Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

Abstract

Aim: To investigate the impact of adipose-derived mesenchymal stem cells (ADSCs) on cell viability and extracellular matrix (ECM) synthesis of corneal stromal cells (CSCs).

Methods: ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly co-cultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate (FITC)/proliferation indices (PI) assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase (MMP), such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot.

Results: ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis (early and late) between the negative control group (6.34% and 2.06%) and the ADCSs-treated group (4.69% and 1.59%). The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type (I, II, III, IV, and V) in CSCs were observed in CSCs/ADSCs group, 3.47-fold, 4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.

Conclusion: ADSCs play a promotive role in CSCs' growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Keywords: adipose-derived mesenchymal stem cell; corneal stromal cells; extracellular matrix; plasticity.

Figure 1. ADSC in vitro culture and identification

Under the microscope, ADSCs were spindle-shaped and grew vigorously. The expression of CD29, CD90, CD34 and CD45 showed as red fluorescence within the ADSCs. DAPI was used to stain the nuclei of the cells as blue fluorescence. Original magnification: 200×. Scale bar represents 50 µm.

Figure 2. Differentiations, proliferation and alteration of cell cycle of ADSCs

A: Flow cytometric analysis of rabbit ADSCs. After culturing for 14d, majority of isolated ADSCs expressing CD29 (98.4%) and CD90 (97.5%) characteristic of mesenchymal stem cells, showing low level of CD34 (3.74%), and CD45 (2.01%). B-D: Adipogenic and osteogenic differentiations of ADSCs. ALP detection (B) and alizarin red staining (C; 200×) and oil red O staining (D; 200×) of ADSC differentiations after day 14 or after day 21 induction of differentiation. E: The proliferation of ADSCs (passage 3, 6, and 9) was assessed by CCK-8 analysis. The ADSCs' growth curve was S-shaped. The increase of growth of ADSCs was observed as early as the third day, especially at the eight day and passage 3. F: Apoptosis phenotype was detected using flow cytometry. P3, P6, P9: Passage 3, 6, 9. ^aP<0.05 and ^bP<0.01.

Figure 3. CSCs in vitro culture and identification

A: Under the microscope, the CSCs were in the spindle-shape. Original magnification: 200×. Scale bar represents 50 µm. B: The expression of the markers vimentin+ showed red fluorescence and CK12+ showed as green fluorescence. The nuclei of the cells were stained blue with DAPI. C: Annexin V-FITC Apoptosis Detection Kit was used to detect the proportions of apoptotic cells under the co-culture conditions of ADSCs. FL1-A: Annexin V; FL3-A: PI. D: CCK-8 detection kit was used to detect the proliferation of CSCs under CSCs cultured alone or ADSCs treatment conditions. The bar graph shows mean±SD in triplicate. ^aP<0.05 and ^bP<0.01.

Figure 4. ADSCs suppressed the expression of MMPs and promoted ECM synthesis in CSCs

The protein expression levels of MMPs (MMP1, MMP-2 and MMP-9), collagen type (I, II, III, IV, and V), ALDH, and fibronectin in CSCs and ADSCs-treated-CSCs determined by Western blot. β-actin were used as the internal control. ^aP<0.05.