Abstract
Maintenance of the epithelium relies on stem cells residing within specialized microenvironments, known as epithelial crypts. Two-photon polymerization (2PP) is a valuable tool for fabricating 3D micro/nanostructures for stem cell niche engineering applications. Herein, biomimetic gelatin methacrylate-based constructs, replicating the precise geometry of the limbal epithelial crypt structures (limbal stem cell "microniches") as an exemplar epithelial niche, are fabricated using 2PP. Human limbal epithelial stem cells (hLESCs) are seeded within the microniches in xeno-free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers. Cell proliferation and a zonation in cell phenotype along the z-axis are observed without the use of exogenous signaling molecules. Significant differences in cell phenotype between cells located at the base of the microniche and those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides. This study provides insight into the influence of the niche's spatial geometry on hLESCs and demonstrates a flexible approach for the fabrication of biomimetic crypt-like structures in epithelial tissues. This has significant implications for regenerative medicine applications and can ultimately lead to implantable synthetic "niche-based" treatments.
Highlights
Maintenance of the epithelium relies on stem cells residing within specialized lated by intrinsic and extrinsic factors, microenvironments, known as epithelial crypts
Human limbal epithelial stem cells medicine-based therapies is being able are seeded within the microniches in xeno-free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers
Significant difto reproduce this instructive microenvironment, or “microniche.” Advancing stem cell niche engineering is important for regenerative medicine applications, including the development of ferences in cell phenotype between cells located at the base of the microniche predictive disease models, stem cell theraand those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides
Summary
Chemicals were purchased from Sigma-Aldrich (UK) unless otherwise stated. GelMA Synthesis: GelMA was synthesized as described previously.[43]. The solution was diluted 5× with PBS and stirred for a further 30 min. The solution was dialyzed against distilled water using 8 kDa cut-off dialysis tubing (BioDesignDialysis Tubing, USA) for 1 week at 50 °C. Quantification of Degree of Crosslinking: Percentage of methacrylation was quantified using 1H NMR (400 MHz; Bruker UK Ltd., UK) at room temperature (RT). GelMA and gelatin were dissolved in deuterium oxide (10 mg mL–1). Presence of a double bond signal (5.35–5.60 ppm) indicated addition of the methacrylate vinyl group. Percentage of methacrylation was calculated using the method described by Hoch et al.[44] The integral of the methylene peak (d = 2.7–2.9 ppm) was used for quantification of the lysine signal. Degree of substitution was determined by comparing lysine signals of modified and unmodified gelatin (Equation (1)). Intensity of the aromatic region (7–7.5 ppm) was used as reference
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