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]

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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 stirred suspension bioreactor, supporting increases of 4.09 ± 0.09 (P1) to 6.90 ± 0.03 (P5) within five days the hypothesis that a mechanical environment plays an important (Fig. 2a and Supplementary Fig. 2a). Exponential growth rates role in maintaining naïve pluripotency. This observed positive were comparable between P1 and P5 naïve hPSCs, but the fold- influence of the bioreactor environment on naïve pluripotency increase was greater for P5 (Fig. 2a). The difference in fold- indicates it may be possible to revert to naïve pluripotency in the increase between P1 and P5 was concomitant with the bioreactor. Further optimization could enhance hPSC biomanu- disappearance of XIST gene expression with subsequent passages facturing for both clinical and research applications. (Supplementary Fig. 1a).

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Primed Naïve mTeSR1 RSeT Reprogram

500μm P0 500μm P1 500μm P2 500μm P3 500μm P4 500μm Passage Passage Passage Passage

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Feed 0 123 45 (FB) 0 123 45 = Primed = Naïve = Counts Feed = Aggregate Collection Feed (FB) = Gene Expression (RT-qPCR) (FB) = Transcriptomic (RNA-seq) = Metabolomics = Cell Surface Protein Marker = Epigenetic Characterization = Differentiation Potential = Chromosomal Stability

Fig. 1 Experimental design. Conversion from a primed to naïve state of pluripotency. Schematic representation of experimental design and morphological changes during transition from primed (flat) towards naïve (dome-shaped) hPSC colonies in static culture (upper panel). Conversion from the primed to naïve pluripotent state was done using RSeT media. Naïve hPSC colonies are shown at several passages (P0–P4) on iMEFs following conversion (upper panel). The arrowheads under the static culture dishes indicate the inoculation of cells from static culture into static suspension and stirred suspension culture for each naïve (P0 and P4) and primed hPSC sample. Following inoculation, naïve hPSCs were counted as passages one and five, respectively. Aliquots of static-cultured cells from naïve (P4) and primed hPSC samples were collected for downstream analyses before inoculation. Batch and fed-batch conditions were used for suspension culture in bioreactors. A fed-batch condition was used for the static suspension culture condition. Sample images of aggregate morphology for naïve and primed hPSCs at days 1 and 5 post-inoculation under batch and fed-batch are shown. The bars below the images of an aggregate’s morphology demonstrate the number of days post-inoculation. Static suspension and stirred suspension bioreactor sample collections for various analyses are shown by different coloured-bullets in the lower-most panel. The days of sample collections for different analyses are shown by the coloured-bullets under the bar. For the fed-batch condition, the media change and feeding were done on day 2 post-inoculation (60% media change, 48-h post-inoculation). Scale bars = 200 µm. iMEFs inactivated mouse embryonic fibroblasts.

To evaluate the effect of feeding regime on growth kinetics in naïve hPSCs (P0 from static culture) (Fig. 2a). Based on our stirred suspension culture, established naïve (P4 from static previous work31, we examined the effect of a 60% media exchange culture) and equivalent primed hPSCs were inoculated into 48 h after cell inoculation. For both primed and naïve hPSCs, bioreactors under batch and fed-batch conditions (Fig. 2b). We there was no significant difference in cell expansion or growth used established naïve hPSCs (P4 from static culture) as they kinetics between batch and fed-batch conditions. This indicates demonstrated a superior fold-increase compared to newly-formed that nutrient limitations or buildup of metabolites and waste

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ab 4.E+05 Naïve P1 Primed - Batch 4.E+05 Naïve P5 *** Primed - Fed Batch 3.E+05 **** Naïve P5 - Batch Primed 3.E+05 Naïve P5 - Fed Batch 2.E+05 ** 2.E+05 ** 1.E+05 1.E+05 Viable Cells/mL Viable Viable Cells/mL Viable 0.E+00 1.E+04 0 20 40 60 80 100 120 020406080100120 Time (h) Time (h)

