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The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates (scRNA-Seq)

ABSTRACT: Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotent state. To investigate the dynamics of this process under spatial confinement we used either PDMS pillars or micro-contact printing of vitronectin. OCT4, E-cadherin, and NANOG were progressively upregulated within about 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interaction seemed to be relevant for the self-organization. After 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of flat iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE184603 | GEO | 2022/08/26

REPOSITORIES: GEO

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The spatial self-organization within pluripotent stem cell colonies is continued in detaching aggregates (bulk RNA-Seq)

Project description:Colonies of induced pluripotent stem cells (iPSCs) reveal aspects of self-organization even under culture conditions that maintain pluripotent state. To investigate the dynamics of this process under spatial confinement we used either PDMS pillars or micro-contact printing of vitronectin. OCT4, E-cadherin, and NANOG were progressively upregulated within about 70 µm from the outer rim of iPSC colonies. Single-cell RNA-sequencing demonstrated that OCT4high subsets have pronounced up-regulation of the TGF-β pathway, particularly of NODAL and its inhibitor LEFTY, at the rim of the colonies. Furthermore, calcium-dependent cell-cell interaction seemed to be relevant for the self-organization. After 5 to 7 days, the iPSC colonies detached spontaneously from micro-contact printed substrates to form 3D aggregates. This new method allowed generation of embryoid bodies (EBs) of controlled size, without any enzymatic or mechanical treatment. Within the early 3D aggregates, the radial organization and differential gene expression continued in analogy to the changes observed during self-organization of flat iPSC colonies. Our results provide further insight into the gradual self-organization within iPSC colonies and at their transition into EBs.

2022-08-26 | GSE160665 | GEO

Surface topography guides morphology and spatial patterning of induced pluripotent stem cell colonies

Project description:The relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs. For more information please check; https://cbit.maastrichtuniversity.nl/

2018-10-31 | E-MTAB-7327 | biostudies-arrayexpress

Nanotopography guides morphology and spatial patterning of induced pluripotent stem cell colonies

Project description:The role of topographic cues in controlling commitment of induced pluripotent stem cells (iPSCs) is largely unknown. Here we demonstrate that groove-ridge nanostructures induce the elongation of iPSC colonies, guide the orientation of apical actin fibers and direct the polarity of cell division. Elongation of iPSC colonies impacts also on the intrinsic molecular patterning which seems to be orchestrated starting from the rim of the colonies. We followed the hypothesis that nanotopography directly modulates the transcriptional program of iPSC, further to guiding the overall spatial organization of the colonies. Single iPSC were seeded on flat (PI flat) and nanostructured polyimide (PI 650) and gene expression profiles were analyzed after three days. No significant differences were observed when cells were kept under culture conditions that sustained pluripotency. Then, we analyzed gene expression changes upon two weeks of multi-lineage differentiation. Many genes revealed significant expression changes in the course of differentiation and this was more pronounced on PI flat as compared to PI 650. Comparison of iPSC that were either differentiated on flat or nanostructured biomaterials revealed differential expression of several genes. Noteworthy, among significantly regulated genes, the biggest fold change on PI 650 versus PI flat after differentiation was observed in ANKRD1, which is one of the best readouts of YAP/TAZ activity. Our study suggests that nanotopography impacts on orientation and organization of iPSC colonies and highlight a possible interaction between mechanosensors and mechanotransducers.

2017-09-18 | GSE84848 | GEO

YAP1 is essential for self-organized differentiation of pluripotent stem cells [RNAseq]

Project description:The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. It has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human induced pluripotent stem cells (iPSCs) remains largely unclear. To gain better insight into the functional relevance of YAP1 in these early cell-fate decisions, we generated iPSC lines with YAP1 knockout (YAP-/-) with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. In YAP-/- iPSCs the expression of several YAP1 targets changed and NODAL, which is an important regulator of cell differentiation, was upregulated. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies toward endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in self-organized embryoid bodies (EBs) germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and in tendency also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the non-directed self-organization of EBs, and this is at least partly attributed to activation of the NODAL pathway.

