June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Functional assessment and xeno-grafting of human induced pluripotent stem cell-differentiated ABCG2-positive limbal stem cells in induced neutropenia mouse model with total limbal stem cell deficiency (LSCD).
Author Affiliations & Notes
  • Heli Skottman
    Faculty of Medicine and Health Technology, Tampereen yliopisto, Tampere, Pirkanmaa, Finland
  • Gary Yam
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Tanja Ilmarinen
    Faculty of Medicine and Health Technology, Tampereen yliopisto, Tampere, Pirkanmaa, Finland
  • Vishal Jhanji
    Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Heli Skottman StemSight oy, Code O (Owner), Tampere University, Faculty of Medicine and Health Technology, Code P (Patent); Gary Yam None; Tanja Ilmarinen StemSight oy, Code E (Employment), StemSight oy, Code O (Owner), Tampere University, Faculty of Medicine and Health Technology, Code P (Patent); Vishal Jhanji None
  • Footnotes
    Support  Academy of Finland (338988), Sigrid Juselius Foundation (1706), Hillman Foundation of Pittsburgh, Unrestricted funds from Research to Prevent Blindness and NEI (P30, EY008098)
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 3807. doi:
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      Heli Skottman, Gary Yam, Tanja Ilmarinen, Vishal Jhanji; Functional assessment and xeno-grafting of human induced pluripotent stem cell-differentiated ABCG2-positive limbal stem cells in induced neutropenia mouse model with total limbal stem cell deficiency (LSCD).. Invest. Ophthalmol. Vis. Sci. 2023;64(8):3807.

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

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Abstract

Purpose : There is an unmet clinical need to treat especially bilateral LSCD. For this allogeneic source of limbal stem cells (LSCs) differentiated from human induced pluripotent stem cells (hiPSC) could provide a solution. In previous studies, we have developed efficient methods to produce an enriched population of ABCG2-positive hiPSC-LSCs and our hypothesis is that these cells including both quiescent (p27 and GPHA2 positive) and proliferative sub-populations could represent high regenerative potential to form corneal epithelium in vivo.

Methods : In this study, hiPSC-LSCs were differentiated, characterized by morphology, immunostaining (PAX6, ABCG2, p40/63, p27, GPHA2), flow cytometry (ABCG2) and colony forming assay and finally cryopreserved. For transplantation, hiPSC-LSCs where thawed and cultured on fibrin membrane. Cells were transplanted to cyclophosphamide-induced neutropenia mouse (Swiss Webster) model with mechanically created LSCD and the eyes received TobraDex gel before tarsorrhaphy. After 5-6 days, eyes were harvested for imaging and histological analyses. Wound only eyes were used as control.

Results : The limbal phenotype of the ABCG2-enriched (around 80%) population of hiPSC-LSCs was confirmed before transplantation assessment. The mechanical scraping with algerbrush followed by thermal cauterization successfully removed both corneal and limbal epithelium for the induction of the LCSD in a mouse model with chemically induced neutropenia. Histological sectioning and staining with human specific antibody (Ku80) confirmed the successful integration and stratification of the hiPSC-LSCs after 5-6 days of transplantation in mice without obvious signs of xeno-rejection. Further staining with markers including PAX6 and p63 confirmed corneal phenotype of the transplanted cells.

Conclusions : The neutropenia mouse model with total LSCD was successfully used for the short-term efficacy assessment of ABCG2-postive hiPSC-LSCs. For the long-term efficacy and safety assessment, immunodeficient animal model will be needed.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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