c 5E+05 d 160 Naïve P5 **** 4E+05 Primed 120 3E+05 ****

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Fig. 2 Bioreactor growth kinetics of naïve and primed hPSCs. a Growth kinetics of naïve and primed hPSCs at different days (D0–D5) post-inoculation under batch condition. Statically cultured, naïve hPSCs were inoculated into bioreactors at passages zero and four following primed to naïve conversion in static culture wherein, they were counted as passages one and five, respectively. b Growth kinetics of naïve and primed hPSCs at different days (D0–D5) post-inoculation under batch and fed-batch (60% media change, 48-h post-inoculation) conditions. Static-cultured, naïve hPSCs were inoculated into bioreactors at passage four following conversion in static culture, wherein they were counted as passage five. c Growth kinetics of naïve hPSCs from H1 and H9 hESC lines at different days (D0–D5) post-inoculation under fed-batch (60% media change, 48-h post-inoculation) conditions. Static-cultured, naïve hPSCs for both cell lines were inoculated into bioreactors at passage four following conversion in static culture, wherein they were counted as passage five. d Bioreactor-cultured aggregate growth. Average aggregate size was measured on days 1–5 post-inoculation to detect bioreactor-cultured aggregate growth in naïve (P5) and primed hPSC samples. e Size distribution of bioreactor-cultured aggregates. Distribution of bioreactor-cultured, naïve (P5) and primed hPSC aggregates were measured on day 5 post-inoculation using the Beckman Coulter Multisizer III. It was defined as multi-cellular spheroids with a mean diameter greater than 50 μm. f Aggregate growth morphology. Representative images show naïve and primed hPSC aggregate morphology at different days (D1, D3, and D5) post-inoculation. The data presented are generated from inoculation density of 50,000 cells/mL. The data are represented as mean ± SEM (n = 4). **** = Adj-P < 0.0001 using GraphPad Prism. Scale bars = 200 µm. products were likely not limiting factors for cell growth. Under at this point could be due to the feeding strategy and might both batch and fed-batch conditions, bioreactor-cultured, naïve indicate that more nutrients may be required for continued hPSCs showed significantly superior growth kinetics compared to expansion of naïve hPSCs seeded at higher densities in the shaken their primed equivalent (Fig. 2b). Both H1 and H9 naïve hPSC suspension well-plates. lines showed comparable average fold-expansion (7.88 and 8.37, We next evaluated the aggregate formation characteristics of respectively) following five days of fed-batch stirred suspension naïve and primed hPSCs in suspension culture, as aggregate size culture (Fig. 2c). is an important bioprocessing variable that has implications on Naïve hPSCs seeded at higher densities in shaken suspension cell health and differentiation32. We assessed aggregate growth by well-plates (1E5 and 2E5 cells/mL) exhibited similar day 3 fold- measuring the average aggregate size for five days post- expansions (1.78 and 2.73-fold, respectively) to the bioreactor inoculation in samples from naïve (P5) and equivalent primed culture seeded at the lower density of 5E4 cells/mL (2.47-fold). hPSCs in stirred suspension culture. Naïve hPSC aggregates After day 3, however, cell growth in the shaken suspension well- showed significantly greater growth compared to their primed plates plateaued (Supplementary Fig. 2b). The expansion plateau counterparts at all time points (Fig. 2d). Primed hPSC aggregates

