(151d) Human Pluripotent Stem Cell Expansion and Endoderm Differentiation in an Automated Stirred-Suspension Bioreactor

Human pluripotent stem cells (hPSCs) are a potentially limitless source of cellular therapeutics and cells for drug screening. Consequently, a robust, scalable, and xeno-free stem cell expansion and differentiation process is necessary to meet clinical demand. We demonstrate the expansion of hPSCs in a controlled stirred-suspension bioreactor as aggregates and their subsequent specification to definitive endoderm (DE) as an example of differentiation towards a germ layer, which can then be differentiated into medically relevant types such as pancreatic cells and hepatocytes. This process allows the production of hPSCs in a homogenous and regulated environment meeting the 10⁸-10⁹ cells/patient demand of most stem cell-based treatment protocols.

H9 human embryonic stem cells (hESCs) and IMR90-4 induced PSCs (hiPSCs) were seeded in a 250 mL instrumented bioreactor followed by the formation of aggregates. Mitigation of shear stress, which was accomplished through the gradual increase in the agitation rate after seeding, was found to be essential for maintenance of appropriate aggregate size, viability and pluripotency. H9 and IMR90 cells were propagated for five and six days, respectively, in xeno-free medium. Culture for longer periods between passages led to decreased pluripotency. The pH in the bioreactor was maintained at 7.4±0.2 and dissolved oxygen was kept constant with controlled headspace aeration. The H9 and IMR90 cells grew 24- and 10-fold, respectively with corresponding viability of greater than 87-90%. Continuous cell proliferation resulted in increases in the mean aggregate size from 29.44±8.91 μm and 35.06±8.39 μm (day 0) to 192.57±72.60 μm and 179.07±57.56 μm (days 5-6), i.e. size that does not hamper oxygen transfer. Cultured aggregates retained their pluripotent state with 70.21±1.48% of H9 cells and 79.31±2.74% of IMR90 cells being NANOG⁺/POU5F1⁺ on day 5 and 6 as determined by flow cytometry. After the expansion stage, the culture was switched to xeno-free priming medium for a day. Cells were further differentiated for three (IMR90) or four (H9) days. By day 3, there were 87.36±6.10% FOXA2/SOX17⁺ IMR90-derived cells, whereas 69.36±2.80% FOXA2/SOX17⁺ cells were noted by day 4 in H9 hESC cultures. H9 aggregate differentiation had to be optimized since IMR90 aggregates differentiate to definitive endoderm more readily while H9 cell require an extra day of differentiation and inhibition of the GSK-3β and still do not achieve the same efficiency of differentiation. Agitation ensured proper mixing without adverse effects on aggregates, which maintained normal morphology through the expansion and differentiation stages. In parallel, data was collected for cells seeded at the same density and cultured in 6-well suspension plates with orbital shaking. While there were differences in the rate of hPSC growth in the two systems, differentiation in the bioreactor yielded a higher percent of DE cells compared to that in well suspension culture.

Ongoing work focuses on further coaxing hPSC-derived DE cells toward PDX1/NKX6.1⁺ cells in the bioreactor. We have successfully differentiated a hESC cell line, INS^GFP/W MEL1, in spinner flasks (as bioreactor surrogates) obtaining 33.34±2.79% GFP⁺ (INS⁺), 43.42±8.58 NKX6.1⁺, and 69.64±3.48 PDX1⁺ cells. Given the scalability of stirred-suspension cultivation systems, the successful expansion and directed differentiation of hPSCs in a fully instrumented stirred-suspension bioreactor paves the way for production of hPSC-derived progeny in commercially relevant scales while minimizing batch-to-batch variation. Moreover, the xeno-free maintenance and specification of hPSCs eliminates the risk of cross contamination with animal-derived components. Our findings will contribute to biomanufacturing technologies for hPSC-based therapies.

Acknowledgements: This work is supported by the grants CBET-1743367 (EST) and CBET-1743407 (MH) from the National Science Foundation (NSF).