title: Rational bioprocess design for human pluripotent stem cell expansion and endoderm differentiation based on cellular dynamics.
abstract: We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation, and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and, with quantitative cell division tracking and fate monitoring, identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion, during directed differentiation, to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Ungrin
authors.forename: Geoff
authors.initials: G
authors.lastname: Clarke
authors.forename: Ting
authors.initials: T
authors.lastname: Yin
authors.forename: Sylvia
authors.initials: S
authors.lastname: Niebrugge
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Nostro
authors.forename: Farida
authors.initials: F
authors.lastname: Sarangi
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wood
authors.forename: Gordon
authors.initials: G
authors.lastname: Keller
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000032:analysis; D061249:Batch Cell Culture Techniques; D002449:Cell Aggregation; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line / Q000166:cytology / Q000187:drug effects; D018920:Coculture Techniques; D003470:Culture Media; D004707:Endoderm / Q000166:cytology*; D005347:Fibroblasts / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D051379:Mice; D010179:Pancreas / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology*; D013535:Suspensions; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D000943:Antigens, Differentiation; D003470:Culture Media; D013535:Suspensions