Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells.
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.
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.
Laura
L
Tomasello
Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy.
Rosa
R
Musso
Department of Ophthalmology, University of Palermo, Palermo, Italy.
Giovanni
G
Cillino
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.
Maria
M
Pitrone
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.|ATeN (Advanced Technologies Network Center), University of Palermo, Palermo, Italy.
Giuseppe
G
Pizzolanti
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.
Antonina
A
Coppola
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy.
Walter
W
Arancio
Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy.
Gianluca
G
Di Cara
Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy.
Ida
I
Pucci-Minafra
Department of Ophthalmology, University of Palermo, Palermo, Italy.
Salvatore
S
Cillino
Laboratory of Regenerative Medicine, Section of Endocrinology, Diabetology and Metabolism, Di.Bi.M.I.S., University of Palermo, Piazza delle Cliniche 2, 90127, Palermo, Italy. carla.giordano@unipa.it.|ATeN (Advanced Technologies Network Center), University of Palermo, Palermo, Italy. carla.giordano@unipa.it.
Carla
C
Giordano
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2 / Q000235:genetics / Q000378:metabolism; D000328:Adult; D000367:Age Factors; D000368:Aged; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D006684:HLA-DR Antigens / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D015415:Biomarkers; D006684:HLA-DR Antigens; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D009363:Neoplasm Proteins; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human
Adult stem cell pluripotency; Fibroblast-like stem cells; Limbal stem cells; Proteomic profile; Regenerative medicine