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Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids II

ABSTRACT: Mouse is the pre-eminent model system for studying genetic regulation of embryonic development. Directed differentiation of pluripotent stem cells (PSCs) has emerged as a powerful complement to in vivo studies of development, allowing for examination of developmental mechanisms at the molecular and cellular level. However, the limited availability of high-quality protocols for directed differentiation of mouse PSCs into defined lineages and organoids has prevented the widespread application of mouse PSC directed differentiation models. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. First, we systematically optimized conditions to generate definitive endoderm that are significantly faster and more efficient than current state-of-the-art protocols for mouse PSCs. Second, we developed a robust protocol for generation of forebrain-patterned organoids. These new models can complement and empower in vivo studies in the mouse and can inform human PSC-based studies of development, which cannot readily be compared to analogous stages in vivo.

ORGANISM(S): Mus musculus

PROVIDER: GSE189870 | GEO | 2021/12/31

REPOSITORIES: GEO

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Rapid and robust directed differentiation of mouse epiblast stem cells into definitive endoderm and forebrain organoids I

Project description:Mouse is the pre-eminent model system for studying genetic regulation of embryonic development. Directed differentiation of pluripotent stem cells (PSCs) has emerged as a powerful complement to in vivo studies of development, allowing for examination of developmental mechanisms at the molecular and cellular level. However, the limited availability of high-quality protocols for directed differentiation of mouse PSCs into defined lineages and organoids has prevented the widespread application of mouse PSC directed differentiation models. Here, we examine the potential of mouse epiblast stem cells (EpiSCs) cultured in media containing Wnt pathway inhibitors (primed ground state conditions) as a starting point for directed differentiation. First, we systematically optimized conditions to generate definitive endoderm that are significantly faster and more efficient than current state-of-the-art protocols for mouse PSCs. Second, we developed a robust protocol for generation of forebrain-patterned organoids. These new models can complement and empower in vivo studies in the mouse and can inform human PSC-based studies of development, which cannot readily be compared to analogous stages in vivo.

2021-12-31 | GSE189869 | GEO

Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules

Project description:Anterior foregut endoderm (AFE) gives rise to many tissue types of interest for therapeutic research including the esophagus, salivary glands, lung, thymus, parathyroid and thyroid. Despite its importance, only few reports describe the generation of AFE from pluripotent stem cells (PSCs) by directed differentiation. Here, we describe a novel protocol to derive a subdomain of AFE, identified by expression of Pax9, from PSCs using small molecules and chemically defined conditions. Generation of a reporter PSC line allows isolation and characterization of Pax9+ AFE cells. When transplanted in vivo, Pax9+ AFE can form several distinct types of complex anterior foregut epithelia including mucosal glands and stratified squamous epithelium. Finally, we show that the directed differentiation protocol can be used to generate AFE from DiGeorge Syndrome patient-specific human induced PSCs, thus creating a platform to produce anterior foregut derivatives for therapy and to enable the study of disorders of the AFE. Total RNA obtained from FACS purified from in vitro dervied mouse definitive endoderm, anterior foregut and ES cells. AFE cells were derived from a 129X1/SvJ background, DE cells from 129X1/SvJ x 129S1/SV-+p+Tyr- cKitlSl-J/+ (R1 ES cells) and non reporter ES cells from a 129P2/OlaHsd background.

2013-04-15 | E-GEOD-42139 | biostudies-arrayexpress

Generation of the organotypic kidney by integrating pluripotent stem cell-derived renal stroma

Project description:Organs consist of not only parenchyma but also stroma, the latter of which coordinates generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulating the complex organ configurations from pluripotent stem cells (PSCs) has remained challenging. For the kidney, the stromal progenitor coordinates differentiation of the two parenchymal progenitors: nephron progenitor and ureteric bud. By identifying the origin and dorsoventral patterning of the renal stromal progenitor, we established its induction protocol from mouse PSCs. When the three types of progenitors were differentially induced from PSCs and assembled, the all PSC-derived organoids reproduced the complex kidney structures, with multiple types of stromal cells distributed around differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived stroma into the conventional organoids enables recapitulation of organotypic architecture.

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A PLURIPOTENT STEM CELL PLATFORM FOR in VITRO SYSTEMS GENETICS STUDIES of MOUSE DEVELOPMENT

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Generation of organized anterior foregut epithelia from pluripotent stem cells using small molecules

