Non-xenogeneic expansion and definitive endoderm differentiation of human pluripotent stem cells in an automated bioreactor

Elena F. Jacobson, Zijing Chen, Demetrios M. Stoukides, Gopika G. Nair, Matthias Hebrok, Emmanuel S. Tzanakakis

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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 and induced pluripotent stem cells 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 an SSB with controlled pH and dissolved oxygen. Cultures displayed a 10- to 12-fold increase in cell number over 5–6 days with the 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 study advances bioprocess development for producing a wide gamut of human DE cell-derived therapeutics.

Original languageEnglish
Pages (from-to)979-991
Number of pages13
JournalBiotechnology and Bioengineering
Issue number2
StatePublished - Feb 2021
Externally publishedYes


  • definitive endoderm (DE) differentiation
  • human pluripotent stem cells (hPSCs)
  • pluripotent expansion
  • stirred suspension bioreactor (SSB)
  • xeno-free


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