July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Human stem cell-derived cells and organoids are relevant in vitro systems to understand maculapothy pathogenesis
Author Affiliations & Notes
  • Han Qin
    Genentech, South San Francisco, California, United States
  • Sehyun Kim
    Genentech, South San Francisco, California, United States
  • Zhe Li
    USC, Los Angeles, California, United States
  • Marion Jeanne
    Genentech, South San Francisco, California, United States
  • Footnotes
    Commercial Relationships   Han Qin, Genentech (E); Sehyun Kim, Genentech (E); Zhe Li, None; Marion Jeanne, Genentech (E)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 4590. doi:https://doi.org/
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      Han Qin, Sehyun Kim, Zhe Li, Marion Jeanne; Human stem cell-derived cells and organoids are relevant in vitro systems to understand maculapothy pathogenesis
      . Invest. Ophthalmol. Vis. Sci. 2018;59(9):4590. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Limited relevant in vitro systems have been developed to understand maculopathies, such as Age-related macular degeneration (AMD). DRAM2 (damage-regulated autophagy modulator 2) mutations cause a monogenic early onset of maculopathy by an unknown cellular mechanism. Our goal was to compare different human in vitro systems to determine if they could help understand the functional consequences of DRAM2 loss. Relevant system(s) might then be used for the study of more complex ocular diseases such as AMD.

Methods : We used lentivirus to knockdown (KD) DRAM2 with shRNA in primary human fetal RPE (hfRPE) cells, and we used CRISPR/Cas to knockout (KO) DRAM2 in human embryonic stem cells (hESCs). hESCs wild-type (WT) and KO were then differentiated into RPE cells and eye organoids. As functional assay, we quantified cell survival after sodium iodate (NaIO3) treatment. We used a non-targeting control shRNA and 2 different shRNAs for the KD experiments. We used 2 independent hESC clones for the KO experiments.

Results : Efficient DRAM2 KD was obtained in the hfRPE cells (remaining DRAM2 expression: 10.8±1.8% and 7±0.9%). After CRISPR/Cas, we obtained 3 hESC clones out of 47 with homozygous DRAM2 KO. We found that hfRPE and hESC-derived RPE cells had similar in vitro differentiation potential, with pigmentation, expression of tight junctions and phagocytosis of photoreceptor outer segment abilities. DRAM2 loss caused increased susceptibility to cell death after NaIO3 treatment in both systems (p<0.001). After 4 days of 5mM NaIO3 treatment, 60.5±1.9% of the hfREP cells with the control shRNA survived, whereas 31.7±1.8% and 39.2±2.7% of the ones with DRAM2 shRNAs survived. In hESC-RPE cells, the same treatment led to the survival of 44.8±2.9% of WT cells and only 6.6±0.8% and 12.6±1.9% of the cells from the KO clones. hESC-derived eye organoids were generated from WT and DRAM2 KO clones and no obvious defect in development was caused by DRAM2 loss. We are differentiating further the organoids to know if DRAM2 loss may affect maturation of photoreceptors or their resistance to stress.

Conclusions : DRAM2 loss increases the susceptibility of human RPE cells to stress-induced cell death. Comparison of DRAM2 loss functional consequences in hfRPE cells and hESC-RPE cells validate the use of stem cell-derived in vitro systems to study maculopathy pathogenesis.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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