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Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

Authors

  • Giovanni G. Giobbe,

    1. Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
    2. Venetian Institute of Molecular Medicine (VIMM), via Orus 2, 35129 Padua, Italy
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  • Monica Zagallo,

    1. Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
    2. Venetian Institute of Molecular Medicine (VIMM), via Orus 2, 35129 Padua, Italy
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  • Massimo Riello,

    1. Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
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  • Elena Serena,

    1. Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
    2. Venetian Institute of Molecular Medicine (VIMM), via Orus 2, 35129 Padua, Italy
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  • Giulia Masi,

    1. Department of Histology, Microbiology and Medical Biotechnologies, via Gabelli 63, 35131 Padua, Italy
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  • Luisa Barzon,

    1. Department of Histology, Microbiology and Medical Biotechnologies, via Gabelli 63, 35131 Padua, Italy
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  • Barbara Di Camillo,

    1. Department of Information Engineering (DEI), University of Padua, via Gradenigo 6B, 35131 Padua, Italy
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  • Nicola Elvassore

    Corresponding author
    1. Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
    2. Venetian Institute of Molecular Medicine (VIMM), via Orus 2, 35129 Padua, Italy
    • Department of Industrial Engineering (DII), University of Padua, via Marzolo 9, 35131 Padua, Italy; telephone: +39-049-8275469; fax: +39-049-8275461
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  • Giovanni G. Giobbe and Monica Zagallo contributed equally to this work.

Abstract

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification. Biotechnol. Bioeng. 2012; 109: 3119–3132. © 2012 Wiley Periodicals, Inc.

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