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. 2023 Oct 18;16(10):1574-1581.
doi: 10.18240/ijo.2023.10.03. eCollection 2023.

Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids

Jin-Lin Du  1   2 , Li-Xiong Gao  3 , Tao Wang  1 , Zi Ye  2 , Hong-Yu Li  1   2 , Wen Li  1   2 , Quan Zeng  4 , Jia-Fei Xi  4 , Wen Yue  4 , Zhao-Hui Li  1   2
Affiliations

Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids

Jin-Lin Du et al. Int J Ophthalmol. .

Abstract

Aim: To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs).

Methods: The hiPSC and a three-dimensional culture method were used for the experiments. Generated embryoid bodies (EBs) were randomly and equally divided into hypoxic and normoxic groups. Photographs of the EBs were taken on days 38, 45, and 52, and the corresponding volume of EBs was calculated. Simultaneously, samples were collected at these three timepoints, followed by fixation, sectioning, and immunofluorescence.

Results: The proportion of Ki67-positive proliferating cells increased steadily on day 38; this proliferation-promoting effect tended to increase tissue density rather than tissue volume. On days 45 and 52, the two groups had relatively similar ratios of Ki67-positive cells. Further immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52 (P<0.05). In contrast, the percentage of PAX6- and CHX10-positive cells significantly decreased following hypoxia treatment at all three timepoints (P<0.01), except for CHX10 at day 45 (P>0.05). Moreover, the proportion of PAX6-/TUJ1+ cells within the neural retinas increased considerably (P<0.01, <0.05, <0.05 respectively).

Conclusion: Low oxygen promotes stemness and proliferation of neural retinas, suggesting that hypoxic conditions can enlarge the retinal progenitor cell pool in hiPSC-derived ROs.

Keywords: hypoxia; retinal ganglion cells; retinal organoid; retinal progenitor cells.

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Figures

Figure 1
Figure 1. The volume of ROs growing under hypoxic treatment was significantly lower than in normoxic treatment
A–F: Optical microscope images of ROs in day 38, 20% O2 (A), day 38, 5% O2 (B), day 45, 20% O2 (C), day 45, 5% O2 (D), day 52, 20% O2 (E), and day 52, 5% O2 (F). G: Schematic diagram measuring the volume of the ROs. Scale bar: 500 µm. They are divided into two basic shapes: an ellipsoid with one short axis (a) and one vertical long axis (b), and the remaining spheres (n) are considered to have a base radius (rn) and a top height (hn). H: Statistical diagram of the volume of ROs. I: Statistical diagram of the cell density within ROs. The volume of ROs growing under hypoxic treatment was significantly lower than that under normoxic treatment (aP<0.05, bP<0.01, cP<0.001). ROs: Retinal organoids.
Figure 2
Figure 2. The effects of hypoxia on the proliferation of NRs and the expression of SOX2-positive cells
A–F: Ki67-immunoreactive proliferating cells (green) of NRs and expression of SOX2-positive cells (red) in day 38, 20% O2 (A), day 38, 5% O2 (B), day 45, 20% O2 (C), day 45, 5% O2 (D), day 52, 20% O2 (E), and day 52, 5% O2 (F). Scale bar: 50 µm. G: Statistical analysis of the Ki67-immunoreactive proliferating cell ratio of NRs in two groups at three timepoints, respectively. H: Statistical analysis of the ratio of SOX2-positive cells in two groups (aP<0.05). NRs: Neural retinas.
Figure 3
Figure 3. The effects of hypoxic treatment on the expression of PAX6- and CHX10-positive cells
A–F: PAX6 (green) and CHX10 (red) double-staining of NRs in day 38, 20% O2 (A); day 38, 5% O2 (B); day 45, 20% O2 (C); day 45, 5% O2 (D); day 52, 20% O2 (E); and day 52, 5% O2 (F). Scale bar: 50 µm. G: Statistical analysis of the ratio of PAX6-positive cells within retinal organoids in two groups at three timepoints, respectively. H: Statistical analysis of the CHX10-positive cell ratio in two groups at three timepoints (bP<0.01, cP<0.001). NRs: Neural retinas.
Figure 4
Figure 4. The effects of hypoxia on the expression of TUJ1- and NESTIN-positive cells
A–F: TUJ1 (red) and NESTIN (green) double-staining of NRs in day 38, 20% O2 (A); day 38, 5% O2 (B); day 45, 20% O2 (C); day 45, 5% O2 (D); day 52, 20% O2 (E); and day 52, 5% O2 (F). Scale bar: 50 µm. G: Statistical analysis of the ratio of TUJ1-positive cells within retinal organoids in two groups at three timepoints, respectively. NRs: Neural retinas.
Figure 5
Figure 5. To further examine the differentiation state of TUJ1-positive cells, double staining of TUJ1 and PAX6 was performed
A–F: TUJ1 (red) and PAX6 (green) double-staining of NRs in day 38, 20% O2 (A); day 38, 5% O2 (B); day 45, 20% O2 (C); day 45, 5% O2 (D); day 52, 20% O2 (E); and day 52, 5% O2 (F). Scale bars: 50 µm. G: Statistical analysis of PAX6-positive cells within TUJ1-positive cells and the ratio of NRs in two groups at three timepoints, respectively. H: Statistical analysis of PAX6-negative cells within the TUJ1-positive cell ratio in two groups at three timepoints, respectively (aP<0.05, bP<0.01). NRs: Neural retinas.
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