to establish a platform of preneurulation-like fate patterning in geometrically confined hPSC colonies in which fates arise because of a BMPs signalling gradient conveying positional information. Our work identifies a Nodal signalling-dependent switch in peri-gastrulation versus preneurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of organized fate specification in differentiating epiblast and ectodermal tissues

holoviz/panel: Version 1.3.1

&lt;p&gt;This micro-release primarily ships a variety of bug and regression fixes focusing on auth, the chat components, and WASM (i.e. PyScript and Pyodide) support. It also adds an enhancement to the auth components that now makes it possible to let users access applications as a guest. Many thanks to our new contributors @art3xa, @polivbr and @tupui as well as our core development team including @MarcSkovMadsen, @maximlt, @ahuang11 and @philippjfr.&lt;/p&gt; &lt;h3&gt;Enhancements&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Add support for authenticating as guest using OAuth and basic auth components (&lt;a href="https://github.com/holoviz/panel/pull/5743"&gt;#5743&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Bug fixes&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Ensure &lt;code&gt;ColorMap&lt;/code&gt; widget correctly handles shared layout and display parameters (&lt;a href="https://github.com/holoviz/panel/pull/5732"&gt;#5732&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix accessing refreshed &lt;code&gt;access_token&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5734"&gt;#5734&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure &lt;code&gt;Markdown&lt;/code&gt; code blocks always wrap (&lt;a href="https://github.com/holoviz/panel/pull/5738"&gt;#5738&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix returning &lt;code&gt;state.user_info&lt;/code&gt; if no &lt;code&gt;id_token&lt;/code&gt; is present in cookies (&lt;a href="https://github.com/holoviz/panel/pull/5747"&gt;#5747&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix &lt;code&gt;Widget.from_param&lt;/code&gt; type annotation (&lt;a href="https://github.com/holoviz/panel/pull/5754"&gt;#5754&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix auto send for &lt;code&gt;ChatInterface&lt;/code&gt; with &lt;code&gt;TextAreaInput&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5762"&gt;#5762&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add support for iframe &lt;code&gt;srcdoc&lt;/code&gt; on &lt;code&gt;Location&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5774"&gt;#5774&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure &lt;code&gt;Tabulator.style&lt;/code&gt; applies correctly with changing data (&lt;a href="https://github.com/holoviz/panel/pull/5757"&gt;#5757&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure &lt;code&gt;panel convert&lt;/code&gt; can correctly detect &lt;code&gt;transformers_js&lt;/code&gt; import (&lt;a href="https://github.com/holoviz/panel/pull/5772"&gt;#5772&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Adjust &lt;code&gt;ReactiveHTML&lt;/code&gt; css resources for relative paths (&lt;a href="https://github.com/holoviz/panel/pull/5779"&gt;#5779&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure invalid query parameters warn instead of erroring (&lt;a href="https://github.com/holoviz/panel/pull/5781"&gt;#5781&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Apply pyscript CSS by default without the splashscreen (&lt;a href="https://github.com/holoviz/panel/pull/5784"&gt;#5784&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure components which require DOM element to be attached can be rendered in &lt;code&gt;Card&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5786"&gt;#5786&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Documentation&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Fix typo in examples/reference/widgets/StaticText.ipynb (&lt;a href="https://github.com/holoviz/panel/pull/5739"&gt;#5739&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add &lt;code&gt;ReactiveExpr&lt;/code&gt; reference docs and change default widget location (&lt;a href="https://github.com/holoviz/panel/pull/5755"&gt;#5755&lt;/a&gt;, &lt;a href="https://github.com/holoviz/panel/pull/5760"&gt;#5760&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix pyscript WASM example (&lt;a href="https://github.com/holoviz/panel/pull/5771"&gt;#5771&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Improve documentation for deep linking docs (&lt;a href="https://github.com/holoviz/panel/pull/5752"&gt;#5752&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt

