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

Author:
Lei, Yuguo, Schaffer, David V.
Source:
Proceedings of the National Academy of Sciences of the United States of America 2013 v.110 no.52 pp. E5039
ISSN:
0027-8424
Subject:
embryonic stem cells, engineering, humans, hydrocolloids, induced pluripotent stem cells, neurons, pharmacology, recombinant proteins, screening, toxicology
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.
Agid:
1784907