Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells

Abstract

Background: In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.

Methods: Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student's unpaired t test and considering p < 0.05 as statistically significant.

Results: The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of "the stem gene profile"; 84 were differentially expressed and were involved in structural activity.

Conclusions: The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.

Keywords: Adult stem cell pluripotency; Fibroblast-like stem cells; Limbal stem cells; Proteomic profile; Regenerative medicine.

Fig. 1

A: a Limbus digested after 2-week plate seeding: f-LSC growth in monolayer (P0 culture passage, 10×); b example of an f-LSC limbosphere in low-adhesion culture conditions (20×); c, d example of an f-LSC limbosphere under adhesion culture condition (c 20×; d 40×). B: SSEA4⁺ immunofluorescence staining: a DAPI on a limbosphere; b SSEA4 detection on a limbosphere (20×); f DAPI on monolayer; g SSEA4 detection in f-LSC monolayer (40×); c, h merge; d, i DAPI/SSEA4-stained BM-MSCs; e, l DAPI/SSEA4-stained HeLa cells. C: Cytofluorimetric assay in total population (presorting, upper panel) and in SSEA4⁺ f-LSCs (postsorting, lower panel). Cells are negative for CD34, CD45, and HLA-DR. All fields are representative of one limbus sample out of at least 12 independent experiments. DAPI 4′,6-diamidino-2-phenylindole, N.C. negative control, P.C. positive control

Fig. 2

a (Upper panel) Cell cycle distribution of f-LSCs (P4 on the left; P30 on the right) performed according to Nicoletti’s protocol. (Lower panel) Increment of expression of CDKN1B in P30 vs. P4 (left); kinetics of f-LSCs at early passage (P4) and late passage (P30) in expansion medium (right). Mean values ± SD of a set of 25 experiments after 6 days. b Cytometric detection of double-positive cell populations for pluripotent stem cell markers at two different culture passages: early passage (P4) and late passage (P30). P passage

Fig. 3

Donor age and long-term culture effect on stem cell profile. a f-LSCs maintain negativity for CD34, CD45, and HLA-DR and are highly positive for SSEA4 in patients < 45 years old (upper graph) and > 45 years old (lower graph). b Real-time quantitative PCR analysis of pluripotent stem cell markers in f-LSC different culture passage obtained from patients of different ages. Mean values ± SD of all limbus donors studied are reported. P passage

Fig. 4

a f-LSC two-dimensional electrophoresis master proteomic map with labeled proteins. Two-dimensional gel electrophoresis assay resulted in a master gel that revealed 164 spots, 78 % of which had an average pixel density of about 0.3. b Densitometry profile of f-LSC proteins. f-LSC fibroblast-like limbal stem cell

Fig. 5

Pie charts representing the GO molecular function of unvaried proteins a and differential expressed proteins b. Protein network of f-LSC unvaried proteins c and differential expressed proteins, performed on the STRING website d. Protein class distribution of unvaried e and differential expressed proteins f, performed on the Gene Ontology website

Fig. 6

a f-LSCs stained with Alizarin red S for detection of calcific deposition in cultured f-LSCs for differentiation towards an osteogenic lineage. b Alcian blue staining to detect chondrogenic differentiation. c Oil Red O-stained neutral triglycerides and lipids on adipose-differentiated cells. The appropriate medium for the multilineage differentiation is reported in Methods