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.

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.

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.

Plating human iPSC lines on micropatterned substrates reveals role for ITGB1 nsSNV in endoderm formation.

Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers.

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.

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.

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.

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.

Stem cell bioengineering: building from stem cell biology.

New fundamental discoveries in stem cell biology have yielded potentially transformative regenerative therapeutics. However, widespread implementation of stem-cell-derived therapeutics remains sporadic. Barriers that impede the development of these therapeutics can be linked to our incomplete understanding of how the regulatory networks that encode stem cell fate govern the development of the complex tissues and organs that are ultimately required for restorative function.

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.

Engineering the haemogenic niche mitigates endogenous inhibitory signals and controls pluripotent stem cell-derived blood emergence.

Efforts to recapitulate haematopoiesis, a process guided by spatial and temporal inductive signals, to generate haematopoietic progenitors from human pluripotent stem cells (hPSCs) have focused primarily on exogenous signalling pathway activation or inhibition. Here we show haemogenic niches can be engineered using microfabrication strategies by micropatterning hPSC-derived haemogenic endothelial (HE) cells into spatially-organized, size-controlled colonies. CD34+VECAD+ HE cells were generated with multi-lineage potential in serum-free conditions and cultured as size-specific haemogenic niches that displayed enhanced blood cell induction over non-micropatterned cultures.