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Scalable processes are requisite for the robust biomanufacturing of human pluripotent stem cell (hPSC)-derived therapeutics. Toward this end, we demonstrate the xeno-free expansion and directed differentiation of human embryonic (hESCs) and induced pluripotent cells (hiPSCs) to definitive endoderm (DE) in a controlled stirred suspension bioreactor (SSB). Based on previous work on converting hPSCs to insulin-producing progeny, differentiation of two hPSC lines was optimized in planar cultures yielding up to 87% FOXA2+ /SOX17+ cells. Next, hPSCs were propagated in a SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5-6 days with maintenance of pluripotency (>85% OCT4+ ) and viability (>85%). For differentiation, SSB cultures yielded up to 89% FOXA2+ /SOX17+ cells or ~ 8 DE cells per seeded hPSC. Specification to DE cell fate was consistently more efficient in the bioreactor compared to planar cultures. Hence, a tunable strategy is established that is suitable for the xeno-free manufacturing of DE cells from different hPSC lines in scalable SSBs. This work advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics. This article is protected by copyright. All rights reserved.

Human pluripotent stem cells (hPSCs) hold great promise as cellular therapeutics and in vitro models of disease. These applications require advances in hPSC biomanufacturing in scalable cultivation systems. Stirred suspension bioreactors (SSBs), which are commonly utilized for the production of biologics, afford real-time monitoring and tight control of the culture environment. Stem cell expansion has been demonstrated in this modality but knowledge is currently limited of how the bioreactor milieu influences hPSC physiology. Here, relevant findings are discussed on the effects of adjustable factors such as dissolved oxygen, hydrodynamic cues, and composition of media on the expansion of hPSCs. Greater understanding of the links between cultivation conditions and hPSC self-renewal and propensity for differentiation will be essential for the rational design of processes to robustly biomanufacture hPSC products for clinical use.