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J Mario Wolosin, Aldo Zamudio, So-Hyang Chung, Zheng Wang; Inhibition of TGFβ-SMAD2/3 Signaling Allows Limbal Explant Outgrowth Culture in Chemically Defined, Xeno-free Medium.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5619.
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© ARVO (1962-2015); The Authors (2016-present)
Expanded population of limbal epithelial precursor cells are needed to treat limbal stem cell deficiency (LSCD). Optimal expansion is achieved by explant culture or by clonal culture under mouse feeder cell support... In both cases the medium requires serum. The purpose of this study was to assess whether the inability to expand the cells in serum-free medium, as it occurs in vivo is due to proliferative arrest caused by endogenously-generated TGFβ.<br />
Rabbit or anonymized cadaver donor limbal biopsies were set on explant culture over permeable Costar inserts in the following media, SHEM (control/reference) consisting of DMEM/F12 - ITS- 5 ng/ml EGF, 5 ng/ml cholera toxin (CT), 5 mg ethanolamine,14 mg phosphoethanol amine and 5 % FBS; sfSHEM = SHEM with FBS replaced by 0.05 % Albumax II ; sfSHEMSB = sfSHEM plus 10 μM of the TGFβ-SMAD2 inhibitor SB431542 (SB); SHEMSB = SHEM plus SB. After culture for up to 2 weeks, biopsies were removed, outgrowths were trypsinized and cell yield, cell morphology (size and granularity) and stem cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. p63, K3 and connexin 43 content were determined by Western blot. To determine whether the absence of FBS affected the proliferative potential of the limbal niche, the biopsies were serially transferred onto new insert for up to six times ( IOVS, 52:4330).
Explant outgrowths cell yields in rabbit, relative to SHEM (in %, for n =10) were, sfSHEM : 3 ±1 (p < 0.01); sfSHEMSB, 94 ±18 (p > 0.95); SHEMSB, 104 ±13 (p >0.95). After outgrowth were established in sfSHEMSB, switching to sfSHEM or sfSHEM+0.2 ng/ml TGFβ caused identical degrees of proliferation inhibition. Removal of EGF from sfSHEMSB, reduced yield by 92±6 % (n=3; p < 0.05) but removal of Albumax and/or CT did not cause a statistically significant change in outgrowth area or cell yield (n= 3, p> 0.95). Outgrowth area, cell size, dye exclusion, p63, Connexin 43 and K3 content, colony formation efficiencies and limbal biopsy extended proliferative potential were essentially identical for SHEM, sfSHEMSB and SHEMSB. Equivalent results of similar cell yield, morphology, transport function and protein expression in human were confirmed in a limited study (n = 3).<br />
Explant outgrowth for limbal transplant can be achieved in xeno-free, chemically defined medium if endogenous TGFβ signaling is blocked.
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