Corneal integrity, transparency, and visual acuity are maintained by corneal epithelial cells (CECs), which are continuously renewed by limbal epithelial stem cells (LESCs). The limbal stem cell deficiency is associated with ocular diseases. This study aimed to develop a novel method to differentiate bone marrow mesenchymal stem cells (BM-MSCs) into LESC-like cells using a culture medium and paired box 6 (Pax6) transfection. The LESC-like cells were confirmed using the LESC markers CK14 and p63 and CEC marker CK12. Pax6 induces BM-MSCs to differentiate into LESC-like cells in vitro. Mouse models of chemical corneal burn were obtained and treated with the LESC-like cells. The transplantation experiment indicated that Pax6-reprogrammed BM-MSCs attached to and replenished the damaged cornea through the formation of stratified corneal epithelium. The proliferation and colony formation abilities of Pax6-overexpressing BM-MSCs were significantly enhanced. These findings provide evidence that BM-MSCs might serve as an excellent candidate for generating bioengineered corneal epithelium and provide a new strategy for the treatment of clinical corneal damage.
Get full access to this article
View all access options for this article.
References
1.
UenoH, KurokawaMS, KayamaM, HommaR, KumagaiY, MasudaC, TakadaE, TsubotaK, UenoS and SuzukiN. (2007). Experimental engraftment of CECs induced by PAX6 gene transfection of mouse embryonic stem cells. Cornea, 26:1220-1227.
2.
HuF, GuanW, ZhangY and PengX. (2022). Herpetic uveitis caused by herpes simplex virus after cataract surgery in a patient without prior viral keratitis or uveitis: A case report. BMC Ophthalmol, 22:104.
3.
KamilS and MohanRR. (2021). Corneal stromal wound healing: major regulators and therapeutic targets. Ocul Surf, 19:290–306.
4.
AhmedF, HouseRJ and FeldmanBH. (2015). Corneal abrasions and corneal foreign bodies. Prim Care Clin Office Pract, 42:363–375.
5.
UtheimTP, ØUtheimA, KhanQE and SehicA. (2016). Culture of oral mucosal epithelial cells for the purpose of treating limbal stem cell deficiency. J Funct Biomater, 7:5.
6.
KatelA and BasuS. (2022). A review of the diagnosis and treatment of limbal stem cell deficiency. Front Med (Lausanne), 9:836009.
7.
GonzálezS, ChenL and DengSX. (2017). Comparative study of xenobiotic-free media for the cultivation of human limbal epithelial stem/progenitor cells. Tissue Eng Part C Methods, 23:219–227.
8.
GhareebAE, LakoM and FigueiredoFC. (2020). Recent advances in stem cell therapy for limbal stem cell deficiency: a narrative review. Ophthalmol Ther, 9:809–831.
9.
ScafettaG, TricoliE, SicilianoC, NapoletanoC, PucaR, VingoloEM, CavallaroG, PolistenaA, FratiG and De FalcoE. (2013). Suitability of human tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells. Stem Cell Rev Rep, 9:847–857.
10.
WangHX, GaoXW, RenB, CaiY, LiW-J, YangY-L and LiY-J. (2017). Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells. Int J Ophthalmol, 10:1021–1027.
11.
SangwanVS, VemugantiGK, IftekharG, BansalAK and RaoGN. (2003). Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury. Cornea, 22:478–481.
12.
RohainaCM, ThenKY, NgAM, HalimWA, ZahidinZM, SaimA and IdrusRBH. (2014). Reconstruction of limbal stem cell deficient corneal surface with induced human bone marrow mesenchymal stem cells on amniotic membrane. Transl Res, 163:200–210.
13.
Bardag-GorceF, HoftRH, WoodA, OlivaJ, NiiharaH, MakalinaoA, ThropayJ, PanD and MeepeI. (2016). The role of e-cadherin in maintaining the barrier function of corneal epithelium after treatment with cultured autologous oral mucosa epithelial cell sheet grafts for limbal stem defficiency. J Ophthalmo, 2016; 4805986.
14.
GalindoS, de la MataA, López-PaniaguaM, HerrerasJM, PérezI, CalongeM and Nieto-MiguelT. (2021). Subconjunctival injection of mesenchymal stem cells for corneal failure due to limbal stem cell deficiency: state of the art. Stem Cell Res Ther, 12:60.
15.
AhmadS, StewartR, YungS, KolliS, ArmstrongL, StojkovicM, FigueiredoF and LakoM. (2007). Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro replication of the corneal epithelial stem cell niche. Stem Cells, 25:1145–1155.
16.
SoleimanifarF, MortazaviY, NadriS and SoleimaniM. (2017). Conjunctiva derived mesenchymal stem cell (CJMSCs) as a potential platform for differentiation into corneal epithelial cells on bioengineered electrospun scaffolds. J Biomed Mater Res A, 105:2703–2711.
17.
KeY, WuY, CuiX, LiuX, YuM, YangC and LiX. (2015). Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats. PLoS One, 10:e0119725.
18.
SunJ, LiuWH, DengFM, LuoY-H, WenK, ZhangH, LiuH-R, WuJ, SuB-Y and LiuY-L. (2018). Differentiation of rat adipose-derived mesenchymal stem cells into corneal-like epithelial cells driven by PAX6. Exp Ther Med, 15:1424–1432.
