Stirred Suspension Bioreactors Maintain Naã¯Ve Pluripotency Of
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ARTICLE https://doi.org/10.1038/s42003-020-01218-3 OPEN Stirred suspension bioreactors maintain naïve pluripotency of human pluripotent stem cells Leili Rohani1, Breanna S. Borys2,3, Golsa Razian1, Pooyan Naghsh1, Shiying Liu1, Adiv A. Johnson4, Pranav Machiraju 5, Heidrun Holland6, Ian A. Lewis7, Ryan A. Groves7, Derek Toms 8, Paul M. K. Gordon9, ✉ Joyce W. Li1, Tania So2,3, Tiffany Dang2,3, Michael S. Kallos2,3 & Derrick E. Rancourt 1 1234567890():,; Due to their ability to standardize key physiological parameters, stirred suspension bior- eactors 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. 1 Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. 2 Pharmaceutical Production Research Facility, Schulich School of Engineering, University of Calgary, Calgary, AB, Canada. 3 Biomedical Engineering Graduate Program, University of Calgary, Calgary, AB, Canada. 4 Nikon Instruments, Melville, New York, NY, USA. 5 Department of Paediatrics and Alberta Children’s Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. 6 Saxonian Incubator for Clinical Translation (SIKT), University of Leipzig, Leipzig, Germany. 7 Department of Biological Sciences, University of Calgary, Calgary, AB, Canada. 8 Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada. 9 CSM Center for Health Genomic and Informatics, University of ✉ Calgary, Calgary, AB, Canada. email: [email protected] COMMUNICATIONS BIOLOGY | (2020) 3:492 | https://doi.org/10.1038/s42003-020-01218-3 | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01218-3 uman pluripotent stem cells (hPSCs) harbor enormous Results Hpotential for regenerative medicine and offer a unique Cell-state conversion enhances hPSC bioprocessing. Previous opportunity to advance the development of cell-based reports have exploited combinations of small molecules, inhibi- therapies1. The development of hPSC biobanks1, clinical trials2, tors, and genetic manipulation20–22,28,29 to convert hPSCs from drug screening3, and disease modeling4 all demand robust stem primed-to-naïve pluripotency. In our study, the conversion was cell manufacturing and expansion. Although the hPSC manu- achieved using commercial RSeTTM medium. This is a defined facturing field is evolving rapidly, a bottleneck is throttling clin- media based on a bFGF- and TGFβ-free version of NHSM20. ical application: existing technologies are limited in their ability to Conversion in static culture produced robust cultures with naïve efficiently scale production of clinically-viable stem cells5. hPSC features, including tightly packed, domed colonies with Previous work suggests that expanding hPSCs as aggregates in refractive edges (Fig. 1). Resulting naïve hPSC colonies were stirred suspension bioreactors offers significant production maintained on inactivated mouse embryonic fibroblasts (iMEFs) advantages compared to static cell culture6,7. Key physiological in RSeT medium for several passages (Fig. 1). From passage (P) parameters can be controlled and standardized in these platforms, four onwards, the resulting established naïve hPSC colonies from allowing high-quality batches with large cell numbers8. Although static culture (P4) showed high expression levels of naïve plur- we8–10 and others11 have demonstrated that murine (m) PSCs are ipotency markers and the disappearance of XIST gene expression expanded robustly in suspension bioreactors, hPSC expansion in (Supplementary Fig. 1a), the latter being a hallmark of naïve these systems have resulted in lower fold-expansion and yields12 pluripotency30. and high costs13–15. Compared to mPSCs, the manufacturing of We have previously shown significant interaction effects conventional hPSCs in stirred suspension bioreactors has proven between inoculation density and agitation rate for hPSCs in more difficult and has resulted in significantly lower fold- stirred suspension culture31. Here, we applied different inocula- expansion14,16. tion cell densities and agitation rates for naïve hPSC in stirred Mouse and human PSCs exhibit distinctive epigenetic features, suspension cultures. We found that uniform-sized aggregates self-renewal signaling requirements, and differentiation potential. formed at inoculation