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Yong-Soo Byun, Lisette Yco, Brittany Shaheen, Abhishek Sharma, Shweta Chaudhary, Sonal Gandhi, Sarmad Jassim, Joy Sarkar, Sapna Tibrewal, Sandeep Jain, Corneal Neurobiology Laboratory; Reprogramming Genes Are Expressed during Spheroidal Culture of Corneal Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5251.
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Corneal stromal cells transform to precursor cells in spheroidal culture. We determined whether reprogramming genes are expressed during this transformation.
Stromal cells were isolated from murine corneas by sequential collagenase digestion and adherent culture to generate a population of mixed stromal cell phenotypes. After trypsinization, spheroidal culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were analyzed for expression of reprogramming genes. Spheroids in culture were induced to differentiate to keratocytes, fibroblasts, myofibroblasts, neural cells, adipocytes, and osteocytes using the appropriate medium.
Sphere formation occurred in ultra-low attachment plates but not in adherent culture of stromal cells. Of the essential reprogramming genes, Oct4 and Sox2 were upregulated in the spheroids, but not c-Myc or Klf4. Myc genes were differentially regulated; c-Myc was downregulated and N-Myc was upregulated. Forced differentiation of spheroids reverted them to keratocytes, fibroblasts, and myofibroblasts and transformed them to neural cells but not to adipocytes or osteocytes.
In spheroidal culture, adherent corneal stromal cells transform to a less differentiated precursor form, possibly due to upregulation of essential reprogramming genes. This finding suggests that terminally differentiated stromal cells possess inherent plasticity and multilineage potential that can be activated.
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