RESEARCH ARTICLE


The Generation of Definitive Endoderm from Human Embryonic Stem Cells on 3D Biodegradable Poly(lactic--glycolic Acid) Scaffolds and its Comparison to those Generated on 2D Monolayer Cultures



Steven Y. Gao*, 1, 4, Jennifer C.Y. Wong1, 4, Justin G. Lees1, 5, Marie B. Best1, Rennian Wang2, Peter A. George3, Justin J. Cooper-White3, Bernard E. Tuch1, 6
Sydney Centre for Developmental and Regenerative Medicine, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, St Leonards, New South Wales, 2065, Australia.


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Gao et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Sydney Centre for Developmental and Regenerative Medicine, Kolling Institute of Medical Research, Royal North Shore Hospital and University of Sydney, St Leonards, New South Wales, 2065, Australia. stevengao@med.usyd.edu.au


Abstract

The generation of insulin producing cells from human embryonic stem cells (hESCs) has shown great promise as a cellular replacement therapy for the treatment of Type 1 Diabetes. Mature functional β-cell surrogates however, have yet to be successfully generated in vivo. One approach to potentially improve current differentiation protocols is the use of 3 dimensional (3D) scaffolds, which has been shown to enhance cellular function and differentiation potential. The present study aimed to explore the feasibility of using single cell preparations of pluripotent hESCs seeded onto laminin or Matrigel coated 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds to derive definitive endoderm, the first vital stage of endoderm tissue differentiation. Our results demonstrated that hESCs which were induced to differentiate on laminin or Matrigel coated 3D scaffolds can be successfully coaxed to differentiate into definitive endoderm. The cells that were cultured on laminin or Matrigel coated 3D scaffolds expressed significantly higher levels of the key endoderm transcription factors SOX17 and FOXA2 in comparison to those differentiated on 2D monolayers. On Matrigel coated 3D scaffolds, the differentiated cells expressed lower levels of the endoderm surface marker CXCR4 and anterior endoderm marker CER in comparison to its monolayer counterpart. Together, the results of this study demonstrated the positive effect of 3D cultures on endoderm commitment from hESCs over traditional monolayer cultures. Furthermore, the definitive endoderm produced on Matrigel coated scaffolds may have a more posterior phenotype in comparison to those derived from monolayers. This may have an effect on later stages of pancreatic differentiation and warrants further detailed investigations.

Keywords: embryonic stem cells , differentiation, Poly(lactic-co-glycolic acid) , scaffold, endoderm, pancrease.