Interleukin-13 maintains the stemness of conjunctival epithelial cell cultures prepared from human limbal explants

Abstract

To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]–P2). Cells were characterized by alcian blue/periodic acid–Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0–P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

The foggy world(s) of p63 isoform regulation in normal cells and cancer

“…Interleukins (ILs) including IL‐1β [184,185], IL‐6 [186], IL‐13 [187], and IL‐17/IL‐22 [188] all upregulate ΔNp63 levels in epithelial cells. ΔNp63 also induces IL‐6 [189], providing positive feedback regulation (Figure 4D).…”

Interleukin-13 increases the stemness of limbal epithelial stem cells cultures

“…The expression of the GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ΔNp63α , BMI-1 , K3 , K7 , K12 , K14 , K17 , mucin 4 ( Muc4 ), and MKI67 genes was detected by qPCR. Cells were collected after each passage (P0-P2), transferred to Eppendorf tubes containing 500 μl of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), and total RNA was extracted according to the manufacturer’s instructions, followed by reverse transcription, which has been described previously [ 18 ]. Briefly, 1 μg of RNA was treated with deoxyribonuclease I (Promega, Madison, WI, USA) and used for reverse transcription (RT).…”

“…Our previous work found a positive effect of interleukin-13 (IL13) on proliferative activity and ΔNp63α gene expression in the conjunctival epithelium generated from limbal explants [ 18 ]. The effect of IL13 on increasing the expression of the ΔNp63α gene in conjunctival cultures leads to the idea of its role in maintaining stemness and using it as part of the medium for the culture of LESCs from limbal explants.…”

Discontinuous transcription of ribosomal DNA in human cells

“…The corneoscleral rims were prepared as described before. [52,53] Shortly, corneoscleral rims were cut into 12 pieces and placed in a 24-well plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland) on Thermanox plastic coverslips (Nunc, Thermo Fisher Scientific, Rochester, NY, USA). Explants were cultured in 1 ml of complete medium [1:1 DMEM/F12, 10% FBS, 1% AA, 10 ng/ml recombinant EGF, 0.5% insulin-transferrin-selenium (Thermo Fisher Scientific), 5 μg/ ml hydrocortisone, 10 μg/ml adenine hydrochloride and 10 ng/ml cholera toxin (Sigma-Aldrich, Darmstadt, Germany)].…”