Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis [bulk RNA-seq]
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PROVIDER: PRJNA674506 | ENA |
REPOSITORIES: ENA
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Project description:It has been notoriously challenging for researchers in the fields of stem cell biology and regenerative medicine to robustly generate uniform populations of differentiated cells from human induced pluripotent stem cells (hiPSCs). In particular, the process of iPSC chondrogenesis has been difficult to reproduce among different laboratories. While several differentiation protocols are being developed, few, if any, yield homogenous hiPSC-derived chondrocytes without any off-target contaminating cells. To improve homogeneity of hiPSC chondrogenesis, we have developed the first application of single-cell RNA sequencing to systemically deconstruct each differentiation step and delineate molecular mechanisms underlying lineage commitment of hiPSCs toward chondrocytes. We identified that MITF as well as several specific canonical and non-canonical WNTs are hub genes of gene regulatory networks governing the off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. By inhibiting these off-target molecules, we significantly enhanced chondrogenic lineage commitment of hiPSCs without cell sorting. Using sequencing data of day 28 (pellet time point) as an example, the percentage of chondrocytes within the pellet was improved from 59.63% (standard protocol) to 87.92% (optimized protocol; note: the other 12.07% belong to mesenchymal cells). Importantly, none of the off-target neural cells and melanocytes were detected, suggesting 100% removal of off-target differentiation by the optimized method. Our optimized protocol was validated by 3 unique hiPSC lines and 3 unique hMSC lines, indicating the protocol can be broadly applied to multiple cell lines. Moreover, therapeutic efficacy of hiPSC-derived chondrocytes was further validated in vivo by subcutaneous implantation and osteochondral defect repair in immunodeficient mice. In addition to the 11 consecutive time points from mesodermal to chondrogenic differentiation stages that were sequenced by bulk RNA-seq, we also sequenced 14 chondrogenic time points at single cell resolution. We believe that our bioinformatic analyses including gene regulatory networks, multicellular signaling models, as well as the large amount of data reported here will provide invaluable resources to investigate cell fate decision during human cartilage development.
2020-11-09 | GSE160786 | GEO
Project description:Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis
| PRJNA674501 | ENA
Project description:Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis [scRNA-seq]
| PRJNA673812 | ENA
Project description:It has been notoriously challenging for researchers in the fields of stem cell biology and regenerative medicine to robustly generate uniform populations of differentiated cells from human induced pluripotent stem cells (hiPSCs). In particular, the process of iPSC chondrogenesis has been difficult to reproduce among different laboratories. While several differentiation protocols are being developed, few, if any, yield homogenous hiPSC-derived chondrocytes without any off-target contaminating cells. To improve homogeneity of hiPSC chondrogenesis, we have developed the first application of single-cell RNA sequencing to systemically deconstruct each differentiation step and delineate molecular mechanisms underlying lineage commitment of hiPSCs toward chondrocytes. We identified that MITF as well as several specific canonical and non-canonical WNTs are hub genes of gene regulatory networks governing the off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. By inhibiting these off-target molecules, we significantly enhanced chondrogenic lineage commitment of hiPSCs without cell sorting. Using sequencing data of day 28 (pellet time point) as an example, the percentage of chondrocytes within the pellet was improved from 59.63% (standard protocol) to 87.92% (optimized protocol; note: the other 12.07% belong to mesenchymal cells). Importantly, none of the off-target neural cells and melanocytes were detected, suggesting 100% removal of off-target differentiation by the optimized method. Our optimized protocol was validated by 3 unique hiPSC lines and 3 unique hMSC lines, indicating the protocol can be broadly applied to multiple cell lines. Moreover, therapeutic efficacy of hiPSC-derived chondrocytes was further validated in vivo by subcutaneous implantation and osteochondral defect repair in immunodeficient mice. In addition to the 11 consecutive time points from mesodermal to chondrogenic differentiation stages that were sequenced by bulk RNA-seq, we also sequenced 14 chondrogenic time points at single cell resolution. We believe that our bioinformatic analyses including gene regulatory networks, multicellular signaling models, as well as the large amount of data reported here will provide invaluable resources to investigate cell fate decision during human cartilage development.
2020-11-09 | GSE160625 | GEO
Project description:The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.
| S-EPMC7806634 | biostudies-literature
Project description:Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but are still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development.
2024-09-11 | E-MTAB-13186 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). However, successfully reprogrammed cells are highly proliferative and escape from cellular senescence - it is therefore conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency and size was higher when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs had indistinguishable in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs M-bM-^@M-^S this is of relevance for standardization and automation of cell culture procedures Gene expression profiles of induced fibroblasts (M-bM-^@M-^\iFM-bM-^@M-^]) from three different donors which were either clonally derived (M-bM-^@M-^\CM-bM-^@M-^]) or bulk-cultured (M-bM-^@M-^\BM-bM-^@M-^]) were compared
2013-06-03 | E-GEOD-42807 | biostudies-arrayexpress
Project description:Characterisation of DNA methylation during mesenchymal stem cell chondrogenesis
| PRJNA530691 | ENA
Project description:Metabolism is vital to cellular function and tissue homeostasis during human lung development. In utero, embryonic pluripotent stem cells undergo endodermal differentiation towards a lung progenitor cell fate that can be mimicked in vitro using induced human pluripotent stem cells (hiPSCs) to study genetic mutations. To identify differences between wild type and surfactant protein B (SFTPB)-deficient cell lines during endoderm specification towards lung, we used an untargeted metabolomics approach to evaluate the developmental changes in metabolites. We found that the metabolites most enriched during the differentiation from pluripotent stem cell to lung progenitor cell, regardless of cell line, were sphingomyelins and phosphatidylcholines, two important lipid classes in fetal lung development. The SFTPB mutation had no metabolic impact on early endodermal lung development. The identified metabolite signatures during lung progenitor cell differentiation may be utilized as biomarkers for normal embryonic lung development.
2021-06-29 | ST002055 | MetabolomicsWorkbench
Project description:We showed that the relation of pluripotent stem cell count to culture volume influences cardiomyogenic differentiation by affecting the concentration of secreted proteins that function as paracrine factors
2018-10-17 | PXD004874 | Pride