fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation

• Published in: Proceedings of the National Academy of Sciences - PNAS 2013-12, Vol.110 (52), p.E5039-E5048
• Main Authors: Lei, Yuguo, Schaffer, David V
• Format: Article
• Language: English
• Subjects: Animals; Biomedical research; Cell Culture Techniques - methods; Cell Differentiation - physiology; Cell Proliferation; Humans; Hydrogels; Karyotyping; Mice; Neural Stem Cells - cytology; Neurons; Physical Sciences; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - physiology; PNAS Plus; Proteins; Stem cells; Teratoma - pathology; Tissue Engineering - methods
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1309408110

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.