Current Issue

Volume 20, Number 4, Jan-Mar(Winter) 2019, Serial Number: 80, Pages: 459-468

Amniotic Membrane Extract Eye Drop Promotes Limbal Stem Cell Proliferation and Corneal Epithelium Healing


Niloufar Shayan Asl, M.Sc., 1, #, Farhad Nejat, M.D, 2, #, Parvaneh Mohammadi, Ph.D, 1, Abdolhossein Nekoukar, Ph.D, 3, Saeed Hesam, Ph.D, 4, Marzieh Ebrahimi, Ph.D, 1, *, Khosrow Jadidi, M.D, 2, *,
Department of Stem Cells and Developmental Biology, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Vision Health Research Center, Semnan University of Medical Sciences, Semnan, Iran
Animal Core Facility, Reproductive Biomedicine Research Centre, Royan Institute for Biotechnology, ACECR, Tehran, Iran
Department of Epidemiology and Reproductive Health, Reproductive Epidemiology Research Centre, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
* Corresponding Address: P.O.Box: 16635-148 Department of Stem Cells and Developmental Biology Cell Science Research Centre Royan Institute for Stem Cell Biology and Technology ACECR TehranIran Email: Mebrahimi@royaninstitute.org, kh.jadidi@gmail.com

The first two authors equally contributed to this article.

Abstract

Objective

Human amniotic membrane (HAM) is used as a supporter for limbal stem cell (LSC) expansion and corneal surgery. The aim of study is to use HAM extracts from healthy donors to enhance proliferation of LSCs in vitro and in vivo.

Materials and Methods

In this interventional experimental study, the effective and cytotoxic doses of the amniotic membrane extract eye drops (AMEED) was assessed by adding different concentrations of AMEED (0-2.0 mg/ml) to LSC cultures for 14 days. Subsequently, the expression levels of ATP-binding cassette sub-family G member 2 (ABCG2, a putative stem cell marker), cytokeratin 3 (K3, corneal maker), K12 and K19 (corneal-conjunctival cell makers) were assessed by real-time polymerase chain reaction (PCR). In the second step, the corneal epithelium of 10 rabbits was mechanically removed, and the right eye of each rabbit was treated with 1 mg/ml AMEED [every 2 hours (group 1) or every 6 hours (group 2)]. The left eyes only received an antibiotic. The corneal healing process, conjunctival infection, degree of eyelid oedema, degree of photophobia, and discharge scores were evaluated during daily assessments. Finally, corneal tissues were biopsied for pathologic evidences.

Results

In comparison to the positive control [10% foetal bovine serum (FBS)], 0.1-1 mg/ml AMEED induced LSC proliferation, upregulated ABCG2, and downregulated K3. There were no remarkable differences in the expression levels of K12 and K19 (P>0.05). Interestingly, in the rabbits treated with AMEED, the epithelium healing duration decreased from 4 days in the control group to 3 days in the two AMEED groups, with lower mean degrees of eyelid oedema, chemosis, and infection compared to the control group. No pathologic abnormalities were observed in either of the AMEED groups.

Conclusion

AMEED increases LSCs proliferation ex vivo and accelerates corneal epithelium healing in vivo without any adverse effects. It could be used as a supplement for LSC expansion in cell therapy.