Abstract
Purpose:
Cell therapy for human ocular surface reconstruction is dependent on autologous limbal stem cell expansion in vitro. However, the effectiveness of current procedure is limited by the scarce of health limbal tissue in patients with bilateral limbal stem cell d(LSC) eficiency diseases. Here we aim to transdifferentiate the mesenchymal adipose derived stem cells (ADSCs) to cells with limbal/epithelial stem cell phenotype by a combination of small molecules and defined cell culture medium for its potential application in the reconstruction of corneal epithelium.
Methods:
Epithelial transdifferentiation was initiated with a combination of small molecules including valporic acid, GSK-3 inhibitor, TGF-β receptor 1 kinase inhibitor, tranylcypromine, Alk5i and retinoic acid, followed by incubation with CNT50 medium. The limbal stem/epithelial cell associated gene expression and phenotype were evaluated by immunostaining, flow cytometry and real-time PCR with a panel of epithelial associated genes/protein such as E-cadherin, Occludin, Connexin 43 and LSC markers, such as ΔNp63 and ABCG2. Cell clone assay were carried out to identify the clonogenicity of transformed stem/progenitor cells.
Results:
After treatment, ADSCs which have fibroblast like multipolar mesenchymal cell morphology were transformed into cells with epithelial like squamous/cuboidal morphology. Quantitative PCR revealed that the expression level of epithelial cell junction associated genes in treated cells, such as E-cad was 1.1-fold higher; ZO-1, 0.8-fold higher and Occludin 4.7-fold higher compared with the untreated cells. The flow cytometry analysis showed that the expression level of epithelial associated cell junction proteins in treated cells, such as E-cadherin was 1.3-fold higher; ZO-1, 2-fold higher and Occludin, 2.2-fold higher than the untreated cells. The induced cells expressed cellular protein which are specific to LSCs and the treated cells still preserved the capability to form clones although their proliferation rate was compromised.
Conclusions:
A combination of small molecules and defined cell culture medium induced the transdifferenitation of mesenchymal ADSCs into cells with limbal epithelial stem/progenitor cell properties. This chemical induced cell population could be potentially utilized as an autologous substitute of limbal stem cells for cornea epithelium reconstruction.
Keywords: 482 cornea: epithelium •
480 cornea: basic science •
721 stem cells