Characterization of ocular surface epithelial and progenitor cell markers in human adipose stromal cells derived from lipoaspirates

Abstract

Purpose: The goal of this study was to characterize and compare mesenchymal stem cells from adult human adipose tissue (ADS cells) with progenitor cell lines from the human corneoscleral limbus and to analyze their potential for the expression of epithelial markers.

Methods: Stem cell markers (CD34, CD90, p63, and ABCG2) and epithelial cell markers (CK3/76, CK12, CK76, CK19, and CK1/5/10/14) were analyzed by immunostaining, flow cytometry, Western blot analysis, and PCR methods. The authors assayed adhesion and proliferation on different extracellular matrix proteins.

Results: ADS cells expressed a set of progenitor cell markers, including p63 and ABCG2. CK12 expression in ADS cell cultures increased spontaneously and progressively by differential adhesion, which demonstrates the cells' potential and capability to acquire epithelial-like cell characteristics. The authors observed an increase in the adhesion and proliferation of ADS cells seeded onto different basement membrane extracellular matrix proteins. Laminin substrates reduced the proliferative state of ADS cells.

Conclusions: The expression of putative stem cell markers (CD90, ABCG2, and p63) and cytokeratins (CK12 and CK76) supports the hypothesis that ADS cells have self-renewal capacity and intrinsic plasticity that enables them to acquire some epithelial-like characteristics. Therefore, adult ADS cells could be a potential source for cell therapy in ocular surface regeneration.

Figure 1.

Primary culture by phase-contrast microscopy. Human LSCs on 3T3-SA mitomycin-inactivated feeder layer exhibited spherical clonal colonies (a). Human lipoaspirate ADS cells presented fibroblast-like morphology after isolation and differential adhesion (b).

Figure 2.

Adipogenic and osteogenic differentiation. Oil-Red-O–positive staining for intracellular fat droplets (b, c) indicated that ADS cells were capable of adipogenic differentiation. Alizarin Red staining (d) and an elution assay confirmed better osteogenic differentiation after 28 days' culture in induction medium. At this stage, alkaline phosphatase activity increased significantly compared with control cultured ADS cells (a). Phase-contrast microscopy (a, b, d); epifluorescence microscopy (c) with nuclear staining carried out with bis-benzamide (blue).

Figure 3.

Indirect immunofluorescence for CKs. ADS cells were positive for CK3/76 (e), which is specific for differentiated epithelia, but were negative for CK1/51014 (f). LSCs were moderate positive for CK3/76 (b) and CK1/5/10/14 (c), which is specific for stratified epithelia. Control conditions (a, d). Nuclear staining was performed with bis-benzamide (blue). Scale bar, 20 μm.

Figure 4.

Western blot analysis for CKs and p63 isoforms. LSCs strongly expressed all isoforms for p63 and a wide panel of several cellular subsets of CKs for differentiated epithelia. ADS cells were negative for p63, with weak expression for CK3/76 and CK12. Normal human corneal epithelial cell (CO) extracts were used as a control for the expression profile. SDS-PAGE experiments were carried out with a twofold concentration of ADS cell protein extract.

Figure 5.

Expression of mRNA by qRT-PCR. Expression of ABCG2, the α-isoform of ΔNp63, and specific corneal epithelial marker CK3 (KRT3), CK12 (KRT12), and CK76 (KRT76) were quantified in normal human corneal epithelial cells (CO), LSCs, and ADS cells immediately isolated (ADS) and after differential adhesion (ADS1). ADS expressed mRNA for ABCG2 and p63α but not for CK3. CK12 mRNA expression in ADS cells increased after differential adhesion in culture, whereas CK76 mRNA expression was maintained. Expression of mRNA is represented by the threshold cycle (Ct) ± SD and normalized for sample loading with 18S rRNA. Note that an increase of 2 in Ct indicates approximately a fourfold decrease in mRNA expression. The number of qPCR cycles (±SD) obtained is represented in an associated table for each condition.

Figure 6.

PCR confirmation for CKs. The presence of CK12 (KRT12) in ADS cells was confirmed by PCR amplification of DNA obtained from reverse-transcribed total RNA. The products were purified, sequenced, and compared with the human CK12 mRNA sequence (mRNA CK12) that exhibited high specificity (≅90%). CK3 (KRT3) was absent in ADS cells. Normal human corneal epithelial cells (CO) and LSCs were used as positive controls. KRT12f, forward PCR product for CK12.

Figure 7.

Adhesion and proliferation assays on extracellular matrix proteins. ADS cells behaved homogenously with improved adhesion (a; 24 hours) and proliferation (b, c; 24 and 72 hours, respectively) on specific extracellular matrix proteins. LN substrates suggested that there was differential behavior between cellular adhesion and proliferation. CTR, control; GEL, gelatin 1%; BSA, bovine serum albumin 1%; Col I, type I collagen 20 μg/mL; FN, fibronectin 10 μg/mL; LN, laminin 10 μg/mL; A, Col IV, type IV collagen 50 μg/mL; Col IV + LN, type IV collagen 50 μg/mL and LN 4 μg/mL. *P < 0.05 and **P < 0.01 compared with control.