June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Pseudotime analysis of human stem cell-derived photoreceptor cell maturation following subretinal transplantation
Ying Liu; Clayton Pio Santiago; Akin Sogunro; Zheng Jiang; Gregory Konar; Ming-wen Hu; Minda McNally; Yuchen Lu; Zhuolin Li; Dzhalal Agakishiev; Sarah Hadyniak; Katarzyna Hussey; Jiang Qian; Robert Johnston; Seth Blackshaw; Mandeep Singh
Author Affiliations & Notes
  • Ying Liu
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Clayton Pio Santiago
    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Akin Sogunro
    Johns Hopkins University Department of Biology, Baltimore, Maryland, United States
  • Zheng Jiang
    Baylor College of Medicine Department of Ophthalmology, Houston, Texas, United States
  • Gregory Konar
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Ming-wen Hu
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Minda McNally
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Yuchen Lu
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Zhuolin Li
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Dzhalal Agakishiev
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Sarah Hadyniak
    Johns Hopkins University Department of Biology, Baltimore, Maryland, United States
  • Katarzyna Hussey
    Johns Hopkins University Department of Biology, Baltimore, Maryland, United States
  • Jiang Qian
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Robert Johnston
    Johns Hopkins University Department of Biology, Baltimore, Maryland, United States
  • Seth Blackshaw
    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States
  • Mandeep Singh
    Retina, Johns Hopkins Medicine Wilmer Eye Institute, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Ying Liu None; Clayton Santiago None; Akin Sogunro None; Zheng Jiang None; Gregory Konar None; Ming-wen Hu None; Minda McNally None; Yuchen Lu None; Zhuolin Li None; Dzhalal Agakishiev None; Sarah Hadyniak None; Katarzyna Hussey None; Jiang Qian Johns Hopkins University, Code P (Patent); Robert Johnston Johns Hopkins University, Code P (Patent); Seth Blackshaw Johns Hopkins University, Code P (Patent); Mandeep Singh Johns Hopkins University, Code P (Patent)
  • Footnotes
    Support  This work was supported by the Foundation Fighting Blindness (Career Development Award to MSS), The Shulsky Foundation, the Juliette RP Vision Foundation, Research to Prevent Blindness (unrestricted grant to the Wilmer Eye Institute), and the National Eye Institute Core Grant EY001765.
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 4513 – F0300. doi:
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      Ying Liu, Clayton Pio Santiago, Akin Sogunro, Zheng Jiang, Gregory Konar, Ming-wen Hu, Minda McNally, Yuchen Lu, Zhuolin Li, Dzhalal Agakishiev, Sarah Hadyniak, Katarzyna Hussey, Jiang Qian, Robert Johnston, Seth Blackshaw, Mandeep Singh; Pseudotime analysis of human stem cell-derived photoreceptor cell maturation following subretinal transplantation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):4513 – F0300.

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Abstract

Purpose : Photoreceptor transplantation is envisioned as a possible treatment for degenerative retinal diseases. In homeostasis, photoreceptor cell maturation and survival are partly regulated by extrinsic cues. However, in the transplantation context, the effect of extrinsic cues on the maturation and survival of donor cells following placement in the recipient niche is incompletely understood. Here, we aimed to investigate the maturation of transplanted human stem cell-derived photoreceptor cells in a mouse model of degenerative retinal disease.

Methods : Retinal organoids derived from Crx.tdTomato+ H9 human embryonic stem cells and cultured for 134 days were microdissected and subretinally transplanted into adult immunodeficient Rd1 mice. Age-matched organoids served as in vitro controls. After three months, we assayed photoreceptor maturation using single-cell RNA sequencing, immunohistochemistry, and patch-clamp electrophysiology.

Results : Genes expressed in mature cone and rod photoreceptor cells were upregulated in transplanted photoreceptor cells compared to the controls. By pseudotime analysis of gene expression changes, we found that the transcriptome of the transplanted photoreceptor cells was more closely aligned with that of normal adult human photoreceptor cells than the transcriptome of the control cells. Photoreceptors that expressed L/M opsin, S opsin, and rhodopsin were more abundant in the transplanted than the cultured organoids. In addition, we detected more abundant photoreceptor inner/outer segment-like structures, and presynaptic terminals, in the transplanted than in the cultured condition. Functionally, patch-clamp electrophysiological responses in the transplanted condition were consistent with cone outer segment maturation.

Conclusions : Human stem cell-derived photoreceptor cells that were transplanted in a mouse model of degenerative retinal disease showed maturation characteristics that exceeded those of in vitro controls. Pseudotime analysis of single-cell RNA sequencing data provides a facile method to assay the maturation of transplanted retinal cells by integrating the expression levels of thousands of genes in multiple cell populations.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Copyright © 2024 Association for Research in Vision and Ophthalmology.
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