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Confined 3D microenvironment regulates early differentiation in Human Pluripotent Stem Cells

ABSTRACT: We aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture. Three different culture conditions of EB culture were analyzed: suspension (standard condition), confinement in microwells of width/depth ratio 1:1 and 1:2. EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture.

ORGANISM(S): Homo sapiens

SUBMITTER: Barbara Di Camillo

PROVIDER: E-GEOD-36480 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Confined 3D microenvironment regulates early differentiation in Human Pluripotent Stem Cells

Project description:We aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture.

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In vitro modeling of primordial germ cell development

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Mapping developmental paths of monkey primordial germ-like cell differentiation from pluripotent stem cells by single cell RNA sequencing analysis

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The expression of microRNAs in embryoid bodies derived from mouse embryonic stem cells

Project description:Mouse embryonic stem cells can spontaneously differentiate and assemble into a spherical embryoid body (EB) during suspension culture. The initial study aims to identify the up-regulated or down-regulated microRNAs during the differentiation process of pluripotent stem cells. From the microRNA profiling, we will focus on the microRNAs associated with mesodermal and endothelial differentiation of embryonic or induced pluripotent stem cells. In this study, the EBs were collected at 0 and 10-day differentiation of ESCs (ATCC: CRL-1934). The miRNA transcripts listed in Sanger miRBase Release 19.0 was detected by microarray.

2016-01-08 | E-GEOD-76662 | biostudies-arrayexpress

Single cell RNA sequencing (scRNA-seq) of human embryoid body (day 4 of culture)

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AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

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2016-03-09 | E-GEOD-77705 | biostudies-arrayexpress

Specific gene expression related to epiblast development

Project description:To explore genes involved in epiblast development, we have employed whole genome microarray expression profiling as a discovery platform to identify genes. Embryoid body (EB) formation induces embryonic stem (ES) cells to differentiate into germ layers via epiblasts and extra-embryonic tissues, whereas ES cells give rise only to extra-embryonic tissues in monolayer culture. We compared gene expression profiles of ES cells in each differentiation culture methods at day 1-3 or 4 of differentiation. The expression of organizer related genes (Lhx1, Cer1 and Foxa2) was specifically induced at day 3 of EBs, but not in monolayer culture. In epiblast model cells, the overexpression of Lhx1 induced gene expression of organizer and meso-endodermal markers, suggested that Lhx1 was related to epiblast differentiation.

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2009-07-02 | E-GEOD-7948 | biostudies-arrayexpress

Transcription profiling of mouse embryoid bodies treated with retinoic acid delivery induces an early streak phenotype in vitro

Project description:During embryogenesis, cell specification and tissue formation is directed by the concentration and temporal presentation of morphogens, and similarly, pluripotent embryonic stem cells differentiate in vitro into various phenotypes in response to morphogen treatment. Embryonic stem cells are commonly differentiated as three dimensional spheroids called embryoid bodies (EBs); however, differentiation within EBs is typically heterogeneous and disordered. Here we show that spatiotemporal control of microenvironmental cues embedded directly within EBs enhances the homogeneity, synchrony and organization of differentiation. Degradable polymer microspheres releasing retinoic acid within EBs induce the formation of cystic spheroids closely resembling the early streak mouse embryo, with an exterior of visceral endoderm enveloping an epiblast layer. These results demonstrate that controlled morphogen presentation to stem cells more efficiently directs cell differentiation and tissue formation, thereby improving developmental biology models and enabling the development of regenerative medicine therapies and cell diagnostics. Experiment Overall Design: ESCs (D3 line) were maintained in an undifferentiated state on gelatin-coated tissue culture plates in LIF-containing media as described previously. EBs were formed from 2x106 ESCs inoculated in LIF-free media and cultured under rotary conditions. EBs containing microspheres were produced by coating CellTracker Red (CONTROLs) or RA-loaded (EXPERIMENTs) microspheres in 0.1% gelatin solution for 3 hours prior to mixing with ESCs in various microsphere to cell ratios and a range of rotary speeds. EB media was exchanged every 1-2 days as needed. Experiment Overall Design: Triplicate control and experiment EBs were processed for microarray.

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