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Liu L, Yu Y, Peng Q, Porsborg SR, Nielsen FM, Jørgensen A, Grove A, Bath C, Hjortdal J, Christiansen OB, Fink T, Zachar V. Distribution of Stromal Cell Subsets in Cultures from Distinct Ocular Surface Compartments. JOVR [Internet]. 2020Oct.25 [cited 2024Apr.24];15(4):493–501. Available from: https://knepublishing.com/index.php/JOVR/article/view/7780
https://doi.org/10.18502/jovr.v15i4.7780
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authors
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Lei Liu
Lei Liu
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
-
Ying Yu
Ying Yu
Department of Neurosurgery, First Hospital of Jilin University, Changchun, China
-
Qiuyue Peng
Qiuyue Peng
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
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Simone R Porsborg
Simone R Porsborg
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
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Frederik M Nielsen
Frederik M Nielsen
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
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Annemette Jørgensen
Annemette Jørgensen
Department of Gynecology and Obstetrics, Aalborg University Hospital, Denmark
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Anni Grove
Anni Grove
Department of Pathology, Aalborg University Hospital, Denmark
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Chris Bath
Chris Bath
Department of Ophthalmology, Aalborg University Hospital, Aalborg, Denmark
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Jesper Hjortdal
Jesper Hjortdal
Department of Ophthalmology, Aarhus University Hospital, Aarhus, Denmark
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Ole B Christiansen
Ole B Christiansen
Department of Gynecology and Obstetrics, Aalborg University Hospital, Denmark
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Trine Fink
Trine Fink
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
-
Vladimir Zachar
Vladimir Zachar
Regenerative Medicine Group, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark
Published Date
- Oct 25, 2020
Distribution of Stromal Cell Subsets in Cultures from Distinct Ocular Surface Compartments
Abstract
Purpose: To reveal the phenotypic differences between human ocular surface stromal cells (hOSSCs) cultured from the corneal, limbal, and scleral compartments.
Methods: A comparative analysis of cultured hOSSCs derived from four unrelated donors was conducted by multichromatic flow cytometry for six distinct CD antigens, including the CD73, CD90, CD105, CD166, CD146, and CD34.
Results: The hOSSCs, as well as the reference cells, displayed phenotypical profiles that were similar in high expression of the hallmark mesenchymal stem cell markers CD73, CD90, and CD105, and also the cancer stem cell marker CD166. Notably, there was considerable variation regarding the expression of CD34, where the highest levels were found in the corneal and scleral compartments. The multi-differentiation potential marker CD146 was also expressed highly variably, ranging from 9% to 89%, but the limbal stromal and endometrial mesenchymal stem cells significantly surpassed their counterparts within the ocular and reference groups, respectively. The use of six markers enabled investigation of 64 possible variants, however, just four variants accounted for almost 90% of all hOSSCs, with the co-expression of CD73, CD90, CD105, and CD166 and a combination of CD146 and CD34. The limbal compartment appeared unique in that it displayed greatest immunophenotype diversity and harbored the highest proportion of the CD146+CD34- pericyte-like forms, but, interestingly, the pericyte-like cells were also found in the avascular cornea.
Conclusions: Our findings confirm that the hOSSCs exhibit an immunophenotype consistent with that of MSCs, further highlight the phenotypical heterogeneity in stroma from distinct ocular surface compartments, and finally underscore the uniqueness of the limbal region.
Keywords:
CD146, CD34, Flow Cytometry, Human Ocular Surface Stromal Cells, Pericytes
References
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11. Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res 1999;18:311–356.
12. Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK. Mesenchymal cells from limbal stroma of human eye. Mol Vis 2008;14:431–442.
13. Wegmeyer H, Broske AM, Leddin M, Kuentzer K, Nisslbeck AK, Hupfeld J, et al. Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 2013;22:2606–2618.
14. Hagmann S, Moradi B, Frank S, Dreher T, Kammerer PW, Richter W, et al. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrowderived mesenchymal stromal cells. BMC Musculoskelet Disord 2013;14:223.
15. Sidney LE, Hopkinson A. Corneal keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with fibroblast growth factor-2, transforming growth factor-beta3 and retinoic acid. J Tissue Eng Regen Med 2018;12:e203–e15.
16. Sidney LE, Branch MJ, Dua HS, Hopkinson A. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells. Cytotherapy 2015;17:1706– 1722.
17. Fink T, Rasmussen JG, Lund P, Pilgaard L, Soballe K, Zachar V. Isolation and expansion of adipose-derived stem cells for tissue engineering. Front Biosci 2011;3:256– 263.
18. Zachar V, Rasmussen JG, Fink T. Isolation and growth of adipose tissue-derived stem cells. Methods Mol Biol 2011;698:37–49.
