Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA674501 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12977706.fastq.gz | Fastqsanger.gz | |||
| SRR12977707.fastq.gz | Fastqsanger.gz | |||
| SRR12977708.fastq.gz | Fastqsanger.gz | |||
| SRR12977709.fastq.gz | Fastqsanger.gz | |||
| SRR12977710.fastq.gz | Fastqsanger.gz |
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Project description:Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis [scRNA-seq]
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Project description:Single Cell Transcriptomic Analysis of Human Pluripotent Stem Cell Chondrogenesis [bulk RNA-seq]
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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 | 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.
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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.
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Project description:Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.
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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:Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.
| S-EPMC5995784 | biostudies-other
Project description:We assessed the pluripotency of human induced pluripotent stem cells (iPSCs) maintained on an automated platform using StemFlexTM and TeSRTM-E8TM media. Single-cell transcriptome sequencing was performed for 20,962 cells from two cell lines grown in the two media. Analysis of transcriptomic profile revealed similar expression of core pluripotency genes, as well as genes associated with naive and primed states of pluripotency. Single cell analysis of the four samples revealed a shared subpopulation structure with three main subpopulations different in pluripotency states. By implementing a machine learning approach, we estimated that 60-96% of the cells in the ground/naive subpopulations and 98-99% of the cells in the primed subpopulations are similar between all four samples. The single cell RNA sequencing analysis of iPSC lines grown in both media is the first report of the molecular pathways modulated in StemFlex medium and how they compare to those modulated in TeSR-E8 medium.
2019-05-08 | E-MTAB-6524 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
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