Summary of the results

a) Effect of high lipid exposure on hiPSCs-derived Müller cells

We evaluate the expression of several genes that were previously demonstrated to play a key role in DR pathogenesis and that were shown to be upregulated in human primary Müller cells.

In this study, as in the previous study regarding high and intermittent glucose exposure, hiPSCs-derived Müller cells were obtained by reprogramming adult human dermal fibroblast (AHDFs).

Regarding inflammatory pathways, we investigated the expression the C-X-C family of chemokines include CXCL1-2 and CXCL8 (or IL-8) in hiPSCs-derived Müller cells under PA exposure. A significant increase of the expression of ATF3, CXL1-2, CXCL8, which is

implicated in neutrophil migration and chemotaxis, has been noted in hiPSCs-derived Müller cells treated with PA for 24 hours.

Regarding WNT signaling, the expression of the tight-junction coding gene CLDN1 was also significantly increased in hiPSCs-derived Müller cells after PA treatment.

Regarding angiogenesis pathway, we evaluated the expression of VEGF and ANGPTL4. The key role of VEGF in angiogenesis and in blood barrier rupture in DR is well known. More recently, ANGPTL4, a well-characterized target of PPAR-b/d signaling, has been demonstrated to play also an important role in the angiogenesis pathway in proliferative DR (Babapoor-Farrokhran et al., 2015, Jee et al., 2015).

The expression of both VEGF and ANGPTL4 was significantly increased in hiPSCs-derived Müller cells treated with PA for 24 hours compared with control condition.

The potential pro-angiogenic role of Müller cell under high lipid exposure was the investigated in aortic ring assays. These assays demonstrated a VEGF-independent pro-angiogenic activity of PA-exposed hiPSCs-derived Müller cells supernatants.

These results are in accordance with previous study of PA effect in primary human Müller cells cultures (Capozzi ME et al., 2018) and in streptozotocin-treated mice (Kandpal RP et al., 2012).

b) Effect of high lipid exposure in other types of hiPSCs-derived Müller cell

We then evaluated if the hiPSCs-derived Müller cell reaction after PA treatment differ depending on their origin. We evaluate the expression of angiogenesis and inflammatory increased gene expression in hiPSCs-derived Müller cells obtained from adult human dermal fibroblasts (A2) used in this study compared with hiPSCs-derived Müller cells generated from two different other sources: post-natal foreskin (F3) and adult retina (5F). We found that the increase of ANGPTL4 and ATF3 expression was similar in these 3 different hiPSCs-derived Müller cell types (Figure 27).

Figure 27: ANGPTL4 and ATF3 expression in hiPSCs-derived Müller cells generated from different sources: adult dermal biopsy (A2), post-natal foreskin (F3) and adult retina (5F); and cultured either in normoglycemic (NG) condition (DMEM medium with a glucose concentration of 5 mmol/l) or hyperglycemic (HG) condition (DMEM medium with a glucose concentration of 25 mmol/l), with (NG-Palm or HG-Palm) or without palmitic acid for 24h.

A) The mean value of ANGPTL4 expression in A2 hiPSCs-derived Müller cells was of 0.6087

± 0.1499 in NG condition vs 1.121 ± 0.1422 in NG-Palm condition (p = 0.0479), and of 0.6488

± 0.01017 in HG condition vs 1.228 ± 0.2613 in HG-Palm condition (p = 0,0457)

B) The mean value of ANGPTL4 expression in F3 hiPSCs-derived Müller cells was 1.629 ± 0.3293 in NG condition vs 4.927 ± 2.2756 in NG-Palm condition (p = 0.2248), and of 3.838 ± 1.768 in HG condition vs 5.889 ± 2.051 in HG-Palm condition (p = 0.4774).

C) The mean value of ANGPTL4 expression in 5F hiPSCs-derived Müller cells was of 1.528 ± 0.2327 in NG condition vs 10.62 ±1.87 in NG-Palm condition (p = 0.0029), and of 3.122 ± 0.5312 in HG condition vs 15.75 ± 0.729 in HG-Palm condition (p < 0.0001)

D) The mean value of ATF3 expression in A2 hiPSCs-derived Müller cells was of 0,8864 in NG condition vs 166.7 in NG-Palm condition (p < 0.0001), and of 0.9309 in HG condition vs 154.8 in HG-Palm condition (p = 0.0126).

E) The mean value of ATF3 expression in F3 hiPSCs-derived Müller cells was of 1.035 in HG condition vs 9.728 in HG-Palm condition (p = 0.0931).

F) The mean value of ATF3 expression in 5F hiPSCs-derived Müller cells was of 1.137 in NG condition vs 156.3 in NG-Palm condition (p < 0.0001), and of 1.289 in HG condition vs 112.8 in HG-Palm condition (p = 0.0005).

All results were normalized with expression of S26 and expressed as mean ± SEM (n = 4 in each group)

c) Effect of high lipid exposure in hiPSCs-derived Müller cell cultured at different passages

We evaluated if the hiPSCs-derived Müller cell reaction after PA treatment differ depending on their passage. We evaluate the expression of ANGPTL4 gene expression in hiPSCs-derived Müller cells obtained after 1, 2 and 3 passages. A significant increase of ANGPTL4 expression in hiPSCs-derived Müller cells was found, independently to the number of passages of the cells (Figure 28).

Figure 28: ANGPTL4 expression in hiPSCs-derived Müller cells cultured either in normoglycemic (NG) condition (DMEM medium with a glucose concentration of 5 mmol/l) or hyperglycemic (HG) condition (DMEM medium with a glucose concentration of 25 mmol/l), with (NG-Palm or HG-Palm) or without palmitic acid for 24h.

A) The mean value of ANGPTL4 expression in hiPSCs-derived Müller cells at passage 1 was of 1.092 in NG condition vs 3.421 in NG-Palm condition (p = 0.0019), and of 1.455 in HG condition vs 3.221 in HG-Palm condition (p = 0,0010).

B) The mean value of ANGPTL4 expression in hiPSCs-derived Müller cells at passage 2 was 1.221 in NG condition vs 5.462 in NG-Palm condition (p = 0.0001), and of 1.498 in HG condition vs 6.616 in HG-Palm condition (p = 0.0006).

C) The mean value of ANGPTL4 expression in hiPSCs-derived Müller cells at passage 3 was of 1.272 in NG condition vs 5.145 in NG-Palm condition (p = 0.0015), and of 0.5096 in HG condition vs 4.427 in HG-Palm condition (p = 0.0059)

All results were normalized with expression of S26 and expressed as mean ± SEM (n = 4 in each group)