Limbal epithelial stem cell sheets from young donors have better regenerative potential

Soonwon Yang; Hyun Jung Lee; Soojung Shin; In Yang Park; So-Hyang Chung

doi:10.1038/s41598-022-17821-9

Limbal epithelial stem cell sheets from young donors have better regenerative potential

Sci Rep. 2022 Aug 19;12(1):14191. doi: 10.1038/s41598-022-17821-9.

Authors

Soonwon Yang^#¹, Hyun Jung Lee^#², Soojung Shin¹, In Yang Park³, So-Hyang Chung⁴

Affiliations

¹ Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, #222 Banpo-daero, Seocho-gu, Seoul, 06591, Korea.
² Department of Biochemical Engineering, Seoil University, Seoul, Republic of Korea.
³ Department of Department of Obstetrics and Gynecology, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
⁴ Department of Ophthalmology and Visual Science, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, #222 Banpo-daero, Seocho-gu, Seoul, 06591, Korea. chungsh@catholic.ac.kr.

^# Contributed equally.

Abstract

To investigate the stemness of limbal epithelial stem cell sheets in relation to the donor's age. Human limbal explants from cadaveric donors were set on human amniotic membrane scaffolds with the xeno-free medium. We evaluated limbal epithelial sheet size, expression of stem/progenitor cell markers, and colony formation efficiency from donors of different age groups (age ≤ 45, age 45-65, and age > 65). Expression of the proliferation marker Ki67, stem/progenitor cell markers p63α and ABCG2, cornea specific marker PANCK, and differentiation marker CK12 were evaluated. To determine the effect of donor age on the storage period of limbal explant sheets, the limbal explant outgrowth sheets were stored in 4 °C for 2 days and analyzed for JC-1, p63α, and PANCK with FACS on each day. From days 6 to 12, the outgrowth area of the limbal epithelial stem cell sheet was significantly larger in the age ≤ 45 groups (296 ± 54.7 mm², day 9) compared to the other two age groups [age 45-65 group (278 ± 62.6 mm²), age > 65 group (257 ± 44.0 mm²), day 9] (p < 0.01). In terms of stemness, outgrowth cells from aged donors (age > 65) showed lower expression of stem/progenitor cell markers p63α and ABCG2 and decreased CFE compared to the other two groups. There were significantly more p63α+ cells in outgrowth cells in the age ≤ 45 group (18.2 ± 3.6%) compared to the age > 65 group (14.1 ± 4.6%; p < 0.01). Limbal explant outgrowth sheet on the age ≤ 45 group (32.7 ± 7.5%) had higher percentages of cells resisting staining by JC-1 compared with sheets under the age > 65 groups (25.7 ± 7.1%, p < 0.01) (JC-1^low). Cells from the age ≤ 45 group showed a higher clonogenic capacity than those from the other two age groups (45 < Age ≤ 65 CFE ratio = 0.7 ± 0.16, p < 0.01; 65 < Age CFE ratio = 0.3 ± 0.06, p < 0.01, vs. Age ≤ 45). In the age > 65 group, positive cells of p63α on D0, 1, and 2 were significantly lower compared to those in the age ≤ 45 group on the storage period (p < 0.01, respectively). Our results imply that donors younger than 65 years of age are a better source of limbal epithelial stem cell sheet generation with high regeneration potential.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Aged
Antigens, Differentiation / metabolism
Cells, Cultured
Epithelial Cells / metabolism
Epithelium, Corneal* / metabolism
Humans
Limbus Corneae*
Middle Aged
Stem Cells

Substances

Antigens, Differentiation