Inhibition of TGFβ cell signaling for limbal explant culture in serumless, defined xeno-free conditions

- Removal of FBS from limbal biopsy explant cultures inhibits limbal explant outgrowth proliferation.
- Inhibition of TGFβ signaling restores growths to its FBS-complemented levels, with preservation of cellular phenotype.
- In the presence of FBS inhibition of TGFβ signaling does not modify growth and differentiation.
- Thus, the only role of FBS in limbal epithelial explant culture is neutralization of cytostatic autocrine TGFβ signaling.
- Results open the door for the production of limbal epithelial sheets for regenerative medicine using serumless media.

Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGFβ. Since TGFβ is a cytostatic effector for epithelial proliferation we examined its effect on these cultures. Limbal biopsies were set on explant culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB = SHEM plus SB431542 an inhibitor of TGFβ signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGFβ inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGFβ act as an autocrine cytostatic agent and that TGFβ inhibitors allow explant culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal explant cultures may result exclusively from neutralization of the TGFβ cytostatic effect.