Gliotic Response and Reprogramming Potential of Human Müller Cell Line MIO-M1 Exposed to High Glucose and Glucose Fluctuations

Abstract

Retinal neurodegeneration (RN), an early marker of diabetic retinopathy (DR), is closely associated with Müller glia cells (MGs) in diabetic subjects. MGs play a pivotal role in maintaining retinal homeostasis, integrity, and metabolic support and respond to diabetic stress. In lower vertebrates, MGs have a strong regenerative response and can completely repair the retina after injuries. However, this ability diminishes as organisms become more complex. The aim of this study was to investigate the gliotic response and reprogramming potential of the human Müller cell line MIO-M1 cultured in normoglycemic (5 mM glucose, NG) and hyperglycemic (25 mM glucose, HG) conditions and then exposed to sustained high-glucose and glucose fluctuation (GF) treatments to mimic the human diabetic conditions. The results showed that NG MIO-M1 cells exhibited a dynamic activation to sustained high-glucose and GF treatments by increasing GFAP and Vimentin expression together, indicative of gliotic response. Increased expression of SHH and SOX2 were also observed, foreshadowing reprogramming potential. Conversely, HG MIO-M1 cells showed increased levels of the indexes reported above and adaptation/desensitization to sustained high-glucose and GF treatments. These findings indicate that MIO-M1 cells exhibit a differential response under various glucose treatments, which is dependent on the metabolic environment. The in vitro model used in this study, based on a well-established cell line, enables the exploration of how these responses occur in a controlled, reproducible system and the identification of strategies to promote neurogenesis over neurodegeneration. These findings contribute to the understanding of MGs responses under diabetic conditions, which may have implications for future therapeutic approaches to diabetes-associated retinal neurodegeneration.

Keywords: Müller cells; dedifferentiation; diabetes; diabetic retinopathy; glucose fluctuations; progenitor cells; retinal neurodegeneration.

Figure 1

Effect of NG and HG culture conditions on GFAP expression in MIO-M1 cells. (A) Representative WB displaying GFAP band obtained from protein extracts of MIO-M1 cells maintained in NG (5 mM glucose, NG cells) and HG (25 mM glucose, HG cells) culture media. α-TUBULIN was used as a loading control. (B) Densitometric analysis of GFAP expression, normalized to α-TUBULIN. The ratio of GFAP protein to α-TUBULIN in HG cells was statistically compared to NG cells. Data are presented as mean ± SEM from three independent experiments. Statistical significance (** p < 0.01 vs. NG cells) was determined using Student’s t-test.

Figure 2

Effect of sustained high-glucose and GF treatments on GFAP expression in NG and HG MIO-M1 cells. NG and HG MIO-M1 cells were exposed to sustained high-glucose and GF treatments as described in M&M and in Supplementary Figure S1. (A) Representative WB analysis showing GFAP protein levels in NG and HG MIO-M1 cells subjected to different glucose treatments (I–V); α-TUBULIN was used as loading control. (B) Densitometric analysis of GFAP expression normalized to α-TUBULIN. The ratio of GFAP protein to α-TUBULIN in NG and HG cells subjected to all different glucose treatments was compared to cells cultured under basal glucose treatment (I). Data are presented as mean ± SEM from three independent experiments. Statistical significance (* p < 0.05) was determined by Student’s t-test.

Figure 3

Effect of NG and HG culture conditions on GFAP expression and morphology in MIO-M1 cells. MIO-M1 were maintained in NG (5 mM glucose, NG cells) and HG (25 mM glucose, HG cells) culture media. (A) Representative photomicrographs of NG (upper panel) and HG (lower panel) MIO-M1 cells showing IF for GFAP (green). Nuclei were stained with Hoechst (blue). Bipolar and radial cells are indicated by an arrow and an arrowhead, respectively. Scale bar: 75 μm. (B) Plots showing the percentage of GFAP-positive cells with bipolar and radial morphology in NG and HG MIO-MI cells. Data were obtained by counting at least 500 cells for each group. Data are presented as mean ± SEM from three independent experiments. Statistical significance (** p < 0.01 vs. bipolar cells of NG cells; ## p  <  0.01 vs. radial cells of NG cells) was determined using Student’s t-test.

Figure 4

Effect of sustained high-glucose and GFs on GFAP expression and morphology in NG and HG MIO-M1 cells. NG and HG MIO-M1 cells were exposed to sustained high-glucose and GF treatments, as described in M&M and in Figure S1. (A) Representative photomicrographs of NG (upper panel) and HG (lower panel) MIO-M1 cells showing IF for GFAP (green). Nuclei were stained with Hoechst (blue). Scale bar: 75 μm. (B,C) Plots showing the percentage of GFAP-positive cells with bipolar and radial morphology in NG and HG MIO-MI cells exposed to different glucose treatments (I–V). Data were obtained by counting at least 500 cells for each group. Data are presented as mean ± SEM from three independent experiments. Statistical significance (* p < 0.05 and ** p < 0.01 vs. bipolar cells of treatment I; # p < 0.05 and ## p < 0.01 vs. radial cells of treatment I) was determined using Student’s t-test.

