Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids.

Introduction: Cell lineage specification is tightly associated with profound morphological changes in the developing human embryo, particularly during gastrulation. The interplay between mechanical forces and biochemical signals is poorly understood.

Methods: Here, we dissect the effects of biochemical cues and physical confinement on a 3D model based on spheroids formed from human induced pluripotent stem cells (hiPSCs).

Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Background: Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by the inheritance of the serpin family A member 1 "Z" genetic variant driving alpha-1 antitrypsin (AAT) protein misfolding in hepatocytes. There are no approved medicines for this disease.

Methods: We conducted a high-throughput image-based small molecule screen using patient-derived induced pluripotent stem cell-hepatocytes (iPSC-hepatocytes).

Cell shape characteristics of human skeletal muscle cells as a predictor of myogenic competency: A new paradigm towards precision cell therapy.

Skeletal muscle-derived cells (SMDC) hold tremendous potential for replenishing dysfunctional muscle lost due to disease or trauma. Current therapeutic usage of SMDC relies on harvesting autologous cells from muscle biopsies that are subsequently expanded in vitro before re-implantation into the patient. Heterogeneity can arise from multiple factors including quality of the starting biopsy, age and comorbidity affecting the processed SMDC.

Hypothesis-free phenotype prediction within a genetics-first framework.

Cohort-wide sequencing studies have revealed that the largest category of variants is those deemed 'rare', even for the subset located in coding regions (99% of known coding variants are seen in less than 1% of the population. Associative methods give some understanding how rare genetic variants influence disease and organism-level phenotypes. But here we show that additional discoveries can be made through a knowledge-based approach using protein domains and ontologies (function and phenotype) that considers all coding variants regardless of allele frequency.

Organoids capture tissue-specific innate lymphoid cell development in mice and humans.

Organoid-based models of murine and human innate lymphoid cell precursor (ILCP) maturation are presented. First, murine intestinal and pulmonary organoids are harnessed to demonstrate that the epithelial niche is sufficient to drive tissue-specific maturation of all innate lymphoid cell (ILC) groups in parallel, without requiring subset-specific cytokine supplementation. Then, more complex human induced pluripotent stem cell (hiPSC)-based gut and lung organoid models are used to demonstrate that human epithelial cells recapitulate maturation of ILC from a stringent systemic human ILCP population, but only when the organoid-associated stromal cells are depleted.

High-content image analysis to study phenotypic heterogeneity in endothelial cell monolayers.

Endothelial cells (ECs) are heterogeneous across and within tissues, reflecting distinct, specialised functions. EC heterogeneity has been proposed to underpin EC plasticity independently from vessel microenvironments. However, heterogeneity driven by contact-dependent or short-range cell-cell crosstalk cannot be evaluated with single cell transcriptomic approaches, as spatial and contextual information is lost.

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.

High-Content Imaging to Phenotype Human Primary and iPSC-Derived Cells.

Increasingly powerful microscopy, liquid handling, and computational techniques have enabled cell imaging in high throughput. Microscopy images are quantified using high-content analysis platforms linking object features to cell behavior. This can be attempted on physiologically relevant cell models, including stem cells and primary cells, in complex environments, and conceivably in the presence of perturbations.

ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor β1, driving expansion of CD44v6 epithelial crypts.

An integrated pipeline for high-throughput screening and profiling of spheroids using simple live image analysis of frame to frame variations.

High-throughput imaging methods can be applied to relevant cell culture models, fostering their use in research and translational applications. Improvements in microscopy, computational capabilities and data analysis have enabled high-throughput, high-content approaches from endpoint 2D microscopy images. Nonetheless, trade-offs in acquisition, computation and storage between content and throughput remain, in particular when cells and cell structures are imaged in 3D.

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.

Induction of Neural Crest Stem Cells From Bardet-Biedl Syndrome Patient Derived hiPSCs.

Neural crest cells arise in the embryo from the neural plate border and migrate throughout the body, giving rise to many different tissue types such as bones and cartilage of the face, smooth muscles, neurons, and melanocytes. While studied extensively in animal models, neural crest development and disease have been poorly described in humans due to the challenges in accessing embryonic tissues. In recent years, patient-derived human induced pluripotent stem cells (hiPSCs) have become easier to generate, and several streamlined protocols have enabled robust differentiation of hiPSCs to the neural crest lineage.

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.

Integrated Multiparametric High-Content Profiling of Endothelial Cells.

Endothelial cells (ECs) are widely heterogeneous at the cell level and serve different functions at the vessel and tissue levels. EC-forming colonies derived from induced pluripotent stem cells (iPSC-ECFCs) alongside models such as primary human umbilical vein ECs (HUVECs) are slowly becoming available for research with future applications in cell therapies, disease modeling, and drug discovery. We and others previously described high-content analysis approaches capturing unbiased morphology-based measurements coupled with immunofluorescence and used these for multidimensional reduction and population analysis.

Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications.

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard.

Corrigendum: Common genetic variation drives molecular heterogeneity in human iPSCs.

This corrects the article DOI: 10.1038/nature22403.

Common genetic variation drives molecular heterogeneity in human iPSCs.

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative.

A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data.

Most image analysis pipelines rely on multiple channels per image with subcellular reference points for cell segmentation. Single-channel phase-contrast images are often problematic, especially for cells with unfavorable morphology, such as induced pluripotent stem cells (iPSCs). Live imaging poses a further challenge, because of the introduction of the dimension of time.

A high-content platform to characterise human induced pluripotent stem cell lines.

Induced pluripotent stem cells (iPSCs) provide invaluable opportunities for future cell therapies as well as for studying human development, modelling diseases and discovering therapeutics. In order to realise the potential of iPSCs, it is crucial to comprehensively characterise cells generated from large cohorts of healthy and diseased individuals. The human iPSC initiative (HipSci) is assessing a large panel of cell lines to define cell phenotypes, dissect inter- and intra-line and donor variability and identify its key determinant components.

Human pluripotent stem cells on artificial microenvironments: a high content perspective.

Self-renewing stem cell populations are increasingly considered as resources for cell therapy and tools for drug discovery. Human pluripotent stem (hPS) cells in particular offer a virtually unlimited reservoir of homogeneous cells and can be differentiated toward diverse lineages. Many diseases show impairment in self-renewal or differentiation, abnormal lineage choice or other aberrant cell behavior in response to chemical or physical cues.

A high-content small molecule screen identifies sensitivity of glioblastoma stem cells to inhibition of polo-like kinase 1.

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors.

High content screening of defined chemical libraries using normal and glioma-derived neural stem cell lines.

Small molecules with potent biological effects on the fate of normal and cancer-derived stem cells represent both useful research tools and new drug leads for regenerative medicine and oncology. Long-term expansion of mouse and human neural stem cells is possible using adherent monolayer culture. These cultures represent a useful cellular resource to carry out image-based high content screening of small chemical libraries.