2013-04-15 | GSE42139 | GEO

Parthenogenetic stem cells for tissue-engineered heart repair

Project description:Uniparental parthenotes are considered an unwanted byproduct of in vitro fertilization. In utero parthenote development is severely compromised by defective organogenesis and in particular by defective cardiogenesis. Although developmentally compromised, apparently pluripotent stem cells can be derived from parthenogenetic blastocysts. Here we hypothesized that nonembryonic parthenogenetic stem cells (PSCs) can be directed toward the cardiac lineage and applied to tissue-engineered heart repair. We first confirmed similar fundamental properties in murine PSCs and embryonic stem cells (ESCs), despite notable differences in genetic (allelic variability) and epigenetic (differential imprinting) characteristics. Haploidentity of major histocompatibility complexes (MHCs) in PSCs is particularly attractive for allogeneic cell-based therapies. Accordingly, we confirmed acceptance of PSCs in MHC-matched allotransplantation. Cardiomyocyte derivation from PSCs and ESCs was equally effective. The use of cardiomyocyte-restricted GFP enabled cell sorting and documentation of advanced structural and functional maturation in vitro and in vivo. This included seamless electrical integration of PSC-derived cardiomyocytes into recipient myocardium. Finally, we enriched cardiomyocytes to facilitate engineering of force-generating myocardium and demonstrated the utility of this technique in enhancing regional myocardial function after myocardial infarction. Collectively, our data demonstrate pluripotency, with unrestricted cardiogenicity in PSCs, and introduce this unique cell type as an attractive source for tissue-engineered heart repair. 8 samples in total. Parthenogenetic stem cells (PSC): - PSC_1 - PSC_2 - PSC_3 Embryoid body (EB) assays of parthenogenetic stem cells: - EB_PSC_1 - EB_PSC_2 Embryonic stem cells (ESC): - ESC_1 - ESC_2 - ESC_3

2013-02-23 | E-GEOD-30868 | biostudies-arrayexpress

Vitamin d receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy

Project description:The poor clinical outcome in pancreatic ductal adenocarcinoma (PDA) has been attributed to intrinsic resistance to chemotherapy and a growth-permissive tumor microenvironment. Quiescent pancreatic stellate cells (PSCs) are neuroendocrine, nestin-positive, lipid-accumulating cells whose homologues in the liver are the principal repository of Vitamin A esters. Upon activation, lipid droplets are lost and via transdifferentiation they become the key cell type responsible for driving the severe desmoplasia that characterizes PDA. Despite their critical role in PDA progression and chemoresistance, therapeutic strategies targeting PSCs are lacking. Here we identified the vitamin D receptor (VDR) as a master genomic regulator of PSC activation and function. In vitro we demonstrate that VDR activation reduces expression in PSCs of genes implicated in activation, inflammation, and extracellular matrix production, as well as restoring lipid droplet integrity. In vivo, the VDR ligand calcipotriol enhances the anti-tumor effects of gemcitabine by increasing intratumoral concentration 5-fold, reducing tumor volume to near baseline and lowering metastases by more than 65%. These findings implicate VDR as a master regulator of PSC activation and identify a novel therapeutic approach for the treatment of pancreatic cancer. RNA-Seq analyses was used to characterize cancer-associated changes between pre-activated (3-day culture) and activated (7-day culture) primary mouse PSCs, as well as control and PDA human PSCs. RNA-Seq was also used to assess the impact of VDR activation (DMSO vs calcipotriol) in a human PSC line (MiaPaCa-2), the mouse primary PSCs

2014-10-03 | E-GEOD-43770 | biostudies-arrayexpress

Gene expression of rat PSCs cultured on plastic, matrigel and collagen

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Project description:Differentiation of neural retina in organoid cultures from human pluripotent stem cells (PSCs) is highly variable and requires an extended period of as many as 200 days. To provide objective assessment of different protocols and culture conditions, we performed transcriptome analysis and molecular staging using RNA-seq data from developing and adult human retina. Using a modified protocol with 9-cis retinal (9CRAL) instead of widely-used all-trans retinoic acid (ATRA) in the culture media, we show comparable differentiation of human embryonic and induced PSCs into a polarized, laminated architecture with all retinal cell types in retinal organoids. Transcriptome comparisons of multiple developing organoids to that of human fetal and adult retina have permitted molecular staging of the differentiation state and uncovered expedited rod photoreceptor maturation by 9CRAL. A direct comparison with the ATRA-organoid cultures revealed higher expression of rhodopsin and other rod genes in 9CRAL-organoids. Our studies thus provide a framework for transcriptome-based staging of retinal organoids to facilitate advances in human disease modeling and evaluation of therapies.

2019-12-17 | GSE129104 | GEO

A Cocktail Factor Approach to Promote the Differentiation of mESC-Derived Oral Epithelium into Salivary Gland Progenitors

Project description:The derivation of salivary gland (SG) progenitors from pluripotent stem cells (PSCs) presents significant potential for developmental biology and regenerative medicine. However, the existing protocols for inducing SG include limited factors, making it challenging to mimic the in vivo microenvironment of embryonic SGs. We reported a cocktail factor approach to enhance the differentiation of mouse embryonic stem cell (mESC)-derived oral epithelium (OE) into SG progenitors through a three-dimensional co-culture method. Upon confirming that the embryonic SG can promote the differentiation of mESC-derived OE, we performed RNA sequence analysis to identify factors involved in the differentiation of SG progenitors. Our findings highlight several efficient pathways related to SG development, with frequent appearances of four factors: IFN-γ, TGF-β2, EGF, and IGF-1. The combined treatment using these cocktail factors increased the expression of key SG progenitor markers, including Sox9, Sox10, Krt5, and Krt14. However, absence of any one of these cocktail factors did not facilitate differentiation. Notably, aggregates treated with the cocktail factor formed SG epithelial-like structures and pre-bud-like structures on the surface. In conclusion, this study offers a novel approach to developing a differentiation protocol that closely mimics the in vivo microenvironment of embryonic SGs. This provides a foundation for generating PSC-derived SG organoids with near-physiological cell behaviors and structures.

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