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

Embigin is a fibronectin receptor that affects sebaceous gland differentiation and metabolism

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism.</p

Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors.

Large cohorts of human induced pluripotent stem cells (iPSCs) from healthy donors are a potentially powerful tool for investigating the relationship between genetic variants and cellular behavior. Here, we integrate high content imaging of cell shape, proliferation, and other phenotypes with gene expression and DNA sequence datasets from over 100 human iPSC lines. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we extracted factors that captured the effects of intrinsic (genetic concordance between different cell lines from the same donor) and extrinsic (cell responses to different fibronectin concentrations) conditions. We identify genes that correlate in expression with intrinsic and extrinsic PEER factors and associate outlier cell behavior with genes containing rare deleterious non-synonymous SNVs. Our study, thus, establishes a strategy for examining the genetic basis of inter-individual variability in cell behavior

Engineered In vitro models of post-implantation human development to elucidate mechanisms of self-organized fate specification during embryogenesis

During embryogenesis, cells in different positions of the embryo acquire different fates in a seemingly autonomous process called ‘fate-patterning’. Fundamental studies have identified important signaling molecules (morphogens) that play crucial roles in coordinating developmental fate-patterning, examples include members of the transforming growth factor beta family – like bone morphogenetic proteins (BMPs), and Nodals. However, mechanistic understanding of how these morphogens coordinate fate-patterning remains unclear. Here we aim to apply bioengineering strategies to develop an in vitro model of developmental fate-patterning and employ it to interrogate the underlying mechanisms that govern this critical process. We first developed a robust, high-throughput platform to enable geometric-confinement of adherent cell types and employed it to screen various BMP4 supplemented defined media to identify conditions that coaxed geometrically-confined human pluripotent stem cell (hPSC) colonies to undergo peri-gastrulation-associated fate-patterning. This screen resulted in identification of defined conditions that spatially segregated compartments in the differentiating hPSC colonies expressing fate markers of trophoblast-like, primitive-streak-like, endoderm-like, mesoderm-like, and ectoderm-like tissues. Using a combination of experimental and computational-modelling approaches, we identified a stepwise mechanism of reaction-diffusion and positional-information underlying the observed peri-gastrulation-like fate-patterning. Here, a BMP4-Noggin reaction-diffusion network self-organized BMP signaling gradient, and this gradient patterned peri-gastrulation-associated fates in a manner consistent with positional-information. Furthermore, we found that Nodal signaling was necessary to induce the expression of the primitive-streak compartment – the precursor of gastrulation-derived fates. Interestingly, we also observed that Nodal signaling dissected gastrulation-associated and neurulation-associated gene expression profiles in differentiating hPSC lines. Specifically, in differentiating hPSCs, upregulation of Nodal signaling was observed in cells that upregulated a gene profile associated with gastrulation whereas absence of Nodal signaling correlated with upregulation of a neurulation-associated gene profile. We hypothesized that treatment of geometrically-confined hPSC colonies with BMP4 in the absence of Nodal signaling would induce fate patterning associated with neurulation. We observed experimental results consistent with this hypothesis and identified a conserved underlying mechanism of a stepwise model of reaction-diffusion and positional-information underlying the pre-neurulation-associated patterning as well. Taken together this work provides deep insight into how morphogens regulate early developmental stages of human embryogenesis – which have been previously inaccessible for experimentation.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

A reductionist approach to determine the effect of cell-cell contact on human epidermal stem cell differentiation

The balance between stem cell renewal and differentiation is determined by the interplay between intrinsic cellular controls and extrinsic factors presented by the microenvironment, or ‘niche’. Previous studies on cultured human epidermis have utilised suspension culture and restricted cell spreading to investigate regulation of differentiation in single keratinocytes. However, keratinocytes are typically adherent to neighbouring cells in vivo. We therefore developed experimental models to investigate the combined effects of cell-ECM adhesion and cell-cell contact. We utilized lipid-modified oligonucleotides to form clusters of keratinocytes which were subsequently placed in suspension to induce terminal differentiation. In this experimental model cell-cell contact had no effect on suspension-induced differentiation of keratinocytes. We next developed a high-throughput platform for robust geometrical confinement of keratinocytes to hexagonal ECM-coated islands permitting direct cell-cell contact between single cells. As in the case of circular islands, differentiation was stimulated on the smallest single hexagonal islands. However, the percentage of involucrin-positive cells on small bowtie islands was significantly lower than on single islands, demonstrating that cell-cell contact reduced differentiation in response to decreased substrate adhesion. None of the small bowtie islands contained two involucrin-positive cells. Rather, if one cell was involucrin-positive the other was involucrin-negative. This suggests that there is intrinsic asymmetry in the effect of cell-cell contact in decreasing differentiation. Thus, our reductionist approaches provide new insights into the effect of the niche on keratinocyte differentiation. STATEMENT OF SIGNIFICANCE: Stem cell behaviour is regulated by a combination of external signals, including the nature of the adhesive substrate and cell-cell interactions. An understanding of how different signals are integrated creates the possibility of developing new biomaterials to promote tissue regeneration and broaden our understanding of skin diseases such as eczema and psoriasis, in which stem cell proliferation and differentiation are perturbed. In this study we have applied two methods to engineer intercellular adhesion of human epidermal stem cells, one involving lipid-modified DNA and the other involving hexagonal micropatterns. We show that the effect of cell-cell adhesion depends on cell-substrate adhesion and uncover evidence that two cells in equivalent environments can nevertheless behave differently

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