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Fulltext A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology

Shaharuddin B, Ahmad S, Md Latar N, Ali S, Meeson A

Stem Cells Transl Med, 2017 03;6(3):761-766.
PMID: 28297580 DOI: 10.5966/sctm.2016-0175

Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766.
Side population cells in anaplastic thyroid cancer and normal thyroid

Mahkamova K, Latar NM, Aspinall S, Meeson A

Exp Cell Res, 2019 01 01;374(1):104-113.
PMID: 30465733 DOI: 10.1016/j.yexcr.2018.11.012

Comparison of studies of cells derived from normal and pathological tissues of the same organ can be fraught with difficulties, particular with cancer where a number of different diseases are considered cancer within the same tissue. In the thyroid, there are 4 main types of cancer, three of which arise from follicular epithelial cells; papillary and follicular which are classified as differentiated, and anaplastic which is classified as undifferentiated. One assay that can be utilised for isolation of cancer stem cells is the side population (SP) assay. However, SP studies have been limited in part due to lack of optimal isolation strategies and in the case of anaplastic thyroid cancer (ATC) are further compounded by lack of access to ATC tumors. We have used thyroid cell lines to determine the optimal conditions to isolate viable SP cells. We then compared SP cells and NSP cells (bulk tumour cells without the SP) of a normal thyroid cell line N-thy ori-3-1 and an anaplastic thyroid cancer cell line SW1736 and showed that both SP cell populations displayed higher levels of stem cell characteristics than the NSP. When we compared SP cells of the N-thy ori-3-1 and the SW1736, the SW1736 SP had a higher colony forming potential, expressed higher levels of stem cell markers and CXCR4 and where more migratory and invasive, invasiveness increasing in response to CXCL12. This is the first report showing functional differences between ATC SP and normal thyroid SP and could lead to the identification of new therapeutic targets to treat ATC.
Human limbal mesenchymal stem cells express ABCB5 and can grow on amniotic membrane

Shaharuddin B, Osei-Bempong C, Ahmad S, Rooney P, Ali S, Oldershaw R, et al.

Regen Med, 2016 Apr;11(3):273-86.
PMID: 26965478 DOI: 10.2217/rme-2016-0009

To isolate and characterize limbal mesenchymal stem cells (LMSCs) from human corneoscleral rings.
Impact of transforming growth factor beta 1 on normal and thyroid cancer side population cells

Latar NM, Mahkamova K, Elson J, Karnik I, Sutherland R, Aspinall S, et al.

Endocrine, 2022 Feb 03.
PMID: 35118633 DOI: 10.1007/s12020-022-02990-4

PURPOSE: To determine the impact of exogenous transforming growth factor beta 1 (TGF-β1) on side population (SP) cells isolated from normal, papillary thyroid cancer and anaplastic thyroid cancer cell lines and from human thyroid tissues.
METHODS: All cell populations were stained with Hoechst 33342 and analysed using dual wavelength flow cytometry to identify SP cells. This SP assay was used to assess the impact of TGF-β1 treatment and withdrawal of treatment on SP percentages. Semi-quantitative and quantitative PCR were used for molecular analysis of cells pre and post TGF-β1 treatment.
RESULTS: All cell lines expressed mRNA for both TGFB1 and its receptors, as well as showing variable expression of CDH1 and CDH2, with expressing of CDH1 being highest and CDH2 being lowest in the normal cell line. Exposure to exogenous TGF-β1 resulted in a reduction in mRNA expression of ABCG2 compared to controls which was significant between control and treated cancer cell lines. SP cells were isolated from primary human thyroid tissues, with numbers being significantly higher in papillary thyroid cancers. Exposure to TGF-β1 decreased the SP percentage in both thyroid cancer cell lines and completely abrogated these cells in the primary papillary thyroid cancer cultures. On withdrawal of TGF-β1 the SP phenotype was restored in the cancer cell lines and SP percentages increased to above that of untreated cells.
CONCLUSIONS: TGF-β1 exposure transiently regulates thyroid cancer SP cells, leading to a reduction in SP percentages, while withdrawal of TGF-β1 results in restoration of the SP phenotype.