RNA-sequencing analysis of naive and primed hPSCs in static and stirred suspension conditions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA515052 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8439650.fastq.gz | Fastqsanger.gz | |||
| SRR8439651.fastq.gz | Fastqsanger.gz | |||
| SRR8439652.fastq.gz | Fastqsanger.gz | |||
| SRR8439653.fastq.gz | Fastqsanger.gz |
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Project description:We have pioneered human pluripotent stem cell (hPSC) manufacturing in stirred suspension bioreactors. Cell therapies require large numbers of quality-controlled hPSCs yet technologies are limited in their ability to efficiently grow and scale clinically-viable hPSCs. We report here that naive hPSCs exhibit superior growth in suspension bioreactors compared to their primed counterpart. Naive hPSCs exhibited a shorter lag phase, and grew into more uniform, homogenous aggregates. Compared to static culture, gene expression analyses revealed that the bioreactor environment promoted the upregulation of naïve- and downregulation of primed-associated transcripts in both primed and naive hPSCs. Bioreactor-cultured naive hPSCs similarly showed more hypomethylated DNA and less primed hPSC-associated surface protein marker compared to statically-cultured naive hPSCs. Gene expression, epigenetic, and cell surface protein marker analyses all suggest that the bioreactor environment promotes the transition from primed-to-naive pluripotent state. Our research shows that reprogramming conventional hPSCs to the naive pluripotent state enhances hPSC manufacturing.
2020-08-31 | GSE125041 | GEO
Project description:Although cell therapies require large numbers of quality-controlled hPSCs, existing technologies are limited in their ability to efficiently grow and scale stem cells. We report here that cell-state conversion from primed-to-naïve pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit superior growth kinetics and aggregate formation characteristics in stirred suspension bioreactors compared to their primed counterparts. Moreover, we demonstrate the role of the bioreactor mechanical environment in the maintenance of naïve pluripotency, through transcriptomic enrichment of mechano-sensing signaling for cells in the bioreactor along with a decrease in expression of lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve hPSCs maintain epigenetic hallmarks of naïve pluripotency, exhibit robust production of naïve pluripotency metabolites, and display reduced expression of primed pluripotency cell surface markers. We also show that these cells retain the ability to undergo targeted differentiation into beating cardiomyocytes, hepatocytes and neural rosettes. They additionally display faster kinetics of teratoma formation compared to their primed counterparts. Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our research corroborates that converting hPSCs to the naïve state enhances hPSC manufacturing and indicates a potentially important role for the bioreactor’s mechanical environment in maintaining naïve pluripotency.
2020-09-18 | GSE144656 | GEO
Project description:Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells (hPSCs)
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Project description:Although cell therapies require large numbers of quality-controlled hPSCs, existing technologies are limited in their ability to efficiently grow and scale stem cells. We report here that cell-state conversion from primed-to-naïve pluripotency enhances the biomanufacturing of hPSCs. Naïve hPSCs exhibit superior growth kinetics and aggregate formation characteristics in stirred suspension bioreactors compared to their primed counterparts. Moreover, we demonstrate the role of the bioreactor mechanical environment in the maintenance of naïve pluripotency, through transcriptomic enrichment of mechano-sensing signaling for cells in the bioreactor along with a decrease in expression of lineage-specific and primed pluripotency hallmarks. Bioreactor-cultured, naïve hPSCs express epigenetic regulatory transcripts associated with naïve pluripotency, and display hallmarks of X-chromosome reactivation. They exhibit robust production of naïve pluripotency metabolites and display reduced expression of primed pluripotency cell surface markers. We also show that these cells retain the ability to undergo targeted differentiation into beating cardiomyocytes, hepatocytes, and neural rosettes. They additionally display faster kinetics of teratoma formation compared to their primed counterparts. Naïve bioreactor hPSCs also retain structurally stable chromosomes. Our research corroborates that converting hPSCs to the naïve state enhances hPSC manufacturing and indicates a potentially important role for the bioreactor’s mechanical environment in maintaining naïve pluripotency.
