June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
YAP1 regulates cell size of limbal BCAM-positive transit amplifying cells.
Author Affiliations & Notes
  • Yuzuru Sasamoto
    Department of Ophthalmology, Boston University Chobanian & Avedisian School of Medicine, Boston, Massachusetts, United States
    Division of Genetics, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
  • Bruce Ksander
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Markus Hermann Frank
    Transplant Research Program, Boston Children's Hospital, Boston, Massachusetts, United States
    Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States
  • Natasha Frank
    Division of Genetics, Brigham and Women's Hospital Department of Medicine, Boston, Massachusetts, United States
    Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Yuzuru Sasamoto Alcon Research Institute, Japan Eye Bank, Code F (Financial Support); Bruce Ksander Ticeba GmbH, Rheacell GmbH & Co. KG, Code P (Patent); Markus Frank Ticeba GmbH, Rheacell GmbH & Co. KG, Code C (Consultant/Contractor), Ticeba GmbH, Rheacell GmbH & Co. KG, Code O (Owner), Ticeba GmbH, Rheacell GmbH & Co. KG, Code P (Patent); Natasha Frank Ticeba GmbH, Rheacell GmbH & Co. KG, Code P (Patent)
  • Footnotes
    Support  NIH/NEI grants 5K99EY031741 to Y.S., 5R01EY025794 and 5R24EY028767 to N.Y.F., B.R.K and M.H.F, NIH/NEI Schepens Core grant P30EY003790 to B.R.K., Alcon Young Investigator Grant to Y.S., Japan Eye Bank Overseas Grant to Y.S., VA R&D Merit Review Award 1I01RX000989 and a Harvard Stem Cell Institute seed grant award to N.Y.F.
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 969. doi:
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    • Get Citation

      Yuzuru Sasamoto, Bruce Ksander, Markus Hermann Frank, Natasha Frank; YAP1 regulates cell size of limbal BCAM-positive transit amplifying cells.. Invest. Ophthalmol. Vis. Sci. 2023;64(8):969.

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

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Abstract

Purpose : Small cell size is a characteristic feature of stem cells and essential for normal stem cell function. However, the molecular mechanisms involved in cell size regulation of corneal epithelial stem cells and their progeny are not fully understood. This study aimed to investigate cell sizes of ABCB5-positive limbal stem cells (LSCs) and BCAM-positive transit-amplifying cells (TACs) and to identify factors involved in cell size maintenance.

Methods : ABCB5-positive LSCs and BCAM-positive TACs were isolated by flow cytometry from donated human corneal tissues obtained from eye banks. Mean cell size was determined by flow cytometry based on forward scatter (FSC) measurements. Immunostaining of human corneas was performed using Yes-associated protein 1 (YAP1) mAb and BCAM pAb. CYR61 mRNA expression was determined by RT-PCR analyses of sorted BCAM-positive and BCAM-negative cells. Gene knockdown (KD) of YAP1 and BCAM was performed using siRNA transfection of in vitro-expanded limbal epithelial cells.

Results : Flow cytometry analyses showed that in the limbus, ABCB5-positive LSCs were 26.4 ± 9.1 % smaller than ABCB5-negative cells (p = 0.0029, n = 5) and BCAM-positive early TACs were 42.6 ± 9.5% smaller than BCAM-negative cells (p < 0.0001, n = 11). In addition, in the central cornea, BCAM-positive late TACs were 11.3 ± 12.7% smaller than BCAM-negative cells (p = 0.0497, n = 11). Immunostaining demonstrated co-expression of YAP1 and BCAM in the basal epithelial cell layer of the limbus and central cornea. Moreover, the YAP1 transcriptional target gene, CYR61, was also highly expressed in BCAM-positive limbal epithelial cells. Knockdown of either YAP1 or BCAM led to significant increases in cell sizes (39.8 ± 17.1 %, p < 0.0001, n = 12 by YAP1 KD#1, 29.2 ± 19.8%, p = 0.0006, n = 12 by YAP1 KD#2, 8.3 ± 5.8%, p = 0.0028, n = 10 by BCAM KD#1, 12.3 ± 11.4%, p = 0.0138, n = 10 by BCAM KD#2).

Conclusions : The current study shows that ABCB5-positive LSCs and BCAM-positive TACs possess small cell sizes, which is consistent with their stem/progenitor cell phenotype. In limbal BCAM-positive early TACs, both the transcription regulator YAP1 as well as BCAM itself are essential for cell size maintenance. Modulation of YAP1 and/or BCAM expression might lead to novel therapeutic approaches to human diseases resulting from corneal epithelial defects in the setting of stem cell deficiency.

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

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