Effects of senescent secretory phenotype acquisition on human retinal pigment epithelial stem cells

Abstract

Regenerative medicine approaches based on mesenchymal stem cells (MSCs) are being investigated to treat several aging-associated diseases, including age-related macular degeneration (AMD). Loss of retinal pigment epithelium (RPE) cells occurs early in AMD, and their transplant has the potential to slow disease progression.The human RPE contains a subpopulation of cells - adult RPE stem cells (RPESCs) - that are capable of self-renewal and of differentiating into RPE cells in vitro. However, age-related MSC changes involve loss of function and acquisition of a senescence-associated secretory phenotype (SASP), which can contribute to the maintenance of a chronic state of low-grade inflammation in tissues and organs.In a previous study we isolated, characterized, and differentiated RPESCs. Here, we induced replicative senescence in RPESCs and tested their acquisition of the senescence phenotype and the SASP as well as the differentiation ability of young and senescent RPESCs.Senescent RPESCs showed a significantly reduced proliferation ability, high senescence-associated β-galactosidase activity, and SASP acquisition. RPE-specific genes were downregulated and p21 and p53 protein expression was upregulated.These findings document the effects of senescence and SASP acquisition on RPESC differentiation ability and highlight the need for a greater understanding of their role in AMD pathogenesis.

Keywords: AMD; RPESCs; age-related diseases; inflammation; senescence.

Figure 1

Proliferation rate, β-gal positivity, telomere length, and cell morphology during RPESC replicative senescence. RPESC replicative senescence. (A) Cumulative number of population doublings (CPD) in RPESCs grown to senescence. (B) Percentage of β-gal-positive cells detected during RPESC replicative senescence from P1 to P16. P11, number of culture passages. Data are reported as mean ± SD. *P =0.039. (C) RPESC telomere length during replicative senescence was analyzed from P1 to P18; data are reported as mean ± SD of 3 independent experiments. (D) Morphological analysis of young (P3) and senescent (P16) RPESCs by the TRIC-phalloidin immunofluorescence assay. Senescent RPESCs appear enlarged and flattened. Magnification 20X, scale bar 200 µm. Pictures are representative of 3 independent experiments.

Figure 2

SASP induction in senescent RPESCs. Young (P3) and senescent (P16) RPESCs were maintained in culture for 48 h. The supernatant was analyzed for IL-6 (A), IL-12 (B), IL-17 (C), TNF-α (D), TGFβ1 (E), INF-γ (F), IL-4 (G), IL-10 (H), and IL-13 (I), by ELISA. Data are mean ± SD of 3 independent experiments. *P = from 0.021 to 0.041.

Figure 3

mRNA expression levels of stemness and RPE-specific genes in senescent and young RPESCs. (A) qRT-PCR analysis of the expression levels of stemness genes in senescent (P16) and young (P3) RPESCs. (B) qRT-PCR analysis of the mRNA levels of RPE-specific genes in senescent and young RPESCs. Data are mean ± SD of 3 independent experiments. *P = from 0.026 to 0.036.

Figure 4

Senescence-associated gene expression profile in senescent and young RPESCs. PCR array performed to analyze the mRNA expression levels of senescence-associated genes in 3 senescent (P16) and 3 young (P3) RPESCs. Data are reported as fold change. A 4-fold difference was considered significant and only mRNAs with a ΔΔCt greater than 4 (A) or lower than -4 (B) are reported. *P = from 0.018 to 0.046.

Figure 5

Human p53 and p21 protein expression levels in senescent and young RPESCs. p53 and p21 protein expression levels (A) Western blot analysis (B) and densitometric analysis of blots. Data are mean ± SD of 3 independent experiments. *P = from 0.022 to 0.046. (C) Relative expression levels of mRNA related to p21 and (D) p53 genes in young (P3), pre-senescent (P11) and senescent (P16) RPESCs cells. Data are mean ± SD of 3 independent experiments. *P = from 0.031 to 0.044.