Preservation of the limbal stem cell phenotype by appropriate culture techniques.
OBJECTIVE
To evaluate the effect of several culture variables on clonal growth and differentiation of limbal stem cells ex vivo and provide an improved culture technique that supports preferential expansion and preservation of stem cells for therapeutic applications.


METHODS
Corneal epithelial stem cells were isolated from human limbal specimens and clonally expanded on a 3T3 feeder layer, followed by subcultivation of holoclones on fibrin gels. The effect of different limbal regions, enzymatic dissociation methods, and culture media supplemented with different calcium, serum, and growth factor concentrations on colony-forming efficiency, colony size, and colony density was compared. A panel of putative stem cell and differentiation markers was used to analyze the epithelial phenotype by morphologic and immunohistochemical methods.


RESULTS
Limbal cells obtained from the superior limbus, isolated by a two-step enzymatic dissociation method (dispase II/trypsin-EDTA), and cultured in low to medium (0.03-0.4 mM) calcium concentrations with proper serum levels (10% FCS) and growth factor combinations (EGF, NGF) yielded the highest clonal growth capacity and an undifferentiated cellular phenotype. Subcultivation of holoclones supported the preservation of stem and progenitor cells in the basal layer of the fibrin-based epithelial sheets, as demonstrated by multiple molecular stem cell markers (p63alpha, Bmi-1, K15, and ABCG2), whereas increased calcium concentrations and air-lifting induced terminal differentiation and gradual loss of stem cells.


CONCLUSIONS
The proposed culture system supports enrichment and survival of limbal stem and progenitor cells during the entire cultivation process and may be essential for long-term restoration of the damaged ocular surface.
Meyer-Blazejewska
Ewa Anna
EA
Department of Ophthalmology, Friedrich-Alexander University of Erlangen-Nürnberg, Erlangen, Germany. ewa.meyer-blazejewska@uk-erlangen.de
Kruse
Friedrich E
FE
Bitterer
Katrin
K
Meyer
Christian
C
Hofmann-Rummelt
Carmen
C
Wünsch
Peter H
PH
Schlötzer-Schrehardt
Ursula
U
D016475:3T3 Cells; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D018920:Coculture Techniques; D003470:Culture Media; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
D015415:Biomarkers; D003470:Culture Media