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A Small-Molecule Wnt Mimic Improves Human Limbal Stem Cell Ex Vivo Expansion.

ABSTRACT: Ex vivo cultured limbal stem/progenitor cells is an effective alternative to other surgical treatments for limbal stem cell deficiency, but a standard xenobiotic-free method for culturing the LSCs in vitro needs to be optimized. Because Wnt ligands are required for LSC expansion and preservation in vitro, to create a small-molecule Wnt mimic, we created a consolidated compound by linking a Wnt inhibitor that binds to the Wnt co-receptor Frizzled to a peptide derived from the N-terminal Dickkopf-1 that binds to Lrp (low-density lipoprotein receptor-related protein) 5/6, another Wnt co-receptor. This Wnt mimic not only enhances cellular Wnt signaling activation, but also improves the progenitor cell phenotype of in vitro cultured limbal epithelial cells. As the maintenance of stem cell characteristics in the process of culture expansion is essential for the success of ocular surface reconstruction, the small molecules generated in this study may be helpful in the development of pharmaceutical reagents for treating corneal wounds.

SUBMITTER: Zhang C

PROVIDER: S-EPMC7200314 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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A Small-Molecule Wnt Mimic Improves Human Limbal Stem Cell Ex Vivo Expansion.

Zhang Chi C Mei Hua H Robertson Sarah Y T SYT Lee Ho-Jin HJ Deng Sophie X SX Zheng Jie J JJ

iScience 20200418 5

Ex vivo cultured limbal stem/progenitor cells is an effective alternative to other surgical treatments for limbal stem cell deficiency, but a standard xenobiotic-free method for culturing the LSCs in vitro needs to be optimized. Because Wnt ligands are required for LSC expansion and preservation in vitro, to create a small-molecule Wnt mimic, we created a consolidated compound by linking a Wnt inhibitor that binds to the Wnt co-receptor Frizzled to a peptide derived from the N-terminal Dickkopf- ...[more]

PMID: 32361505

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Project description:The gold standard substrate for the ex vivo expansion of human limbal stem cells (LSCs) remains the human amniotic membrane (HAM) but this is not a defined substrate and is subject to biological variability and the potential to transmit disease. To better define HAM and mitigate the risk of disease transmission, we sought to determine if decellularisation and/or ?-irradiation have an adverse effect on culture growth and LSC phenotype. Ex vivo limbal explant cultures were set up on fresh HAM, HAM decellularised with 0.5M NaOH, and 0.5% (w/v) sodium dodecyl sulfate (SDS) with or without ?-irradiation. Explant growth rate was measured and LSC phenotype was characterised by histology, immunostaining and qRT-PCR (ABCG2, ?Np63, Ki67, CK12, and CK13). ?-irradiation marginally stiffened HAM, as measured by Brillouin spectromicroscopy. HAM stiffness and ?-irradiation did not significantly affect the LSC phenotype, however LSCs expanded significantly faster on ?-irradiated SDS decellularised HAM (p<0.05) which was also corroborated by the highest expression of Ki67 and putative LSC marker, ABCG2. Colony forming efficiency assays showed a greater yield and proportion of holoclones in cells cultured on ?-irradiated SDS decellularised HAM. Together our data indicate that SDS decellularised HAM may be a more efficacious substrate for the expansion of LSCs and the use of a ?-irradiated HAM allows the user to start the manufacturing process with a sterile substrate, potentially making it safer. STATEMENT OF SIGNIFICANCE:Despite its disadvantages, including its biological variability and its ability to transfer disease, human amniotic membrane (HAM) remains the gold standard substrate for limbal stem cell (LSC) culture. To address these disadvantages, we used a decellularised HAM sterilised by gamma-irradiation for LSC culture. We cultured LSCs on fresh HAM, HAM decellularised with NaOH, HAM decellularised with sodium dodecyl sulfate (SDS) and HAM decellularised with SDS and sterilised with gamma-irradiation. We demonstrated that although HAM decellularised with SDS and sterilised with gamma-irradiation is significantly stiffer this does not affect LSC culture growth rate or the phenotype of cultured LSCs. We therefore recommend the use of SDS decellularised gamma-irradiated HAM in future LSC clinical trials.

Dataset Information

A Small-Molecule Wnt Mimic Improves Human Limbal Stem Cell Ex Vivo Expansion.

Publications

A Small-Molecule Wnt Mimic Improves Human Limbal Stem Cell Ex Vivo Expansion.

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