2020-05-08 | ST001377 | MetabolomicsWorkbench
Project description:Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and cardiac regeneration. Challenges with quality, inter-batch consistency, cryopreservation and scale remain, reducing experimental reproducibility and clinical translation. Here, we report a robust stirred suspension cardiac differentiation protocol with careful functional characterization of the resulting hiPSC-CMs. In a bioreactor, the protocol produced 1.2E6/mL hiPSC-CMs with ～94% purity from 14 iPSC lines. Bioreactor-differentiated CMs (bCMs) showed high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs (mCMs), bCMs had greater reproducibility and more mature functional properties, including pacing capture to 4 Hz and greater force production in 3D engineered heart tissues. In more readily available magnetically stirred spinner flasks, the protocol yielded 1.8E6/mL spinner-differentiated CMs (sCMs) with 94% purity. Differentiation scaled readily in spinner flasks, as a 3.8-fold increase in cultured volume yielded 3.4E6/ml sCMs. sCMs had intermediate functional properties between mCMs and bCMs. Minor protocol modifications generated the first bioreactor-derived cardiac organoids (bCOs) fully generated in suspension. These reproducible, scalable, and resource efficient approaches to generate cardiac cells and organoids with well-characterized properties will expand the applications of hiPSC-CMs.
2024-06-05 | GSE263372 | GEO
Project description:Overview: RNA-seq was used to profile the whole-transcriptome gene expression of highly replicated schizont-stage cultures of W2mef and Dd2 grown under static and suspended conditions. Methods: Transcript profiles of schizont stages of static and suspended W2mef and Dd2 were generated by RNA sequencing. Two to eight replicates were sequenced per sample. Illumina stranded TruSeq libraries were sequenced using an Illumina MiSeq. Hisat2 was used to align paired-end fastQ files and indexed bam files generated with samtools. Bam files were filtered to exclude reads with MAPQ scores below 60, reads counted using the SummarizeOverlaps feature of the GenomicAlignments package in R and differential expression analysis conducted using DESeq2 in R. Results: We show that in addition to invasion-related genes, parasites that have been induced to switch invasion phenotype upon culturing in suspension increase the expression of a number of genes most of which belong to gene families that code for exported proteins. Conclusions: Our data suggest that in addition to invasion phenotype switch, suspension cultures may select for other parasite properties that may be essential for parasite survival in vivo.
2019-04-18 | GSE129949 | GEO
Project description:Due to their ability to standardize key physiological parameters, stirred suspension bioreactors can potentially scale the production of quality-controlled pluripotent stem cells (PSCs) for cell therapy application. Because of differences in bioreactor expansion efficiency between mouse (m) and human (h) PSCs, we investigated if conversion of hPSCs, from the conventional "primed" pluripotent state towards the "naïve" state prevalent in mPSCs, could be used to enhance hPSC production. Through transcriptomic enrichment of mechano-sensing signaling, the expression of epigenetic regulators, metabolomics, and cell-surface protein marker analyses, we show that the stirred suspension bioreactor environment helps maintain a naïve-like pluripotent state. Our research corroborates that converting hPSCs towards a naïve state enhances hPSC manufacturing and indicates a potentially important role for the stirred suspension bioreactor's mechanical environment in maintaining naïve-like pluripotency.
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Project description:Various culture systems have been used to derive and maintain human pluripotent stem cells (hPSCs), but they are inefficient in sustaining cloning and suspension expansion of hPSCs. Through systematically modulating Wnt and Activin/Nodal signaling, we developed a defined medium (termed AIC), which enables efficient cloning and long-term expansion of hPSCs (AIC-hPSCs) through single-cell passage on feeders, matrix or in suspension (25-fold expansion in 4 days) and maintains genomic stability of hPSCs over extensive expansion. Moreover, the AIC medium supports efficient derivation of hPSCs from blastocysts or somatic cells under feeder-free conditions. Compared to conventional-culture hPSCs, AIC-hPSCs have similar gene expression profiles but down-regulated differentiation genes and display higher metabolic activity. The AIC medium shows a good compatibility for suspension expansion of different hPSC lines. Our study provides a robust culture system for derivation, cloning and suspension expansion of high-quality hPSCs that benefits GMP production and processing of therapeutic hPSC products
2020-04-23 | GSE145163 | GEO
Project description:Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems
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