Growth Factor-Based Manufacturing of Human Pluripotent Stem Cell-Derived Cardiomyocytes Using the Vertical Wheel Bioreactor System
Bibliographic record
Abstract
Abstract Background Multiple protocols have been reported for the large-scale generation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in bioreactors using small molecules; however, no comparable bioreactor-based methods have been established using growth factors. This is despite evidence that differentiation with optimized concentrations of BMP4, Activin A, and bFGF offers finer control of cardiomyocyte phenotype. Here, we develop scalable hPSC expansion and growth factor-based cardiac differentiation protocols using the vertical wheel bioreactor (VWBR) system. Methods and Results The expansion of undifferentiated hPSCs was first optimized in 100 mL VWBRs by varying parameters, including starting cell seeding density, agitation rate, and media exchange schedule. Proliferation, viability, aggregate diameter, media metabolites, and pluripotency were assessed during hPSC expansion. Next, we evaluated the effects of undifferentiated hPSC culture conditions on subsequent cardiomyocyte differentiation potential. We found that hPSCs expanded in static culture or in VWBRs at different densities and agitation rates all differentiated into hPSC-CM populations of similar cardiac purity; however, cardiomyocyte yields were initially lower when VWBR-expanded hPSCs were used. We compared the differentiation kinetics of hPSCs expanded in VWBRs to conventional 2D culture and found that the former had accelerated mesodermal commitment and significantly greater cKit + /CXCR4 + /PDGRFα - cell formation during differentiation. Modifying our aggregation and mesoderm induction steps improved cell yields and enabled reliable production of >1x10 6 cells/mL cardiac troponin T + (cTnT) hPSC-CMs. Highlighting the versatility of our growth factor-based system, variation in the BMP4:Activin A ratio enabled a second heart field-like differentiation and generation of atrial-like cardiomyocytes in VWBRs. We further show that our expansion and differentiation protocols are reproducible and economical in 500 mL VWBRs, yielding on average 1.11x10 6 hPSC-CMs/mL at a mean purity of 93% cTnT + . Characterization of VWBR produced hPSC-CM force generation, action potentials, and intercellular calcium transients confirmed the expected phenotype of ventricular-like cells. Lastly, VWBR produced hPSC-CMs robustly engrafted in the infarcted guinea pig myocardium, supporting use as a cell therapy product. Conclusions This novel bioreactor-based protocol will enable cardiac cell therapy and tissue engineering applications by providing scalable and consistent production of hPSC-derived cardiac cell products.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".