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. 2023 Sep 11;13(1):15012.
doi: 10.1038/s41598-023-42359-9.

An inspired microenvironment of cell replicas to induce stem cells into keratocyte-like dendritic cells for corneal regeneration

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An inspired microenvironment of cell replicas to induce stem cells into keratocyte-like dendritic cells for corneal regeneration

Mahsa Fallah Tafti et al. Sci Rep. .

Abstract

Corneal stromal disorders due to the loss of keratocytes can affect visual impairment and blindness. Corneal cell therapy is a promising therapeutic strategy for healing corneal tissue or even enhancing corneal function upon advanced disorders, however, the sources of corneal keratocytes are limited for clinical applications. Here, the capacity of cell-imprinted substrates fabricated by molding human keratocyte templates to induce differentiation of human adipose-derived stem cells (hADSCs) into keratocytes, is presented. Keratocytes are isolated from human corneal stroma and grown to transmit their ECM architecture and cell-like topographies to a PDMS substrate. The hADSCs are then seeded on cell-imprinted substrates and their differentiation to keratocytes in DMEM/F12 (with and without chemical factors) are evaluated by real-time PCR and immunocytochemistry. The mesenchymal stem cells grown on patterned substrates present gene and protein expression profiles similar to corneal keratocytes. In contrast, a negligible expression of myofibroblast marker in the hADSCs cultivated on the imprinted substrates, is observed. Microscopic analysis reveals dendritic morphology and ellipsoid nuclei similar to primary keratocytes. Overall, it is demonstrated that biomimetic imprinted substrates would be a sufficient driver to solely direct the stem cell fate toward target cells which is a significant achievement toward corneal regeneration.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Graphical illustration of (I) Isolation and culture of primary corneal stromal cells (keratocytes), to prepare keratocyte-imprinted PDMS substrate, (II) characterization of the imprinted substrate by SEM and AFM, (III) culture of stem cells on imprinted PDMS, (IV) characterization of the cells on the patterned substrate using SEM and AFM, (V) evaluations of gene and protein expression by differentiated cells.
Figure 2
Figure 2
Keratocytes and ADSCs characterization. (a) Light microscopy photographs of cultured keratocytes with dendritic morphology. (b) The gene expression profile demonstrated higher expression of positive keratocyte markers, including Lumican (LUM), Keratocan (KERA), ALDH3A1, and CD34 and minimal expression of myofibroblast marker (ACTA2) in keratocytes and ADSCs. (c,d) Immunofluorescence images of human keratocytes cultured for Keratocan (green)/lumican (red)/nuclei (DAPI, blue), and α-SMA (green)/nuclei (DAPI, blue). (e) Representative of average protein levels. (f) Light inverted microscopy photographs of ADSCs with spindle shape morphology. (g) Flow cytometry results showing up-expression of CD105, CD73, CD90, and down-expression of CD45 in hADSCs.
Figure 3
Figure 3
Observation of imprinted substrates by optical, SEM, and AFM. (a) Preparation process of patterned PDMS. (b) Light image of the cell-imprinted replicas on surface of PDMS. (c) The SEM image of keratocyte-imprinted substrate. (d,e) 2D and 3D AFM images of the patterned substrates.
Figure 4
Figure 4
Evaluation of surface roughness of PDMS substrates. (a,b) Profilometer maps of plain PDMS and patterned substrate. (c,d) AFM images of plain PDMS and patterned substrate. (e,f) Statistical analysis of surface parameters that shows significant increase of Ra and Rq in patterned substrate as compared to plain substrate. Data are mean ± SD. ***p < 0.001.
Figure 5
Figure 5
Characterization of cells on patterned substrates. (a) SEM image illustrates the ADSCs cultured on keratocytes-imprinted substrate. (b) AFM images of the ADSCs cultivated on keratocytes-imprinted substrate are shown in 2D and (c) 3D.
Figure 6
Figure 6
2D AFM topographic images of substrates for height profile. (a) A height profile of patterned substrate without cell (between A-A points). (b) Height profile after cell seeding on the imprinted substrate. (c) an irregulate height profile after emplacing the cell horns on the PDMS surface.
Figure 7
Figure 7
Quantitative analysis of gene expression by ADSCs after 7, 14 and 21 days of culture on cell-imprinted substrate compared to the plain PDMS and TCP as controls. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. ns non significant.
Figure 8
Figure 8
Immunocytochemistry of seeded cells for protein expression after 2 and 3 weeks’ cultivation on patterned PDMS (KBM), patterned PDMS (DMEM/F12) and plain PDMS (KBM) at 40 X magnification. (a,d) Double staining for lumican (red) and keratocan (green) in ADSCs cultivated on patterned PDMS (KBM), patterned PDMS (DMEM/F12), and plain PDMS (KBM) after 14 and 21-days. (b,e) Expression of α-SMA protein (green) in the ADSCs on patterned PDMS (KBM and DMEM/F12), and plain PDMS (KBM). (c,f) Statistical analyses for cultured ADSCs on the imprinted substrates (KBM and DMEM/F12), compared to plain PDMS (KBM) at day 14 and 21, respectively. Data are mean ± SD. ***p < 0.001. ns nonsignificant.
Figure 9
Figure 9
Confocal microscopy of keratocyte-like induced cells compared to ADSCs and primary keratocytes at 40 X magnification. (a) Representative confocal images of ADSCs, (b) keratocyte like induced cells, and (c) primary keratocytes which were double stained by phalloidin (red) and keratocan (green). The DAPI stained cells nuclei (blue) that indicated the changes in the morphology of the ADSCs from long spindle to dendritic morphology in keratocyte-like induced cells and primary keratocytes. Notable expression of keratocan protein is observed in (b) keratocyte-like induced cells and (c) primary keratocytes.
Figure 10
Figure 10
Confocal images of nuclei shape of ADSCs, primary keratocyte, keratocyte-like induced cells at 40X. (a) The nucleus images of ADSCs, (b) keratocyte-like induced cells and (c) primary keratocytes. (df) The nucleus changes of cells by (d) circularity, (e) roundness, and (f) aspect ratio. Statistical analysis by Kruskal–Wallis test indicated significantly higher values for circularity and roundness of the stem cell nuclei than keratocyte and keratocyte-like induced cells. In contrast, significant elongation is illustrated in the keratocyte-like induced cell nuclei compared to ADSCs. Data are mean ± SD. ***p < 0.001. ns non significant.

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