3D ECM-rich environment sustains the identity of naïve human iPSCs
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PROVIDER: PRJNA677974 | ENA |
REPOSITORIES: ENA
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Project description:The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signalling pathways during maintenance of human naïve pluripotency have been characterized. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining transcriptional, proteomic and secretome analyses, we found that naïve, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naïve hiPSCs, and also allows direct and timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in 3D starting from a single cell.
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Project description:The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. In the last 5 years the signalling environment promoting human naïve pluripotency has been extensively investigated. However, little is known about the role exerted by the extracellular matrix and three-dimensional organization in naïve maintenance. Here, by using an unbiased and integrated approach we found that naïve, but not primed, hiPSC colonies are characterized by an extracellular matrix-rich microenvironment, and by a specific extracellular matrix fingerprint at transcriptional, proteomic and secretome levels. Based on this, we developed a 3D culture system which supports robust long-term feeder-free expansion of naïve hiPSCs. In this same 3D ECM-rich environment, we can recapitulate timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in three-dimensions starting from a single cell.
2021-11-11 | GSE161371 | GEO
Project description:The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways during maintenance of human naïve pluripotency has been characterized. However, little is known about the role exerted by the extracellular matrix and its three-dimensional organization. Here, using an unbiased and integrated approach combining transcriptional, proteomic and secretome analyses, we found that naïve, but not primed, hiPSC colonies are characterized by a self-organized extracellular matrix (ECM)-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naïve hiPSCs, and also allows direct and timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in 3D starting from a single cell
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Project description:The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways during maintenance of human naïve pluripotency has been characterized. However, little is known about the role exerted by the extracellular matrix and its three-dimensional organization. Here, using an unbiased and integrated approach combining transcriptional, proteomic and secretome analyses, we found that naïve, but not primed, hiPSC colonies are characterized by a self-organized extracellular matrix (ECM)-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naïve hiPSCs, and also allows direct and timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in 3D starting from a single cell
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Project description:The establishment of in vitro naïve human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. In the last 5 years the signalling environment promoting human naïve pluripotency has been extensively investigated. However, little is known about the role exerted by the extracellular matrix and three-dimensional organization in naïve maintenance. Here, by using an unbiased and integrated approach we found that naïve, but not primed, hiPSC colonies are characterized by an extracellular matrix-rich microenvironment, and by a specific extracellular matrix fingerprint at transcriptional, proteomic and secretome levels. Based on this, we developed a 3D culture system which supports robust long-term feeder-free expansion of naïve hiPSCs. In this same 3D ECM-rich environment, we can recapitulate timely developmental morphogenesis simply by modulating the signalling environment. Our study opens new perspectives for future applications of naïve hiPSCs to study critical stages of human development in three-dimensions starting from a single cell.
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Project description:3D ECM-rich environment sustains the identity of naïve human iPSCs [Bulk RNA-seq in triplicate after 1, 4, and 7 days of culture]
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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.
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