June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Abcb5-positive retinal pigment epithelial cells are required for normal retinal development and ageing
Author Affiliations & Notes
  • Bruce R Ksander
    Schepens Eye Research Institute, Harvard Medical School, Boston, MA
  • Ruth Lewis
    Schepens Eye Research Institute, Harvard Medical School, Boston, MA
  • Brian J Wilson
    Transplant Research Program, Boston Children's Hospital, Harvard Medical School, Boston, MA
  • Gretchen Berg
    Transplant Research Program, Boston Children's Hospital, Harvard Medical School, Boston, MA
  • Markus Hermann Frank
    Transplant Research Program, Boston Children's Hospital, Harvard Medical School, Boston, MA
  • Natasha Frank
    Division of Genetics, Brigham & Women's Hospital, Harvard Medical School, Boston, MA
  • Footnotes
    Commercial Relationships Bruce Ksander, Boston Children's Hosptial (P), TICEBA / Rhea Cell (F); Ruth Lewis, None; Brian Wilson, None; Gretchen Berg, None; Markus Frank, Boston Children's Hospital (P), Boston Children's Hospital (P); Natasha Frank, Boston Children's Hospital (P), VA Hospital, Boston (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1486. doi:
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      Bruce R Ksander, Ruth Lewis, Brian J Wilson, Gretchen Berg, Markus Hermann Frank, Natasha Frank; Abcb5-positive retinal pigment epithelial cells are required for normal retinal development and ageing. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1486.

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

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Abstract

Purpose: <br /> It is controversial whether there is a subpopulation of stem cells that is required for normal homeostasis and aging of RPE cells. Our previously published data using transplantation experiments, and functional analysis of murine and human specimens indicated that the ABCB5 gene identifies a subpopulation of stem cells in the limbus that are required to maintain normal corneal epithelial homeostasis. Based on reports that Abcb5 is also expressed in the retina, our current studies examined the presence and function of Abcb5 positive RPE cells.

Methods: C57BL/6 Abcb5 knockout mice were generated that lacked exon 10 of the murine gene (GenBank accession number JQ655148), which encodes a functionally critical extracellular domain homologous to amino acids 493-508 of human ABCB5 (GenBank accession number NM_178559). Polymerase chain reaction analysis confirmed deletion. Abcb5 protein loss was demonstrated using an exon-10-encoded epitope-targeted monoclonal antibody, an amino-terminus- targeted antibody, and a specific extracellular-loop- associated peptide-targeted human immunoglobulin (Ig)G1 monoclonal antibody (clone 3B9). WT and KO tissues were also analyzed via RNA in situ hybridization. Expression of Abcb5 in RPE was determined by IHC and flow cytometry. Morphology of RPE in groups of age-matched WT and KO retinas were examined by H&E.

Results: WT B6 mouse RPE cells express a small subpopulation of Abcb5+ cells (2-8% of total RPE). In the absence of Abcb5, there was an age-related loss of the normal RPE morphology. As compared with age-matched 9 month old wild-type mice, Abcb5 KO mice displayed areas where RPE were enlarged and distended, due to cytoplasmic vacuoles. The areas of abnormal RPE in Abcb5 KO mice coincided with a significant thinning and attenuation of overlaying photoreceptor outer and inner segments, which was associated with a loss of cells in the outer nuclear layer. The appearance of abnormal RPE also coincided with a loss of underlying choriocapillaris.

Conclusions: A small subpopulation of Abcb5+ RPE cells is required to maintain the normal function of the RPE layer in the aging retina. Since the ABCB5 gene is known to maintain normal stem cell quiescence, these data imply there is an RPE subpopulation with stem cell characteristics.

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