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An RNAi Screen Reveals an Essential Role for HIPK4 in Human Skin Epithelial Differentiation from iPSCs.

ABSTRACT: Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells, we identified candidate kinases involved in their differentiation from hiPSCs. Among these, we found HIPK4 to be an important inhibitor of this process. Indeed, its silencing increased the amount of generated skin epithelial precursors at an early time point, increased the amount of generated keratinocytes at a later time point, and improved growth and differentiation of organotypic cultures, allowing for the formation of a denser basal layer and stratification with the expression of several keratins. Our data bring substantial input regarding regulation of human skin epithelial differentiation and for improving differentiation protocols from pluripotent stem cells.

SUBMITTER: Larribere L

PROVIDER: S-EPMC5639458 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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An RNAi Screen Reveals an Essential Role for HIPK4 in Human Skin Epithelial Differentiation from iPSCs.

Larribère Lionel L Galach Marta M Novak Daniel D Arévalo Karla K Volz Hans Christian HC Stark Hans-Jürgen HJ Boukamp Petra P Boutros Michael M Utikal Jochen J

Stem cell reports 20170928 4

Molecular mechanisms responsible for the development of human skin epithelial cells are incompletely understood. As a consequence, the efficiency to establish a pure skin epithelial cell population from human induced pluripotent stem cells (hiPSCs) remains poor. Using an approach including RNAi and high-throughput imaging of early epithelial cells, we identified candidate kinases involved in their differentiation from hiPSCs. Among these, we found HIPK4 to be an important inhibitor of this proce ...[more]

PMID: 28966120

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Project description:IntroductionThe extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes.MethodsWe developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance.ResultsWe identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein-coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype-independent overall survival differences.ConclusionDiverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.

Dataset Information

An RNAi Screen Reveals an Essential Role for HIPK4 in Human Skin Epithelial Differentiation from iPSCs.

Publications

An RNAi Screen Reveals an Essential Role for HIPK4 in Human Skin Epithelial Differentiation from iPSCs.

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