4 COMMUNICATIONS BIOLOGY | (2020) 3:492 | https://doi.org/10.1038/s42003-020-01218-3 | www.nature.com/commsbio COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-020-01218-3 ARTICLE that formed on day 1 had an average diameter of 38.8 ± 6.5 µm preserved more distance from the stirred suspension culture and expanded minimally, reaching an average diameter of only (PC1) (Supplementary Fig. 3c). 57.3 ± 13.7 µm by day 5. Aggregates of naïve hPSCs increased Transcriptomic comparison of naïve hPSCs cultured under from an average diameter of 54.0 ± 6.5 µm on day 1 to 134.4 ± static suspension and static conditions revealed that 4,039 18.6 µm on day 5 (Fig. 2d). The naïve hPSC aggregates had a transcripts were differentially expressed (Supplementary Data 1). relatively uniform size distribution showing a single defined peak We used Ingenuity Pathway Analysis (IPA) and its z-score at an average diameter of 130–200 µm only four days following algorithm to identify enriched canonical pathways that were more bioreactor seeding, which is an ideal aggregate size based on our or less active (positive or negative z-score) according to the IPA data and data from others31,34. By contrast, primed hPSC database. To enhance the stringency of our analysis, we aggregates had a larger standard deviation in diameter size in considered z-score ≥2or≤−2, and –log10(Benjamini–Hochberg the range of 50–90 µm (Fig. 2e). Primed hPSCs showed poor multiple testing corrected p value of differential expression) ≥2as aggregation and were smaller than naïve hPSC aggregates, which the filter criteria for pathway enrichment significance. The results showed few single cells one day post-inoculation, and further of IPA showed enrichment of 9 canonical pathways in static formed larger uniform aggregates past days 3 and 5 in stirred suspension compared to static condition (Supplementary Data 2). suspension bioreactors (Fig. 2f). We found negative enrichment (negative z-score, implying We further attempted to serial passage naïve hPSCs in 6-well downregulation) of the Mouse ESC Pluripotency pathway (z- shaken suspension plates (seeded at 2E5 cells/mL, high density) in score: −2.302) in naïve hPSCs cultured in static suspension addition to 100 mL stirred suspension bioreactors (seeded at 5E4 culture, corroborating the decrease of pluripotency in these cells cells/mL, low density). The cells were passaged every five days. In (Fig. 3b, Supplementary Table 1). Furthermore, negative enrich- both systems, following the first few days of passage two, the ment of pathways related to cell–cell connection and adhesion aggregates began to fall apart (Supplementary Figs. 2c, d). In the such as Actin Cytoskeleton and RhoA signalling were shown in first three days of passaging, percent viability declined from 52 ± static suspension culture (Fig. 3b, Supplementary Table 1). 1.65% on day 1 to 39 ± 2.2% on day 3 (Supplementary Fig. 2e). Positive enrichment (positive z-score, implying upregulation) of Although aggregates still were able to form (Supplementary AMPK signaling (Fig. 3b, Supplementary Table 1) matched our Fig. 2f), the growth was reduced with each passage. observations that aggregates formed in static suspension condi- tion demonstrated necrotic or apoptotic cores (Supplementary Fig. 1c). Previous reports showed that AMPK is activated in Bioreactors support a naïve pluripotency transcriptomic state. response to stressors such as ischemia and necrotic cores37. In-line with previous naïve hPSC reports24–26,28,29,35, our RT- Transcriptomic comparison of naïve hPSCs cultured under qPCR analysis showed elevated expression levels of naïve hPSC- stirred suspension and static culture revealed that 10,181 associated genes (i.e., KLF2 and KLF5) in bioreactor-cultured, transcripts were differentially expressed (Supplementary Data 3). naïve hPSCs (Fig. 3a). Consistent with previously published IPA analysis showed enrichment of 31 canonical pathways in findings24,25,35,36, we also observed significantly reduced expres- stirred suspension compared to static culture (Supplementary sion of the primed hPSC-associated genes (i.e., OTX2, ZIC2, and Data 4). Substantially, we found positive enrichment of the DUSP6) in bioreactor-cultured, naïve hPSCs (Fig. 3a, Supple- mechano-sensing HIPPO signaling pathway in stirred suspension mentary Fig. 3a). culture (z-score: 3.162) (Fig. 4a, Supplementary Table 2). We also Increased levels of naïve (i.e., KLF2, KLF4, and STELLA) and found negative enrichment for several lineage specific pathways decreased levels of primed (i.e., CD24) pluripotency genes were with neural bias such as Synaptogenesis as well as primed observed in naïve hPSCs cultured in the bioreactor (Supplemen- pluripotency related pathways such as Stearate Biosynthesis, in tary Fig. 3b). We also saw an intermediate level of KLF4 stirred suspension culture (Fig. 4a, Supplementary Table 2). Like expression in bioreactor-cultured, naïve hPSCs, which is static suspension culture, pathways related to cell–cell connection, consistent with previous reports showing that KLF4 is inter- adhesion, and extracellular matrix (ECM) such as Integrin mediately expressed in naïve hPSCs converted by RSeT exhibited negative enrichment in stirred suspension culture medium35,36. Regardless, the expression level of KLF4 was high (Fig. 4a, Supplementary Table 2). More enriched canonical in bioreactor-cultured, naïve hPSCs compared to their primed pathways are shown in Supplementary Data 4 and Supplementary counterparts (Supplementary Fig. 3b). Our finding that bior- Table 2. eactor-cultured, naïve hPSCs display strong KLF2 expression Finally, we compared the transcriptomes of