2023-02-17 | GSE214401 | GEO

YAP1 is essential for self-organized differentiation of pluripotent stem cells [DNAm]

2023-02-17 | GSE214400 | GEO

A compound-based therapy approach for epidermolysis bullosa simplex (EBS)

Project description:Keratin cytoskeletal proteins are crucial for the maintenance of skin integrity. Mutations in genes coding for K5 and K14 cause the human skin disorder epidermolysis bullosa simplex (EBS) leading to substantial alterations in keratin assembly and collapse of keratin filaments into cytoplasmic protein aggregates. The phenotypic consequences of K5 and K14 mutations comprise fragility of basal keratinocytes and skin blistering upon mild mechanical trauma. Treatment of EBS is only supportive and consists primarily of wound care and avoidance of mechanical stress. Besides symptomatic care, no efficient therapeutic treatment is available for EBS. In the present study, we used patient-derived keratinocytes carrying the most frequent K14.R125C mutation as a reproducible EBS model to understand EBS pathomechanisms and to develop a therapy approach aimed to restore a functional keratin network. Numerous post-translational modifications (PTMs) such as phosphorylation have been reported to occur on keratins, which affect the organization of keratin networks. Whether keratin mutations affect the occurrence of PTMs and thereby keratin aggregation in EBS is yet unknown. We find that the K14.R125C mutation alters keratin and keratin-associated protein PTMs in distinct ways and suggest that disease mutations and altered PTMs aggravate keratin aggregation. We reason that chemical compounds affecting the interplay of mutations and PTMs enable the reformation of a keratin cytoskeleton from aggregates are potential candidates for combating EBS.

2022-06-29 | PXD030939 | Pride

kidney organoids derived from human induced pluripotent stem cells (iPSC)

Project description:iPSCs were maintained on Geltrex coated flat culture dish in E8 media according to manufacturer’s guidelines. Colonies were manually harvested at 60-80% confluence. Cells were then collected and dissociated into single cells using EDTA. Cells were put on to ultra low attachment 24 well or 96 well plate to allow them to form aggregated in suspension with ROCK inhibitor. Cell aggregates 191were cultured in E8 medium with daily medium change for 6-7 days. Control iPSC-A (iPSC-aggregates) were plated on a Geltrex in 96 well plate or 8 well culture chamber. And then aggregates were treated E8 medium with daily medium change for 12-14 days.

2021-04-20 | GSE172319 | GEO

Human embryoid bodies as a novel system for genomic studies of functionally diverse cell types

Project description:Embryoid bodies, 3D aggregates of spontaneously differentiating iPSCs, have the potential to be a useful model system for genomic studies of diverse cell types. We first collected single-cell RNA-sequencing data from embryoid bodies (EBs) generated from 3 Yoruba individuals (18858, 18511, and 19160) in 3 replicates. We classify functional heterogeneity in EB cells using unsupervised clustering, differential expression analysis, reference annotation, and topic modelling. We then inferred differentiation trajectories that recapitulate known developmental patterns of gene expression. We later collected Day 21 EBs from a single replicate of 5 additional Yoruba iPSC lines (18856, 18912, 19140,19159, and 19210) and use this data to demonstrate robustness of EB cell type composition across iPSC lines. Our results establish EBs as a powerful model system to facilitate discovery of QTLs for cell type composition, as well as eQTLs and dynamic eQTLs across a multitude of human cell types and developmental trajectories.

2021-06-16 | GSE178274 | GEO

Effects of 4-phenylbutyrate on human keratinocytes

Project description:Keratins 5 and 14 are critical for cytoskeletal integrity, as shown by missense mutations in these genes, which cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS). The complexity of the pathomechanisms in EBS is not fully understood and no effective management exists. In addition to fragility, EBS keratinocytes are characterized by aggregates of misfolded keratin. Here, we tested the chemical chaperone 4-phenylbutyrate (4-PBA) as a putative novel therapy, using keratinocytes from patients with severe generalized EBS due to distinct KRT5 and KRT14 mutations.

2019-12-03 | PXD010028 | Pride

Self-Renewing Tri-Potent Stem/Progenitor-like Cells from Adult Human Pancreas

Project description:Stem and progenitor cells in the adult human pancreas provide an under-explored resource for regenerative medicine. Using micro-manipulation and methylcellulose-containing colony/organoid assays, we identified cells within the human cadaveric exocrine pancreas that fulfill the definition of a stem cell: able to self-renew and differentiate. Exocrine tissues were collected after the isolation of endocrine cells, dissociated into single cells, and plated into a 3-dimensional semisolid medium. We found that some pancreatic ductal cells gave rise to cystic colonies/organoids containing pancreatic duct, acinar, and endocrine lineage cells. These cells self-renewed and expanded approximately 300-fold over 9 weeks. When transplanted into diabetic mice, colonies/organoids lowered blood glucose levels and gave rise to insulin-expressing endocrine cells. Thus, stem/progenitor-like cells capable of self-renewal and differentiation either preexist in the adult human pancreas or readily adapt in culture. These human cells have implications for therapy in diabetes patients.

2021-07-02 | GSE153834 | GEO

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