title: A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation.
abstract: Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, are promising for numerous biomedical applications, such as cell replacement therapies, tissue and whole-organ engineering, and high-throughput pharmacology and toxicology screening. Each of these applications requires large numbers of cells of high quality; however, the scalable expansion and differentiation of hPSCs, especially for clinical utilization, remains a challenge. We report a simple, defined, efficient, scalable, and good manufacturing practice-compatible 3D culture system for hPSC expansion and differentiation. It employs a thermoresponsive hydrogel that combines easy manipulation and completely defined conditions, free of any human- or animal-derived factors, and entailing only recombinant protein factors. Under an optimized protocol, the 3D system enables long-term, serial expansion of multiple hPSCs lines with a high expansion rate (~20-fold per 5-d passage, for a 10(72)-fold expansion over 280 d), yield (~2.0 × 10(7) cells per mL of hydrogel), and purity (~95% Oct4+), even with single-cell inoculation, all of which offer considerable advantages relative to current approaches. Moreover, the system enabled 3D directed differentiation of hPSCs into multiple lineages, including dopaminergic neuron progenitors with a yield of ~8 × 10(7) dopaminergic progenitors per mL of hydrogel and ~80-fold expansion by the end of a 15-d derivation. This versatile system may be useful at numerous scales, from basic biological investigation to clinical development.
authors.affiliation: Departments of Bioengineering and Chemical Engineering, California Institute for Quantitative Biosciences, and Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720.
authors.forename: Yuguo
authors.initials: Y
authors.lastname: Lei
authors.forename: David V
authors.initials: DV
authors.lastname: Schaffer
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D006801:Humans; D020100:Hydrogels; D007621:Karyotyping; D051379:Mice; D058953:Neural Stem Cells / Q000166:cytology; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D013724:Teratoma / Q000473:pathology; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D020100:Hydrogels