Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precu...

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Veröffentlicht in:Biotechnology journal 2015-10, Vol.10 (10), p.1612-1624
Hauptverfasser: Miranda, Cláudia C., Fernandes, Tiago G., Pascoal, Jorge F., Haupt, Simone, Brüstle, Oliver, Cabral, Joaquim M.S., Diogo, Maria Margarida
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Sprache:eng
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Zusammenfassung:3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically‐defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single‐cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC‐derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling‐up. 3D suspension culture is considered a promising approach to better mimic the in vivo cellular microenvironment. In this work, the authors developed a scalable integrated process, combining expansion and neural commitment of hPSCs with optimized spatial and temporal control of culture conditions, using suspension aggregates in a chemically‐defined environment. This approach resulted in a robust method for production of hPSC‐derived neural precursors, minimizing processing steps, and may constitute a promising alternative to the traditional planar adherent culture systems due to high potential for scaling‐up.
ISSN:1860-6768
1860-7314
DOI:10.1002/biot.201400846