Figure 5

Effect of sustained high-glucose and GFs on Vimentin filaments of NG and HG MIO-M1 cells. NG and HG MIO-M1 cells were exposed to sustained high-glucose and GF treatments (I–V), as described in M&M and in Figure S1. Representative photomicrographs of NG (upper panel) and HG (lower panel) MIO-M1 cells showing IF for Vimentin (green). Nuclei stained with Hoechst (blue). The insets show the organization of Vimentin filaments. Scale bar: 75 μm.

Figure 6

Effect of NG and HG culture conditions on SHH expression in MIO-M1 cells. (A) Representative WB displaying basal SHH levels in MIO-M1 cells cultured either in NG (5 mM glucose) or HG (25 mM glucose) media. HSP90 was used as a loading control. (B) Densitometric analysis of SHH expression normalized to HSP90. The ratio of SHH protein to HSP90 in HG cells was statistically compared to NG cells. Data are represented as mean ± SEM from three independent experiments. Statistical significance (** p < 0.01 vs. NG cells) was determined using Student’s t-test. (C) Representative micrographs showing SHH (red) in MIO-M1 cultured under NG and HG conditions. Hoechst was used for nuclei staining. The inset highlight SHH inside the cells, with spots marked by an arrow. Scale bar: 20 μm. (D) Quantitative analysis of SHH distribution inside the cells was performed using the ImageJ program v1.53k. Data were obtained by counting at least 500 cells for each group. Data are presented as mean ± SEM from three independent experiments. Statistical significance (** p < 0.01 vs. widespread NG cells; ## p < 0.01 vs. punctate NG cells) was determined using Student’s t-test.

Figure 7

Effect of sustained high-glucose and GFs on SHH expression in NG and HG MIO-M1 cells. NG and HG MIO-M1 cells were exposed to sustained high-glucose and GF treatments as described in M&M and in Figure S1. (A) Representative Western blot analysis showing SHH protein levels in MIO-M1 cells cultured in NG and HG conditions subjected to different glucose treatments (I–V). HSP90 was used as a loading control. (B) Densitometric analysis of SHH expression normalized to HSP90 for both NG and HG cells in the different treatments (I–V). The ratio of SHH to HSP90 in NG and HG cells subjected to all different glucose treatments was compared to cells cultured under the basal glucose treatment (I). Data are presented as mean ± SEM from three independent experiments. Statistical significance (* p < 0.05; ** p < 0.01; **** p < 0.0001) was determined by Student’s t-test. (C) Representative micrographs showing SHH (red) in MIO-M1 cells cultured in NG (upper panel) and HG (lower panel) and exposed to different glucose treatments (I–V). Scale bar: 20 μm. (D) Quantitative analysis of SHH distribution within the cells was performed using the ImageJ program v1.53k. Plots represent the percentage of SHH-positive cells exhibiting a widespread and punctate morphology among all SHH-positive cells counted in the NG and HG cells exposed to different glucose treatments (I–V). Data were obtained by counting at least 500 cells for each group. Data are presented as mean ± SEM from three independent experiments. Statistical significance (* p < 0.05 and ** p < 0.01 vs. widespread NG cells in the basal glucose treatment (I); # p < 0.05; ## p < 0.01 vs. punctate NG cells in the basal glucose treatment (I)) was determined using Student’s t-test.

Figure 8

Effect of NG and HG culture conditions and of sustained high-glucose and GF treatments in NG and HG MIO-M1 cells on SOX-2 expression. MIO-M1 Müller cells cultured in NG and HG conditions and under different glucose treatments for 96 h (I–V), as described in Figure S1. (A) Representative micrographs of MIO-M1 cultured in NG (upper panel) and HG (lower panel) conditions showing the immunostaining for SOX-2 (red). Nuclei were stained with Hoechst (blue). Scale bar: 20 μm. (B) Quantitative analysis of SOX-2-positive cells was performed using the ImageJ program v 1.53k. Plots represent the percentage of SOX-2-positive cells relative to all Hoechst-positive cells counted in NG and HG culture conditions. Data were obtained by counting at least 500 cells for each group. Data are presented as mean ± SEM from three independent experiments. (C) qRT-PCR was performed on MIO-M1 cells cultured in NG and HG conditions and SOX-2 transcript analysis was obtained with 2^−ΔCt methods. (D) qRT-PCR was performed on MIO-M1 cells cultured in NG and HG media under different glucose treatments (I–V), and SOX-2 transcript analysis was obtained with 2^−ΔCt methods. Data are presented as mean ± SEM from three independent experiments. Statistical significance (* p < 0.05, ** p < 0.01) was determined by Student’s t-test.