Ex vivo expanded SSEA-4+ human limbal stromal cells are multipotent and do not express other embryonic stem cell markers.
OBJECTIVE
The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.


METHODS
Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition, expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2), tumour protein p63 (p63), paired box 6 (Pax6), cytokeratin 3 (AE5), cytokeratin 10, and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes, osteocytes, and chondrocytes.


RESULTS
Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2, p63, Pax6, AE-5, and keratocan sulfate. After passaged, a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60, Tra-1-81, and transcription factors like octamer-binding transcription factor 4 (Oct4), SRY(sex determining region Y)-box 2 (Sox2), and Nanog. Early passaged cells when induced were able to differentiate into adipocytes, osteocytes and chondrocytes.


CONCLUSIONS
The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However, despite the expression of SSEA-4, these cells did not express any other markers of ESC. Therefore, we conclude that the cells did not show properties of ESC.
Stem Cell Laboratory, Haematology Unit, Cancer Research Centre, Institute for Medical Research, Kuala Lumpur, Malaysia. limmn@imr.gov.my
Moon Nian
MN
Lim
Noor Hamidah
NH
Hussin
Ainoon
A
Othman
Thiageswari
T
Umapathy
Puteri
P
Baharuddin
Rahman
R
Jamal
Zubaidah
Z
Zakaria
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005434:Flow Cytometry; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D053558:Keratin-3 / Q000378:metabolism; D016850:Limbus Corneae / Q000166:cytology* / Q000378:metabolism; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D005136:Eye Proteins; D018398:Homeodomain Proteins; C490948:KERA protein, human; D053558:Keratin-3; D009363:Neoplasm Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; D011509:Proteoglycans; D012097:Repressor Proteins; D055746:Stage-Specific Embryonic Antigens; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C066584:stage-specific embryonic antigen-4