holoviz/panel: Version 1.3.2

&lt;p&gt;This micro-release focuses on a number of performance improvements, speeding up the initial rendering of simple apps by 2x in many cases. In addition it includes a number of smaller enhancements for various widgets, better support for async and threading and a number of bug fixes related to authentication, the &lt;code&gt;Tabulator&lt;/code&gt; widget and a few other items. We are very pleased to welcome @isumitjha and @fohria as new contributors and want to thank our core team including @maximlt, @Hoxbro, @MarcSkovMadsen, @ahuang11 and @philippjfr for their continuing contributions.&lt;/p&gt; &lt;h3&gt;Performance&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Speed up &lt;code&gt;Pane.clone&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5848"&gt;#5848&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Speed up &lt;code&gt;config&lt;/code&gt; attribute access (&lt;a href="https://github.com/holoviz/panel/pull/5851"&gt;#5851&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Cache templates loaded from string (&lt;a href="https://github.com/holoviz/panel/pull/5854"&gt;#5854&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Only load extension entrypoints once (&lt;a href="https://github.com/holoviz/panel/pull/5855"&gt;#5855&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Do not freeze document models unless needed (&lt;a href="https://github.com/holoviz/panel/pull/5864"&gt;#5864&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Enhancements&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Use the compiled version of Pyodide by default (&lt;a href="https://github.com/holoviz/panel/pull/5808"&gt;#5808&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add support for &lt;code&gt;AutocompleteInput.search_strategy&lt;/code&gt; parameter (&lt;a href="https://github.com/holoviz/panel/pull/5832"&gt;#5832&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Use &lt;code&gt;stdlib_module_names&lt;/code&gt; when determining pyodide dependencies (&lt;a href="https://github.com/holoviz/panel/pull/5818"&gt;#5818&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add &lt;code&gt;Tabulator.sortable&lt;/code&gt; parameter (&lt;a href="https://github.com/holoviz/panel/pull/5827"&gt;#5827&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add delay for tooltip to show up for buttons (&lt;a href="https://github.com/holoviz/panel/pull/5860"&gt;#5860&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add &lt;code&gt;serialize&lt;/code&gt; method on &lt;code&gt;ChatMessage&lt;/code&gt; and &lt;code&gt;ChatFeed&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5764"&gt;#5764&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Allow running onload and defer_load tasks on threads (&lt;a href="https://github.com/holoviz/panel/pull/5865"&gt;#5865&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Allow Image panes for &lt;code&gt;ChatMessage.avatar&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5870"&gt;#5870&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Allow async callbacks on &lt;code&gt;FileDownload&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5878"&gt;#5878&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Allow running scheduled tasks on threads (&lt;a href="https://github.com/holoviz/panel/pull/5879"&gt;#5879&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Bug fixes&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Fix Google OAuth default scopes (&lt;a href="https://github.com/holoviz/panel/pull/5823"&gt;#5823&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix logic for cleaning up OAuth user (&lt;a href="https://github.com/holoviz/panel/pull/5824"&gt;#5824&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Set &lt;code&gt;text_align&lt;/code&gt; correctly if &lt;code&gt;Tabulator&lt;/code&gt; is given Bokeh &lt;code&gt;formatter&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5866"&gt;#5866&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix &lt;code&gt;FileDownload&lt;/code&gt; &lt;code&gt;embed=True&lt;/code&gt; style for &lt;code&gt;Fast&lt;/code&gt; design (&lt;a href="https://github.com/holoviz/panel/pull/5875"&gt;#5875&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Enable &lt;code&gt;Tabulator&lt;/code&gt; selection across pages with &lt;code&gt;pagination=&quot;remote&quot;&lt;/code&gt; and &lt;code&gt;selectable=&quot;checkbox&quot;&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5889"&gt;#5889&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix rendering of &lt;code&gt;VTK&lt;/code&gt; colorbar (&lt;a href="https://github.com/holoviz/panel/pull/5902"&gt;#5902&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Ensure HoloViews &lt;code&gt;DynamicMap&lt;/code&gt; updates when widget dimension has unit (&lt;a href="https://github.com/holoviz/panel/pull/5904"&gt;#5904&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add space between &lt;code&gt;MaterialTemplate&lt;/code&gt; app and site title separator (&lt;a href="https://github.com/holoviz/panel/pull/5905"&gt;#5905&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Consistently handle errors during authentication (&lt;a href="https://github.com/holoviz/panel/pull/5909"&gt;#5909&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Compatibility&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Updates for Numpy 2.0 compatibility (&lt;a href="https://github.com/holoviz/panel/pull/5817"&gt;#5817&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Improve notebook handling when loading bokeh dev versions (&lt;a href="https://github.com/holoviz/panel/pull/5820"&gt;#5820&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Support for rendering into DOM from PyScript Next worker (&lt;a href="https://github.com/holoviz/panel/pull/5911"&gt;#5820&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt; &lt;h3&gt;Documentation&lt;/h3&gt; &lt;ul&gt; &lt;li&gt;Enhance &lt;code&gt;extension&lt;/code&gt; and &lt;code&gt;config&lt;/code&gt; docs (&lt;a href="https://github.com/holoviz/panel/pull/5790"&gt;#5790&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Document setting a &lt;em&gt;multiselect&lt;/em&gt; header filter on &lt;code&gt;Tabulator&lt;/code&gt; (&lt;a href="https://github.com/holoviz/panel/pull/5825"&gt;#5825&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add missing &lt;code&gt;GridSpec&lt;/code&gt; docs (&lt;a href="https://github.com/holoviz/panel/pull/5840"&gt;#5840&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix for getting_started widgets example (&lt;a href="https://github.com/holoviz/panel/pull/5859"&gt;#5859&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Add more references to Panel Chat Examples (&lt;a href="https://github.com/holoviz/panel/pull/5881"&gt;#5881&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Document &lt;code&gt;Tabulator&lt;/code&gt; fontawesome css (&lt;a href="https://github.com/holoviz/panel/pull/5892"&gt;#5892&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;Fix broken link to panel tagged items in holoviz blog (&lt;a href="https://github.com/holoviz/panel/pull/5903"&gt;#5903&lt;/a&gt;)&lt;/li&gt; &lt;li&gt;MenuButton docs improvements (&lt;a href="https://github.com/holoviz/panel/pull/5907"&gt;#5907&lt;/a&gt;)&lt;/li&gt; &lt;/ul&gt