Keratocytes derived from spheroid culture of corneal stromal cells resemble tissue resident keratocytes.
OBJECTIVE
Corneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes.


METHODS
Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes.


RESULTS
Stromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids.


CONCLUSIONS
Stemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America; Catholic Institute for Visual Science, Department of Ophthalmology and Visual Science, College of Medicine, Catholic University of Korea, Seoul, Korea.
Yong-Soo
YS
Byun
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Sapna
S
Tibrewal
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Eunjae
E
Kim
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America; Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
Lisette
L
Yco
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Joy
J
Sarkar
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Yair
Y
Ivanir
Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America.
Chia-Yang
CY
Liu
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Cecile M
CM
Sano
Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America.
Sandeep
S
Jain
D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D060527:Corneal Keratocytes / Q000166:cytology / Q000378:metabolism*; D003319:Corneal Stroma / Q000166:cytology* / Q000502:physiology; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins / Q000378:metabolism*; D051379:Mice; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D015854:Up-Regulation
C558396:Acta2 protein, mouse; D000199:Actins; D016895:Culture Media, Serum-Free; D018398:Homeodomain Proteins; C099280:Kera protein, mouse; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D011509:Proteoglycans