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Background <p>Age-related macular degeneration (AMD) is characterized by progressive retinal pigment epithelium (RPE) dysfunction driven by oxidative stress and chronic inflammation, in which NLRP3 inflammasome activation plays a critical role. Mesenchymal stem cells (MSCs) exhibit therapeutic potential, but their efficacy is limited by poor survival and reduced paracrine activity in hostile microenvironments. Here, we investigated whether three-dimensional (3D) spheroid culture enhances the protective effects of umbilical cord-derived MSCs (UC-MSCs) on RPE cells by promoting autophagy and suppressing inflammasome activation.</p> Methods <p>Human UC-MSCs were cultured as 3D spheroids or conventional 2D monolayers and applied in sodium iodate (NaIO<sub>3</sub>)-induced oxidative injury models both in vitro and in vivo. Retinal morphology and function were assessed via histology and electroretinography, while NLRP3/caspase-1 activation, LC3-II/I ratios, and autophagy flux were quantified using immunofluorescence and Western blot. GO/KEGG enrichment was performed to identify pathways associated with 3D MSCs efficacy. Mechanistic involvement of autophagy was validated using 3-methyladenine (3-MA) and rapamycin.</p> Results <p>3D MSCs formed compact spheroids exhibiting enhanced paracrine potential and significantly outperformed 2D MSCs in protecting RPE cells against NaIO<sub>3</sub>-induced injury. In vivo, 3D MSC treatment preserved retinal structure, reduced RPE cell loss, and improved retinal function. In vitro, co-culture with 3D MSCs markedly improved ARPE-19 viability, reduced apoptosis, and modulated autophagy-related marker expression, as evidenced by increased LC3-II/I ratios. 3D MSCs significantly inhibited NLRP3 inflammasome activation and pro-inflammatory cytokine release, effects reversed by 3-MA and further enhanced by rapamycin.</p> Conclusions <p>3D spheroid culture substantially augments the therapeutic efficacy of UC-MSCs by boosting autophagy and suppressing NLRP3 inflammasome signaling, resulting in enhanced protection of RPE cells from oxidative and inflammatory injury. These findings provide preclinical evidence supporting 3D MSCs as a promising therapeutic strategy for AMD.</p> Graphical Abstract <p></p>

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3D spheroids of umbilical cord-derived MSCs protect retinal pigment epithelium against oxidative and inflammatory injury by activating autophagy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月2日

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  • Qian Xu,
  • Mengyao Li,
  • Dong Li,
  • Xiaoyu Dai,
  • Yifei Zhang,
  • Yi Qu

摘要

Background

Age-related macular degeneration (AMD) is characterized by progressive retinal pigment epithelium (RPE) dysfunction driven by oxidative stress and chronic inflammation, in which NLRP3 inflammasome activation plays a critical role. Mesenchymal stem cells (MSCs) exhibit therapeutic potential, but their efficacy is limited by poor survival and reduced paracrine activity in hostile microenvironments. Here, we investigated whether three-dimensional (3D) spheroid culture enhances the protective effects of umbilical cord-derived MSCs (UC-MSCs) on RPE cells by promoting autophagy and suppressing inflammasome activation.

Methods

Human UC-MSCs were cultured as 3D spheroids or conventional 2D monolayers and applied in sodium iodate (NaIO3)-induced oxidative injury models both in vitro and in vivo. Retinal morphology and function were assessed via histology and electroretinography, while NLRP3/caspase-1 activation, LC3-II/I ratios, and autophagy flux were quantified using immunofluorescence and Western blot. GO/KEGG enrichment was performed to identify pathways associated with 3D MSCs efficacy. Mechanistic involvement of autophagy was validated using 3-methyladenine (3-MA) and rapamycin.

Results

3D MSCs formed compact spheroids exhibiting enhanced paracrine potential and significantly outperformed 2D MSCs in protecting RPE cells against NaIO3-induced injury. In vivo, 3D MSC treatment preserved retinal structure, reduced RPE cell loss, and improved retinal function. In vitro, co-culture with 3D MSCs markedly improved ARPE-19 viability, reduced apoptosis, and modulated autophagy-related marker expression, as evidenced by increased LC3-II/I ratios. 3D MSCs significantly inhibited NLRP3 inflammasome activation and pro-inflammatory cytokine release, effects reversed by 3-MA and further enhanced by rapamycin.

Conclusions

3D spheroid culture substantially augments the therapeutic efficacy of UC-MSCs by boosting autophagy and suppressing NLRP3 inflammasome signaling, resulting in enhanced protection of RPE cells from oxidative and inflammatory injury. These findings provide preclinical evidence supporting 3D MSCs as a promising therapeutic strategy for AMD.

Graphical Abstract