Cell and Organ Transplantology. 2017; 5(1):62-66.
Morphological and functional characteristics of cell culture derived from the mouse nail unit
Kalmukova O. O.1, 2, Ustymenko A. M.1, Lutsenko T. M.1, Klymenko P. P.1, Kyryk V. M.1
1State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Kyiv, Ukraine
2Educational and Scientific Centre “Institute of Biology”, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
Nail unit is a complex anatomical structure that is capable of rapid growth and regeneration throughout the life. Such significant reparative potential is associated with the presence different types of stem and progenitor cells, whose biology remains one of the fundamental issues today. Taking into account the active search for new stem cell sources for cell therapy, the view of the nail unit as a potential site for the localization of undifferentiated cells with stem potency is topical problem.
Purpose. The study was conducted with an objective to establish the morphological, morphometric and proliferative characteristics of cultured cells isolated from the mouse nail unit.
Materials and methods. Primary cultures of cells were obtained from tissue sampling, which included areas of the proximal nail fold, nail matrix and onychodermis of the FVB mouse nail organ. Cells were cultured in DMEM:F12 medium with 15 % fetal bovine serum during 6 passages. We determined the colony-forming activity, the population growth rate and doubling time, measured the area of cells, nuclei, and calculated the nuclear-cytoplasmic ratio. For cell morphology analysis, we used staining with Bemer’s hematoxylin and eosin, Heidenhain’s iron hematoxylin and May-Grünwald stain.
Results. According to the morphological analysis in vitro the cells from mouse nail unit are heterogeneous with high synthetic activity and a low nuclear-cytoplasmic ratio – the features characteristic of the low-differentiated cells. The population doubling time of the culture was 80 ± 6.5 hours on average, the fastest growing cells were at the 4th passage (63 ± 7 hours). The specific growth rate for cell culture is low (0.01 ± 0.0007).
The colony forming efficiency at the 5th passage was only 4 %. A significant number of colonies was small with large poorly proliferative cells, which may indicate a production of large numbers of transitional progenitor cells.
Conclusion. The obtained cell culture from the mouse nail unit according to the analysis of their morphology, morphometry and proliferative potential is heterogeneous and requires the further development of pure culture technologies for the detailed characterization of separate subpopulations of cells.
Key words: nail unit; nail matrix; onychodermis; cell culture; clonogenic assayFull Text PDF (eng) Full Text PDF (ua)
|1. Sorock G, Lombardi D, Hauser R, et al. Acute traumatic occupational hand injuries: type, location, and severity. J Occup Environ Med. 2002; 44: 345-51.
|2. Rinkevich Y, Maan Z, Walmsley G, et al. Injuries to appendage extremities and digit tips: A clinical and cellular update. Dev Dyn. 2015; 244(5): 641-50.
|3. Lehoczky J. Are fingernails a key to unlocking the puzzle of mammalian limb regeneration? Experimental Dermatolog. 2016; doi: 10.1111/exd.13246
|4. Kalmukova OO. Stem cells in nail unit of mammalians. Cell and Organ Transplantology. 2016; 4(1):138-143. doi: 10.22494/COT.V4I1.1
|5. Ito T, Ito N, Saathoff M, et al. Immunology of the human nail apparatus: the nail matrix is a site of relative immune privilege. J Invest Dermatol. 2005; 125: 1139-148.
|6. Sellheyer K, Nelson P. The concept of the onychodermis (specialized nail mesenchyme): an embryological assessment and a comparative analysis with the hair follicle. J Cutan Pathol. 2013; 40: 463-471.
|7. Lee KJ, Kim WS, Lee JH, et al. CD10, a marker for specialized mesenchymal cells (onychofibroblasts) in the nail unit. J Dermatol Sci. 2006; 42: 65-67.
|8. Sellheyer K. Nail stem cells. JDDG. 2013; 1103: 235-39.
|9. Nakamura M, Ishikawa O. The localization of label-retaining cells in mouse nails. J Invest Dermatol. 2008; 128: 728-30.
|10. Takeo M, Chou W, Sun Q, et al. Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature. 2013; 499: 228-32.