19. Prasad SM, Czepiel M, Cetinkaya C, Smigielska K, Weli SC, Lysdahl H, et al. Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif 2009;42:63–74.
20. Gargett CE, Schwab KE, Zillwood RM, Nguyen HP, Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod 2009;80:1136–1145.
21. Nielsen FM, Riis SE, Andersen JI, Lesage R, Fink T, Pennisi CP, et al. Discrete adipose-derived stem cell subpopulations may display differential functionality after in vitro expansion despite convergence to a common phenotype distribution. Stem Cell Res Ther 2016;7:177.
22. Peng Q, Alipour H, Porsborg S, Fink T, Zachar V. Evolution of ASC immunophenotypical subsets during expansion in vitro. Int J Mol Sci 2020;21:1408.
23. Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 2001;45:194–205.
24. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 2006;69:1037–1042.
25. Zimmerlin L, Donnenberg VS, Rubin JP, Donnenberg AD. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A 2013;83:134–140.
26. Lv FJ, Tuan RS, Cheung KM, Leung VY. Concise review: the surface markers and identity of human mesenchymal stem cells. Stem Cells 2014;32:1408–1419.
27. West-Mays JA, Dwivedi DJ. The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol 2006;38:1625–1631.
28. Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells 2014;32:1380–1389.
29. Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, Okano H, et al. Isolation of multipotent neural crestderived stem cells from the adult mouse cornea. Stem Cells 2006;24:2714–2722.
30. Fayazi M, Salehnia M, Ziaei S. Differentiation of human CD146-positive endometrial stem cells to adipogenic-, osteogenic-, neural progenitor-, and glial-like cells. In Vitro Cell Dev Biol Anim 2015;51:408–414.
31. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–313.
32. Ainscough SL, Linn ML, Barnard Z, Schwab IR, Harkin DG. Effects of fibroblast origin and phenotype on the proliferative potential of limbal epithelial progenitor cells. Exp Eye Res 2011;92:10–19.
33. Bray LJ, Heazlewood CF, Munster DJ, Hutmacher DW, Atkinson K, Harkin DG. Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas. Cytotherapy 2014;16:64–73.
34. Corselli M, Chen CW, Sun B, Yap S, Rubin JP, Peault B. The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells. Stem Cells Dev 2012;21:1299– 1308.
35. Fernandez-Perez J, Binner M, Werner C, Bray LJ. Limbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro. Sci Rep 2017;7:6377.
36. Li GG, Chen SY, Xie HT, Zhu YT, Tseng SC. Angiogenesis potential of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 2012;53:3357–3367.
37. Sa da Bandeira D, Casamitjana J, Crisan M. Pericytes, integral components of adult hematopoietic stem cell niches. Pharmacol Ther 2017;171:104–113.
2. Pellegrini G, Rama P, Mavilio F, De Luca M. Epithelial stem cells in corneal regeneration and epidermal gene therapy. J Pathol 2009;217:217–228.
3. Bath C, Muttuvelu D, Emmersen J, Vorum H, Hjortdal J, Zachar V. Transcriptional dissection of human limbal niche compartments by massive parallel sequencing. PLOS ONE 2013;8:e64244.
4. Bath C, Yang S, Muttuvelu D, Fink T, Emmersen J, Vorum H, et al. Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation. Stem Cell Res 2013;10:349– 360.
5. Liu L, Nielsen FM, Emmersen J, Bath C, Ostergaard Hjortdal J, Riis S, et al. Pigmentation is associated with stemness hierarchy of progenitor cells within cultured limbal epithelial cells. Stem Cells 2018;36:1411–1420.
6. Branch MJ, Hashmani K, Dhillon P, Jones DR, Dua HS, Hopkinson A. Mesenchymal stem cells in the human corneal limbal stroma. Invest Ophthalmol Vis Sci 2012;53:5109–5116.
7. Hashmani K, Branch MJ, Sidney LE, Dhillon PS, Verma M, McIntosh OD, et al. Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation. Stem Cell Res Ther 2013;4:75.
8. Vereb Z, Poliska S, Albert R, Olstad OK, Boratko A, Csortos C, et al. Role of human corneal stroma-derived mesenchymal-like stem cells in corneal immunity and wound healing. Sci Rep 2016;6:26227.
9. Tsai CL, Wu PC, Fini ME, Shi S. Identification of multipotent stem/progenitor cells in murine sclera. Invest Ophthalmol Vis Sci 2011;52:5481–5487.
10. Carlson EC, Wang IJ, Liu CY, Brannan P, Kao CW, Kao WW. Altered KSPG expression by keratocytes following corneal injury. Mol Vis 2003;9:615–623.
