Retinoic Acid Enhances the Differentiation of Adipose-Derived Stem Cells to Keratocytes In Vitro

Abstract

Purpose: All-trans retinoic acid (RA) supplementation was investigated as a method of enhancing the differentiation of human adipose-derived stem cells (ASCs) to corneal keratocytes in vitro, in combination with a chemically defined serum-free medium.

Methods: Adipose-derived stem cells were cultured in monolayer and supplemented with 0.1, 1, or 10 μM RA for 14 days. The effects of RA on cell proliferation, migration, and extracellular matrix (ECM) accumulation were evaluated. In addition, the expression of phenotypic keratocyte markers was examined by reverse transcription polymerase chain reaction (PCR), immunocytochemistry, and Western blotting.

Results: Adipose-derived stem cells cultured with RA showed improved cell proliferation and ECM production. In addition, RA enhanced the expression of keratocyte-specific markers, keratocan, aldehyde dehydrogenase 3A1, lumican, and decorin, when compared to serum-free media alone. Furthermore, the presence of RA increased the amount of collagen type I while reducing the expression of fibrotic marker, α-smooth muscle actin.

Conclusions: These findings indicate that RA is a useful supplement for promoting a keratocyte phenotype in ASC.

Translational relevance: This study is particularly important for the generation of biological corneal substitutes and next generation cell based therapies for corneal conditions.

Keywords: differentiation; keratocytes; retinoic acid; stem cells.

Figure 1

Cell proliferation as determined by Presto Blue assay (n = 3 ± SD; significant difference compared to KM at *day 7 and ‡day 14, P < 0.05).

Figure 2

(A) Cell migration over 6 days after scratch at day 0. Scale bar: 500 μm. (B) Quantification of cell migration (n = 3 ± SD).

Figure 3

(A) sGAG as quantified by DMMB (n = 3 ± SD; *significant difference compared to KM at day 14, P < 0.05). (B) sGAG/DNA (n = 3 ± SD).

Figure 4

Fold change gene expression analysis of corneal-specific markers KERA, ALDH3A1, LUM, and DCN as quantified by rtPCR (n = 3 ± SD; *significant difference comparing days 7 to 14; ‡significant difference compared to KM at day 7; †significant difference compared to KM at day 14, P < 0.05).

Figure 5

Fold change gene expression analysis of ECM markers αSMA, COL1A1, and COL3A1 as quantified by rtPCR (n = 3 ± SD; *significant difference comparing days 7 to 14l; ‡significant difference compared to KM at day 7; †significant difference compared to KM at day 14, P < 0.05).

Figure 6

Immunofluorescent staining of protein markers keratocan, ALDH3A1, αSMA (all green) and f-actin (red) after 14 days in culture. Nuclei were stained with DAPI (blue). Scale bar: 200 μm.

Figure 7

Expression of keratocyte and fibrotic markers keratocan, ALDH3A1, and αSMA at the protein level. Lysates from serum-free KM, RA supplemented a,nd DMSO control cells extracted at day 14 and analyzed by Western blotting.