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Transcriptomically Guided Mesendoderm Induction of Human Pluripotent Stem Cells Using a Systematically Defined Culture Scheme.

ABSTRACT: Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation.

SUBMITTER: Carpenedo RL

PROVIDER: S-EPMC6915803 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Project description:Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient and broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple yet robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. After 14 days of differentiation, BA-treated cells expressed genes indicative of mesoderm and endoderm-derived tissue types, including lung, kidney and adipose tissue. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. Teratomas formed from BA-treated cells contained higher ratios of mesoderm and endoderm to ectoderm tissue compared to PSC-derived teratomas. Collectively, these data demonstrate that single-cell seeding with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

2019-04-11 | GSE129570 | GEO

Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Project description:Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

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Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

Project description:Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

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Generation of Keratinocytes from Human Induced Pluripotent Stem Cells Under Defined Culture Conditions.

Project description:Differentiation of keratinocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has become an important tool for wound healing research and for studying skin diseases in instances where patient cells are not available. Several keratinocyte differentiation protocols using hiPSC colony fragments or embryoid bodies have been published with some requiring prolonged time for differentiation or extended use of reagent cocktails. In this study, we present a simplified method to efficiently generate large numbers of uniformly differentiated keratinocytes in less than 4 weeks from singularized hiPSCs with differentiation factors, retinoic acid and bone morphogenetic protein 4 (BMP4). Low seeding density of singularized iPSCs results in keratinocyte cultures with minimum cell death during differentiation and up to 96% homogeneity for keratin 14-positive cells and low percentage of keratinocyte maturation markers, comparable to early passage primary keratinocytes. hiPSC-derived keratinocytes remain in a proliferative state, can be maintained for prolonged periods of time, and can be terminally differentiated under high calcium conditions in the same way as primary human keratinocytes. Moreover, coculturing hiPSC-derived fibroblasts and keratinocytes consistently formed organotypic 3D skin equivalents. Therefore, keratinocytes generated by this method are a viable source of cells for downstream applications.

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Chemically defined and xeno-free culture condition for human extended pluripotent stem cells.

Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

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Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

Project description:The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

| S-EPMC3219112 | biostudies-literature

Generation of stable integration-free pig induced pluripotent stem cells under chemically defined culture condition.

Project description:Genome integration-free pig induced pluripotent stem cells (iPSCs) bring tremendous value in pre-clinical testing of regenerative medicine, as well as conservation and exploitation of endangered or rare local pig idioplasmatic resources. However, due to a lack of appropriate culture medium, efficient induction and stable maintenance of pig iPSCs with practical value remains challenging. Here, we established an efficient induction system for exogenous gene-independent iPSCs under chemically defined culture condition previously used for generation of stable pig pre-gastrulation epiblast stem cells (pgEpiSCs). WNT suppression was found to play an essential role in establishment of exogenous gene-independent iPSCs. Strikingly, stable integration-free pig iPSCs could be established from pig somatic cells using episomal vectors in this culture condition. The iPSCs had pluripotency features and transcriptome characteristics approximating pgEpiSCs. More importantly, this induction system may be used to generate integration-free iPSCs from elderly disabled rare local pig somatic cells and the iPSCs could be gene-edited and used as donor cells for nuclear transfer. Our results provide novel insights into potential applications for genetic breeding of livestock species and pre-clinical evaluation of regenerative medicine.

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Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds.

Project description:Reprogramming of mouse and human somatic cells can be achieved by ectopic expression of transcription factors, but with low efficiencies. We report that DNA methyltransferase and histone deacetylase (HDAC) inhibitors improve reprogramming efficiency. In particular, valproic acid (VPA), an HDAC inhibitor, improves reprogramming efficiency by more than 100-fold, using Oct4-GFP as a reporter. VPA also enables efficient induction of pluripotent stem cells without introduction of the oncogene c-Myc.

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Random migration of induced pluripotent stem cell-derived human gastrulation-stage mesendoderm.

Project description:Gastrulation is the initial systematic deformation of the embryo to form germ layers, which is characterized by the placement of appropriate cells in their destined locations. Thus, gastrulation, which occurs at the beginning of the second month of pregnancy, is a critical stage in human body formation. Although histological analyses indicate that human gastrulation is similar to that of other amniotes (birds and mammals), much of human gastrulation dynamics remain unresolved due to ethical and technical limitations. We used human induced pluripotent stem cells (hiPSCs) to study the migration of mesendodermal cells through the primitive streak to form discoidal germ layers during gastrulation. Immunostaining results showed that hiPSCs differentiated into mesendodermal cells and that epithelial-mesenchymal transition occurred through the activation of the Activin/Nodal and Wnt/beta-catenin pathways. Single-cell time-lapse imaging of cells adhered to cover glass showed that mesendodermal differentiation resulted in the dissociation of cells and an increase in their migration speed, thus confirming the occurrence of epithelial-mesenchymal transition. These results suggest that mesendodermal cells derived from hiPSCs may be used as a model system for studying migration during human gastrulation in vitro. Using random walk analysis, we found that random migration occurred for both undifferentiated hiPSCs and differentiated mesendodermal cells. Two-dimensional random walk simulation showed that homogeneous dissociation of particles may form a discoidal layer, suggesting that random migration might be suitable to effectively disperse cells homogeneously from the primitive streak to form discoidal germ layers during human gastrulation.

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Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture.

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