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Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells

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Abstract

Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

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Authors and Affiliations

  1. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 Xian Lie Nan Road, Guangzhou, 510060, People’s Republic of China

    Minghai Huang, Bowen Wang, Xuanwei Liang, Xiaoran Wang, Ying Liu, Qiang Zhou & Zhichong Wang

  2. Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, People’s Republic of China

    Pengxia Wan

  3. Nanning Aier Eye Hospital, Nanning, 530015, People’s Republic of China

    Minghai Huang

Authors
  1. Minghai Huang
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  2. Bowen Wang
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  3. Pengxia Wan
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  4. Xuanwei Liang
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  5. Xiaoran Wang
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  6. Ying Liu
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  7. Qiang Zhou
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  8. Zhichong Wang
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Corresponding author

Correspondence to Zhichong Wang.

Additional information

Minghai Huang and Bowen Wang contributed equally to this work.

This study was supported by grant no. 2012AA020507 from the National High Technology Research and Development Program (863 project) of China, grant no. 81270971 from the National Natural Science Foundation of China, grant no. S2012010009113 from the Natural Science Foundation of Guangdong Province of China and grant no. 2012PI05 from the Fundamental Research Funds of State Key Laboratory of Ophthalmology of China.

The authors declare no potential conflicts of interest.

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Figure S1

Representative time-course of changes in the limbal microvascular net in rabbit transplantaion models observed by the slit lamp method. (A) In the limbal autograft orthotopic transplantation model, the autografts showed ischemia at postoperative day 0 but the limbal microvascular net of the ischemic autografts re-vascularized at postoperative day 3 and restored a regular vasculature loop pattern at postoperative month 1. (B) In the model of the heterotopic transplantation of a limbal autograft into the central cornea, the limbal microvascular net disappeared at postoperative days 3-7. However, in the zone of lamellar limbal excision, an irregular limbal microvascular net showed neo-vascularization on the side of the sclera and even some mild vascular invasion of the cornea at postoperative month 1 (white arrow microvasculature, black arrow ischemic or disappeared microvasculature). Magnification: ×16 (GIF 253 kb)

High resolution image (TIFF 2687 kb)

Figure S2

(A) In the limbal autograft orthotopic transplantation model, epithelial cells in the peripheral corneal part of the limbal autograft were Muc5AC-. (B) In the zone of lamellar limbal excision, some epithelial cells in the peripheral cornea with vascular invasion were Muc5AC+ and exhibited deficient limbal barrier function (blue nuclei, red Muc5AC). Cells overlying the dotted line represent basal cells (white arrow Muc5AC+ cells, namely conjunctival goblet cells). Bars 10 μm (GIF 24 kb)

High resolution image (TIFF 530 kb)

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Huang, M., Wang, B., Wan, P. et al. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells. Cell Tissue Res 359, 547–563 (2015). https://doi.org/10.1007/s00441-014-2032-4

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  • DOI: https://doi.org/10.1007/s00441-014-2032-4

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