11. Jester JV, Petroll WM, Cavanagh HD. Corneal stromal wound healing in refractive surgery: the role of myofibroblasts. Prog Retin Eye Res 1999;18:311–356.
12. Polisetty N, Fatima A, Madhira SL, Sangwan VS, Vemuganti GK. Mesenchymal cells from limbal stroma of human eye. Mol Vis 2008;14:431–442.
13. Wegmeyer H, Broske AM, Leddin M, Kuentzer K, Nisslbeck AK, Hupfeld J, et al. Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 2013;22:2606–2618.
14. Hagmann S, Moradi B, Frank S, Dreher T, Kammerer PW, Richter W, et al. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrowderived mesenchymal stromal cells. BMC Musculoskelet Disord 2013;14:223.
15. Sidney LE, Hopkinson A. Corneal keratocyte transition to mesenchymal stem cell phenotype and reversal using serum-free medium supplemented with fibroblast growth factor-2, transforming growth factor-beta3 and retinoic acid. J Tissue Eng Regen Med 2018;12:e203–e15.
16. Sidney LE, Branch MJ, Dua HS, Hopkinson A. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells. Cytotherapy 2015;17:1706– 1722.
17. Fink T, Rasmussen JG, Lund P, Pilgaard L, Soballe K, Zachar V. Isolation and expansion of adipose-derived stem cells for tissue engineering. Front Biosci 2011;3:256– 263.
18. Zachar V, Rasmussen JG, Fink T. Isolation and growth of adipose tissue-derived stem cells. Methods Mol Biol 2011;698:37–49.
19. Prasad SM, Czepiel M, Cetinkaya C, Smigielska K, Weli SC, Lysdahl H, et al. Continuous hypoxic culturing maintains activation of Notch and allows long-term propagation of human embryonic stem cells without spontaneous differentiation. Cell Prolif 2009;42:63–74.
20. Gargett CE, Schwab KE, Zillwood RM, Nguyen HP, Wu D. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod 2009;80:1136–1145.
21. Nielsen FM, Riis SE, Andersen JI, Lesage R, Fink T, Pennisi CP, et al. Discrete adipose-derived stem cell subpopulations may display differential functionality after in vitro expansion despite convergence to a common phenotype distribution. Stem Cell Res Ther 2016;7:177.
22. Peng Q, Alipour H, Porsborg S, Fink T, Zachar V. Evolution of ASC immunophenotypical subsets during expansion in vitro. Int J Mol Sci 2020;21:1408.
23. Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 2001;45:194–205.
24. Maecker HT, Trotter J. Flow cytometry controls, instrument setup, and the determination of positivity. Cytometry A 2006;69:1037–1042.
25. Zimmerlin L, Donnenberg VS, Rubin JP, Donnenberg AD. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A 2013;83:134–140.
26. Lv FJ, Tuan RS, Cheung KM, Leung VY. Concise review: the surface markers and identity of human mesenchymal stem cells. Stem Cells 2014;32:1408–1419.
27. West-Mays JA, Dwivedi DJ. The keratocyte: corneal stromal cell with variable repair phenotypes. Int J Biochem Cell Biol 2006;38:1625–1631.
28. Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells 2014;32:1380–1389.
29. Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, Okano H, et al. Isolation of multipotent neural crestderived stem cells from the adult mouse cornea. Stem Cells 2006;24:2714–2722.
30. Fayazi M, Salehnia M, Ziaei S. Differentiation of human CD146-positive endometrial stem cells to adipogenic-, osteogenic-, neural progenitor-, and glial-like cells. In Vitro Cell Dev Biol Anim 2015;51:408–414.
31. Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–313.
32. Ainscough SL, Linn ML, Barnard Z, Schwab IR, Harkin DG. Effects of fibroblast origin and phenotype on the proliferative potential of limbal epithelial progenitor cells. Exp Eye Res 2011;92:10–19.
33. Bray LJ, Heazlewood CF, Munster DJ, Hutmacher DW, Atkinson K, Harkin DG. Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas. Cytotherapy 2014;16:64–73.
34. Corselli M, Chen CW, Sun B, Yap S, Rubin JP, Peault B. The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells. Stem Cells Dev 2012;21:1299– 1308.
35. Fernandez-Perez J, Binner M, Werner C, Bray LJ. Limbal stromal cells derived from porcine tissue demonstrate mesenchymal characteristics in vitro. Sci Rep 2017;7:6377.
36. Li GG, Chen SY, Xie HT, Zhu YT, Tseng SC. Angiogenesis potential of human limbal stromal niche cells. Invest Ophthalmol Vis Sci 2012;53:3357–3367.
37. Sa da Bandeira D, Casamitjana J, Crisan M. Pericytes, integral components of adult hematopoietic stem cell niches. Pharmacol Ther 2017;171:104–113.