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Martha L Funderburgh, Irona Khandaker, James L Funderburgh; Novel gene expression identifies regenerative potential in adult corneal stem cells. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4123.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Many gene products are used to identify mesenchymal stem cells, but few have been linked to stem cell functions such as multi-potent differentiation and tissue regeneration. Recent clinical trials have documented the ability of corneal stromal stem cells (CSSC) to suppress corneal inflammation and to regress corneal scarring. This study examines the hypothesis that gene expression patterns by CSSC can identify cell lines with high potential for regenerative property.
Methods: CSSC lines were isolated from corneal tissue from 24 human donors and used at passage 3 as previously reported. The effect of conditioned media from confluent CSSC on immune response was demonstrated on cultured RAW264.7 macrophages stimulated by RANKL. The response of the CSSC medium on macrophages was assayed at 3 days by SYBR qPCR for Mmp9, Apc5 and Ctsk. Regenerative potential of CSSC was previously published in a mouse corneal wound healing model (PCMID4398334). Gene expression was examined by RNAseq, Nanostring Array and TaqMan qPCR. Protein expression was examined by immunostaining of cultured CSSC. Image analysis of nuclear staining was scored using FIJI software in >10 images, and differences were determined by student’s t-test (p<0.05).
Results: Comparison of conditioned media from 24 CSSC cell lines showed varying abilities to inhibit differentiation of RAW 264.7 macrophages in response to an inflammatory stimulus. Cell lines with strong anti-inflammatory properties showed strong potential to prevent scarring in a mouse wound healing, whereas lines with weak anti-inflammatory ability showed little potential for scar-free healing. Gene expression analyses identified several genes with expression correlating to anti-inflammatory potential, including ALX1, TGX18, MMP16 and FDG5. Immunostaining of CSSC in culture found expression of ALX1, a homeobox protein, to be differentially expressed in CSSC nuclei. Cell lines with strong anti-inflammatory and regenerative properties had significantly more nuclei staining for this protein.
Conclusions: Previous reports have shown that mesenchymal cells vary in their ability to exhibit useful stem cell functions, particularly stimulation of regeneration of damaged tissues. Identification of genes and proteins expressed in stem cells correlating to regenerative potential could represent an important tool in choosing cell lines for clinical use.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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