19.
Mohamed-AhmedS, YassinMA, RashadA, EspedalH, IdrisSB, Finne-WistrandA, MustafaK, VindenesH and FristadI. (2021). Comparison of bone regenerative capacity of donor-matched human adipose-derived and bone marrow mesenchymal stem cells. Cell Tissue Res, 383:1061–1075.
20.
BakshD, YaoR and TuanRS. (2007). Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells, 25:1384–1392.
21.
LennaS, BrozovichA, HiraseT, ParadisoF, WeinerBK and TaraballiF. (2022). Comparison between cancellous trabecular and cortical specimens from human lumbar spine samples as an alternative source of mesenchymal stromal cells. Stem Cells Dev, 31:21–22.
22.
LaroyeC, GibotS, ReppelL and BensoussanD. (2017). Mesenchymal stromal/stem cells: a new treatment for sepsis and septic shock?. Stem Cells, 35:2331–2339.
23.
BasuS, AliH and SangwanVS. (2012). Clinical outcomes of repeat autologous cultivated limbal epithelial transplantation for ocular surface burns. Am J Ophthalmol, 153:643–650.
24.
HeH and YiuSC. (2014). Stem cell-based therapy for treating limbal stem cells deficiency: a review of different strategies. Saudi J Ophthalmol, 28:188–194.
25.
PinnamaneniN and FunderburghJL. (2012). Concise review: stem cells in the corneal stroma. Stem Cells, 30:1059–1063.
26.
Markitanova IuV, IuSmirnovaA, PanovaIG, SukhikhGT, Zinov'evaRD and MitashovVI. (2006). Analysis of expression of regulatory genes Pax6, Prox1, and Pitx2, in differentiating eye cells in human fetus. Izv Akad Nauk Ser Biol, 33:339–346.
27.
OuyangH, XueY, XueY, LinY, ZhangX, XiL, PatelS, CaiH, LuoJ and ZhangM. (2014). WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature, 511:358–361.
28.
LinHF, LaiYC, TaiCF, TsaiJ-L, HsuH-C, HsuR-F, LuS-N, FengN-H, ChaiC-Y and LeeC-H. (2013). Effects of cultured human adipose-derived stem cells transplantation on rabbit cornea regeneration after alkaline chemical burn. Kaohsiung J Med Sci, 29:14–18.
29.
JiangTS, CaiL, JiWY, HuiY-N, WangY-S, HuD and ZhuJ. (2010). Reconstruction of the corneal epithelium with induced marrow mesenchymal stem cells in rats. Mol Vision, 16:1304–1316.
30.
CrumCP, MckeonFD. (2010). p63 in epithelial survival, germ cell surveillance, and neoplasia. Annu Rev Pathol, 5:349–371.
31.
ChenB, MiS, WrightB and ConnonCJ. (2012). Investigation of K14/K5 as a Stem Cell Marker in the Limbal Region of the Bovine Cornea. PLoS One, 5:e13192.
32.
ChenWYW, MuiMM, KaoWY, LiuCY and TsengSC. (1994). Conjunctival epithelial cells do not transdifferentiate in organotypic cultures: expression of K12 keratin is restricted to corneal epithelium. Curr Eye Res, 13:765–778.
33.
Ashery-PadanR and GrussP. (2001). Pax6 lights-up the way for eye development. Curr Opin Cell Biol, 13:706–714.
34.
JingfaZ, JingxiangZ, ChaoyangZ, JingtingZ, LiminG, Dawei L and QQinghua. (2022). Diabetic macular edema: current understanding, molecular mechanisms and therapeutic implications. Cells, 11:3362.
35.
ApteRS, ChenDS and FerraraN. (2019). VEGF in signaling and disease: beyond discovery and development. Cell, 176:1248–1264.
36.
RoshandelD, Medi EslaniM, Baradaran-RafiiA, CheungAY, KurjiK, JabbehdariS, MaizA, JalaliS, DjalilianAR and HollandEJ. (2018). Current and upcoming therapies for corneal neovascularization. Ocul Surf, 16:398–414.
37.
HuangW, WnagL, YangR, HuR, ZhengQ and ZanX. (2022). Combined delivery of small molecule and protein drugs as synergistic therapeutics for treating corneal neovascularization by a one-pot coassembly strategy. Mater Today Bio, 17:100456.
38.
ZhongY, WangK., ZhangY, YinQ, LiS, WangJ, ZhangX, HanH and YaoK. (2021). Ocular Wnt/β-catenin pathway inhibitor XAV939-loaded liposomes for treating alkali-burned corneal wound and neovascularization. Front Bioeng Biotechnol, 9:753879.
39.
HiungJ, WangW, YuJ, YuX, ZhengQ, PengF, HeZ, ZhaoW, ZhangZ, LiX and WangQ. (2017). Combination of dexamethasone and Avastin by supramolecular hydrogel attenuates the inflammatory corneal neovascularization in rat alkali burn model. Colloids Surf B Biointerfaces, 159:241–250.
40.
YinH, LuQ, WangX, MajumdarS, JunAS, StarkWJ, GrantMP and ElisseeffJH. (2019). Tissue-derived microparticles reduce inflammation and fibrosis in cornea wounds. Acta Biomaterialia, 85:192–202.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
