March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Directed Differentiation Of Limbal Stem Cells Into Scleral Fibroblasts
Author Affiliations & Notes
  • Lidia Tekie
    Biology, San Francisco State University, San Francisco, California
  • Christine F. Wildsoet
    Ctr for Ocular Disease & Dvlpmt, Univ of California, Berkeley, Berkeley, California
  • Footnotes
    Commercial Relationships  Lidia Tekie, None; Christine F. Wildsoet, None
  • Footnotes
    Support  CIRM bridges grant number TB1-01194
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3509. doi:
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      Lidia Tekie, Christine F. Wildsoet; Directed Differentiation Of Limbal Stem Cells Into Scleral Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3509.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Limbal stem cells (LSC) are adult multipotent stem cells with multi-lineage differentiation potential. The purpose of this study was to investigate the capacity of limbal stem cells to be isolated, cultured, and directed to differentiate into a scleral fibroblast lineage to regenerate lost sclera tissue in degenerative myopia.

Methods: : Guinea pigs and white leghorn chickens, which are common myopia animal models, were used as our source of tissue. Primary LSC culture was obtained by two methods: by plating limbal explants on 3T3 fibroblasts, or by enzymatically isolating the LSCs from the limbal tissue. The cells were cultured in equal amounts of (DMEM/F12), supplemented with 10% FBS, 10 ng/ml bFGF, 100 IU/ml penicillin, and 100 mg/ml streptomycin. When the cells had grown to over 80% confluence, they were sorted by flow cytometry. Flow-cytometry, RT-PCR, and immunofluorescence staining were used for analysis. These LSC were then cultured in cell culture media preconditioned by scleral fibroblasts for 7 days. After day 7 transforming growth factor beta 1 (TGF-β1) was added to the preconditioned culture medium for 14 days. The cells were then treated with fibroblast growth factor 2 (FGF2) for 14 days. The cell culture was then observed for morphological similarities to scleral fibroblasts.

Results: : The expression of ABCG2 and CD34 in freshly cultured primary limbal stem cells was evaluated by flow cytometry using anti-ABCG2 and -CD34 mAbs. Cells were stained for P63, a LSC specific-positive marker, after they were grown for 7 days in DMEM/F12 medium. The expression pattern for LSC associated markers was examined by RTPCR. P63, ABCG2, CD34 were highly expressed, and the differentiation markers, CK3, CK12, and Cx43, were undetectable. LSCs cultured in conditioned media with TGF-β1 obtain scleral fibroblast precursor morphology after 21 days in culture. With the addition of FGF2 after 14 days the precursor cells obtain differentiated scleral fibroblast morphology.

Conclusions: : Cell culture media preconditioned by scleral fibroblasts that is also supplemented with TGF-β1 and FGF2 appear to contain factor(s) that are beneficial to differentiating LSCs toward a scleral fibroblast lineage. The next steps of this project will be, to optimize this differentiation protocol and attempt it on human LSCs. This will be a significant contribution in facilitating the development of transplantation therapies for ocular diseases and advance cell-based therapy for degenerative myopia.

Keywords: differentiation • myopia • sclera 

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