Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems
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ABSTRACT:
PROVIDER: PRJNA1096957 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28574978_1.fastq.gz | Fastqsanger.gz | |||
| SRR28574978_2.fastq.gz | Fastqsanger.gz | |||
| SRR28574979.fastq.gz | Fastqsanger.gz | |||
| SRR28574980_1.fastq.gz | Fastqsanger.gz | |||
| SRR28574980_2.fastq.gz | Fastqsanger.gz |
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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:Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and 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, and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines, the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs, bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks, which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible, scalable, and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications, and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols.
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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 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.
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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.
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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.
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