Giovanni G. Giobbe and Monica Zagallo contributed equally to this work.
Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells†
Article first published online: 20 JUN 2012
Copyright © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 109, Issue 12, pages 3119–3132, December 2012
How to Cite
Giobbe, G. G., Zagallo, M., Riello, M., Serena, E., Masi, G., Barzon, L., Di Camillo, B. and Elvassore, N. (2012), Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells. Biotechnol. Bioeng., 109: 3119–3132. doi: 10.1002/bit.24571
- Issue published online: 25 OCT 2012
- Article first published online: 20 JUN 2012
- Accepted manuscript online: 4 JUN 2012 08:23AM EST
- Manuscript Accepted: 23 MAY 2012
- Manuscript Revised: 24 APR 2012
- Manuscript Received: 28 DEC 2011
- human embryonic stem cells;
- human induced pluripotent stem cells;
- embryoid bodies;
- hydrogel microwells;
- endogenous factors
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.