|11. Lee DY, Park JH, Shin HT, et al. The presence and localization of onychodermis (specialized nail mesenchyme) containing onychofibroblasts in the nail unit: a morphological and immunohistochemical study. Histopathology. 2012; 61(1): 123-30.
|12. Park JH, Kim JH, Lee JH, et al. Onychodermis (specialized nail mesenchyme) is present in ectopic nails. J Cutan Pathol. 2013; 40: 600-602.
|13. Perrin C. The nail dermis: from microanatomy to constitutive modelling. Histopathology. 2015; 66(6): 864-72.
|14. Naveau A, Seidel K, Klein OD. Tooth, hair and claw: comparing epithelial stem cell niches of ectodermal appendages. Experimental cell research. 2014; 325(2): 96-103.
|15. Narytnyk A, Gillinder K, Verdon B, et al. Neural crest stem cell-specific deletion of the Pygopus2 gene modulates hair follicle development. Stem Cell Reviews and Reports. 2014; 10(1): 60-68.
|16. Soukup V, Epperlein HH, Horacek I, et al. Dual epithelial origin of vertebrate oral teeth. Nature. 2008; 455(7214): 795-98.
|17. Okazaki M, Yoshimura K, Fujiwara H, et al. Induction of hard keratin expression in non-nail-matrical keratinocytes by nail-matrical fibroblasts through epithelial-mesenchymal interactions. Plastic and reconstructive surgery. 2003; 111(1): 286-90.
|18. Achten G. Normale Histologie und Histochemie des Nagels. In: Jadassohn J: Handbuch der Haut- und Geschlechtskrankheiten.Bd 1. Berlin: Springer, 1968: 339-76.
|19. Sieber-Blum M, Grim M, Hu YF, et al. Pluripotent neural crest stem cells in the adult hair follicle. Developmental Dynamics. 2004; 231(2): 258-69.
|20. Fernandes KJ, McKenzie IA, Mill P, et al. A dermal niche for multipotent adult skin-derived precursor cells. Nature cell biology. 2004; 6(11): 1082-93.
|21. Anbar T, Hay RA, Abdel-Rahman AT, et al. Clinical study of nail changes in vitiligo. Journal of cosmetic dermatology. 2013; 12(1): 67-72.
|22. Haber R, Khoury R, Kechichian E, et al. Splinter hemorrhages of the nails: a systematic review of clinical features and associated conditions. International Journal of Dermatology. 2016; 55(12): 1304-310.
|23. Schons KR, Knob CF, Murussi N, et al. Nail psoriasis: a review of the literature. An Bras Dermatol. 2014; 89(2): 312-17.
|24. Available: http://www.doubling-time.com/compute.php|
|25. Cell cloning by serial dilution in 96 well plates, protocol. John A. Ryan. Corning. Available: http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/34963/3-2Single_cell_cloning_protocol.pdf|
|26. Kitahara T, Ogawa H. Cultured nail keratinocytes express hard keratins characteristic of nail and hair in vivo. Arch Dermatol Res. 1992; 284(4): 253-56.
|27. Picardo M, Tosti A, Marchese C, et al. Characterization of cultured nail matrix cells. J Am Acad Dermatol. 1994; 30: 434-40.
|28. Quintanilla RHJr, Asprer JS, Vaz C, et al. CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLoS One. 2014; 9(1): e85419.
|29. Kyryk V, Kuchuk O, Butenko G. Cultivation of cells with stem potential from nail matrix. Abstracts of the World Conference on Regenerative Medicine, November 2-4, 2011, Leipzig, Germany. Regenerative Medicine. 2011; 6(6), Suppl. 2:216-217.|
|30. Rinkevich Y, Lindau P, Ueno H, et al. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature. 2011; 476(7361): 409-13.
Kalmukova OO, Ustymenko AM, Lutsenko TM, Klymenko PP, Kyryk VM. Morphological and functional characteristics of cell culture derived from the mouse nail unit. Cell and Organ Transplantology. 2017; 5(1):62-66. doi:10.22494/cot.v5i1.69