Induced pluripotent stem cell-derived limbal epithelial cells (LiPSC) as a cellular alternative for in vitro ocular toxicity testing

Abstract

Induced pluripotent stem cells hold great potential to produce unlimited amount of differentiated cells as cellular source for regenerative medicine but also for in vitro drug screening and cytotoxicity tests. Ocular toxicity testing is mandatory to evaluate the risks of drugs and cosmetic products before their application to human patients by preventing eye irritation or insult. Since the global ban to use animals, many human-derived alternatives have been proposed, from ex-vivo enucleated postmortem cornea, primary corneal cell culture and immortalized corneal epithelial cell lines. All of them share limitations for their routine use. Using an improved protocol, we derived limbal epithelial cells from human induced pluripotent stem cells, named LiPSC, that are able to be passaged and differentiate further into corneal epithelial cells. Comparative RT-qPCR, immunofluorescence staining, flow cytometry analysis and zymography assays demonstrate that LiPSC are morphologically and molecularly similar to the adult stem cells. Moreover, contrary to HCE, LiPSC and primary limbal cells display similarly sensitive to cytotoxicity treatment among passages. Our data strongly suggest that LiPSC could become a powerful alternative cellular model for cosmetic and drug tests.

The Spectrum of PAX6 Mutations and Genotype-Phenotype Correlations in the Eye

“…Mutations in SOX2 are the most common cause of bilateral anophthalmia and severe microphthalmia [89,90]. Accordingly, a recent report has linked novel missense mutations in PAX6 (c.160A>C, p.Ser54Arg and c.372C>A, p.Asn124Lys) to severe bilateral microphthalmia, with phenotype resembling SOX2-associated MAC [139]. The authors showed that these mutations significantly reduce the binding affinity of PAX6 to a specific regulatory sequence in the EE, LE9, which also synergistically responds to SOX2 [48].…”

“…MAC phenotypes are significantly associated with mutations that are likely to disrupt the PAX6-DNA interaction, with mutations being more common in the PD, which is the domain that is known to interact with SOX2 [67,[138][139][140]. Mutations in SOX2 are the most common cause of bilateral anophthalmia and severe microphthalmia [89,90].…”

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein

“…Routine subcultures were obtained by detaching cells with StemPro Accutase Cell Dissociation Reagent (Gibco, Life Technologies) and replating at 2,000 cells/cm 2 (T‐LSCs) or 3,000 cells/cm 2 ( PAX6 +/– T‐LSCs). Primary LSCs and corneal fibroblasts (COFs) were cultivated as described elsewhere . All cells were negative for mycoplasma contamination (monthly tested).…”

Corneal cell therapy: with iPSCs, it is no more a far-sight

“…Protocol to derive corneal epithelial cells from iPSCs [ 43 – 46 ] has provided critical insights into the role of each of the exogenous factors incorporated in the culture process. For example, BMP4 has been shown to be critical in the directed differentiation process of PSCs to corneal epithelial progenitors (CEPs) [ 44 , 47 ] while Hayashi et al [ 24 ] demonstrated BMP4 treatment suppressed CE differentiation from iPSCs. Kamarudin et al [ 43 ] recently reported differences in the activity of the endogenous bone morphogenetic protein (BMP) signaling between hiPSC lines and how it impacts their differentiation fate.…”

“…From these studies, we learn about two crucial aspects: one being the density of cells plated for differentiation, as it affects the differentiation efficiency [ 44 ], and the second being the choice of extracellular matrix for directed differentiation. In case of differentiation of the iPSCs towards CE fate, collagen IV [ 47 ] has been demonstrated to be an ideal substrate. Interestingly, Zhang et al demonstrated that slightly elevated CO 2 was conductive to the differentiation of CE progenitors from hESCs [ 49 ].…”