densities of 1E4, 2.5E4, and 5E4 cells/mL in Conventional hPSCs display a “primed” state of pluripotency the bioreactor (Supplementary Fig. 1b). We also observed that an similar to mouse epiblast stem cells derived from the post- agitation rate of 100 RPM (maximum fluid shear 6 dynes/cm2) implantation epiblast17. Conversely, mPSCs reside in the so- facilitated the formation of aggregates with a healthy morphology called “naïve” pluripotent state resembling the pre-implantation and an average diameter below levels where we would expect epiblast18. Prior reports have shown that specific culture condi- necrosis to occur32. We increased seeding densities of naïve tions can either generate naïve hPSCs directly from embryos or hPSCs in suspension culture through agitated well plate systems. convert primed hPSCs to the naïve state19. Compared to the Naïve hPSCs were cultured in 24- and 6-well shaken suspension primed state, naïve hPSCs have advantageous bioprocessing plates at seeding densities of 1E5 and 2E5 cells/mL for five days, properties, including a higher single-cell cloning efficiency20,an and cultures underwent a single 60% media exchange on day 3. enhanced differentiation potential21, an advanced adherent Unlike bioreactor-cultured, naïve hPSCs, shaken suspension growth rate19,22, bivalent metabolic activity22,23, an altered tran- culture in well-plates resulted in less uniform aggregate sizes scriptome, and a hypomethylated epigenome20,22,24,25. Currently, with dark centers that may represent partly necrotic or apoptotic the field has a limited understanding of how the hPSC state cores (Supplementary Fig. 1c). influences bioprocessing and whether this can resolve existing cell Having identified a successful combination of inoculation cell manufacturing gaps. density and agitation rate in stirred suspension bioreactors, we Recently, Lipsitz et al. reported that the conversion of hPSCs to assessed growth kinetics and aggregate formation characteristics an alternative pluripotent state enhanced suspension culture in both naïve and primed hPSCs. To evaluate each cell type’s yield26. However, pluripotency maintenance of this “high-sus- growth kinetics in stirred suspension culture, newly-converted pension-yield” state was dependant on ERK inhibitory cytokine naïve hPSCs (P0 from static culture), established naïve hPSCs (P4 withdrawal from suspension culture. This suggests that the cell’s from static culture), and equivalent primed hPSCs, were each interaction with its mechanical environment and media compo- inoculated into separate 100 mL stirred suspension bioreactors nents plays a critical role in maintaining naïve-like pluripotency agitated at 100 rpm. Naïve hPSCs were then counted at passages within the bioreactor. one (P1) and five (P5), respectively. Batch conditions were used to Considering that study26, the robust expansion of mPSCs in determine growth kinetics for five days post-inoculation without bioreactors8–11,16, and knowing that naïve hPSCs resemble the effect of a feeding regime. Here, growth rates and multi- mPSCs, we report here that cell-state conversion significantly plication ratios were significantly greater, while exponential enhances hPSC bioprocessing in stirred suspension culture. Since doubling times were significantly shorter for both P1 and P5 naïve hPSCs are metastable27, we sought to assess the relationship naïve hPSCs compared to their primed counterparts (Fig. 2a, between naïve pluripotency and bioreactors. Specifically, we Supplementary Fig. 2a). The exponential growth rates for P1 aimed to identify whether or not the bioreactor mechanical naïve hPSCs) and P5 naïve hPSCs were 1.5X greater than those environment influences the naïve pluripotency state. Through observed in primed hPSCs. transcriptomics, assessing the expression of epigenetic regulators, In agreement with previous studies, primed hPSCs experienced metabolomics, and cell-surface protein marker analyses, we show extensive cell death upon inoculation and an extended lag period that the bioreactor environment helps maintain the naïve plur- in bioreactor culture12,26,33. By contrast, naïve hPSCs recovered ipotent state. We observed enrichment of the mechano-sensing and expanded in unoptimized batch culture conditions with fold- HIPPO signalling in the