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Ladan Espandar, Shijia Zhang, Kathleen M. Imhof, Bruce A. Bunnell; Co-culturing Adipose-derived Stem Cells (ASCs) with Adult Keratocytes, a Novel Method of Differentiation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3506. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the efficiency of co-culture system for ASCs to differentiate to keratocytes.
Co-culture Methods: Transwell inserts with 0.4-micron pores were placed into the fibronectin-coated 6-well plates that contained passage 3~4 of h-ASCs in basal keratocyte differentiation medium (KDM); and human keratocytes were inoculated into the inserts in KDM medium. The cells were allowed to culture for 14 days before RNA isolation.Control Groups: Cultured keratocytes in KDM medium and cultured h-ASCs in KDM and complete culture medium (CCM) were used as control groups for co-cultured keratocyts and h-ASCs respectively.q-RT PCR: After two weeks of cell culturing, total RNA was isolated using an RNeasy mini kit. The real-time PCR reactions were performed as previously described with slight modification. Human keratocan and aldehyde dehydrogenase (ALDH3A1) mRNA levels were assayed using Taq-Man Gene Expression Assays.
q-RT PCR was performed for ALDH3A1 and human keratocan in h-ASCs and keratocytes in co-cultured and control groups separately. Results are shown in Figure 1 A, B and Figure 2 A, B.
Co-culture system may enhance and facilitate differentiation of h-ASCs. There is an absolute need for more studies to explore interaction between stem cells and adult cells in co-culture system.
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