FREE SHIPPING over $40
OVER 10000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Stem cell research & therapy2018; 9(1); 181; doi: 10.1186/s13287-018-0918-x

Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis.

Abstract: Skin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium. Conditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium. Conditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers. Our results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation.
Get Full Text
Publication Date: 2018-07-04 PubMed ID: 29973296PubMed Central: PMC6032561DOI: 10.1186/s13287-018-0918-xGoogle Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research describes a possible solution to chronic wound healing in equine species by developing a method for long-term expansion of primary equine keratinocytes (cells that constitute skin) in a lab setting. These expanded cells, when transplanted, can differentiate into skin layers, offering potential healing benefits.

Objective of the Study

  • The study aims to test conditional reprogramming (CR) as a method to rapidly expand primary equine keratinocytes while ensuring the cells maintain their ability to differentiate into skin layers (stratified epidermis). This could potentially present a solution for chronic wound healing in horses.

Methods Used in the Study

  • Conditional reprogramming was used to isolate and propagate equine keratinocyte cultures.
  • To ensure the cells isolated were of equine origin, PCR (Polymerase Chain Reaction) and FISH (Fluorescent In-Situ Hybridization) were used.
  • A chromosomal count was run using Karyotyping.
  • FACS (Fluorescence-Activated Cell Sorting) analysis and immunofluorescence were used to confirm the quality of the cells and their proliferative state.
  • A three-dimensional air-liquid interphase method was used to test the ability of the cells to differentiate and form a stratified epidermis.

Results of the Study

  • The study found that conditional reprogramming method was efficient for isolating and propagating equine keratinocyte cultures.
  • The cells were propagated at an impressive rate and a feeder-free culture method was developed that allowed their long-term expansion.
  • Karyotyping showed normal equine chromosomes, validating that the cells maintained their genetic make-up.
  • FACS results indicated a pure population of keratinocytes.
  • When the cells were transferred to a three-dimensional air-liquid culture, they were successful in forming a well-differentiated stratified squamous epithelium, replicating the anatomy of skin layers.

Implications of the Study

  • The results suggest that conditional reprogramming can be used to create large volumes of primary equine keratinocytes in the lab.
  • When transplanted, these cells can effectively differentiate into skin layers, offering a potential solution for treating chronic wounds in horses.
  • The use of autologous cells (cells from the same individual) reduces the risk of immune rejection when transplanted back into the horse.

Cite This Article

APA
Alkhilaiwi F, Wang L, Zhou D, Raudsepp T, Ghosh S, Paul S, Palechor-Ceron N, Brandt S, Luff J, Liu X, Schlegel R, Yuan H. (2018). Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis. Stem Cell Res Ther, 9(1), 181. https://doi.org/10.1186/s13287-018-0918-x

Publication

Stem cell research & therapy
ISSN: 1757-6512
NlmUniqueID: 101527581
Country: England
Language: English
Volume: 9
Issue: 1
Pages: 181

Researcher Affiliations

Alkhilaiwi, Faris
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
  • Department of Oncology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
  • Department of Biochemistry and Molecular Biology, Georgetown University Medical School, Washington, DC, 20057, USA. faa50@georgetown.edu.
  • College of Pharmacy, King Abdul Aziz University, Jeddah, Saudi Arabia. faa50@georgetown.edu.
Wang, Liqing
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Zhou, Dan
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Raudsepp, Terje
  • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
Ghosh, Sharmila
  • Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, USA.
Paul, Siddartha
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Palechor-Ceron, Nancy
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Brandt, Sabine
  • Equine Clinic, VetOMICs Core Facility, Veterinary University Vienna, Vienna, Austria.
Luff, Jennifer
  • Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.
Liu, Xuefeng
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA.
Schlegel, Richard
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. richard.schlegel@georgetown.edu.
Yuan, Hang
  • Department of Pathology, Georgetown University Medical School, Washington, DC, 20057, USA. Hang.Yuan@georgetown.edu.

MeSH Terms

  • Animals
  • Cell Differentiation / physiology
  • Cells, Cultured
  • Epidermal Cells / cytology
  • Epidermal Cells / metabolism
  • Epidermis / metabolism
  • Horses
  • Keratinocytes / cytology
  • Keratinocytes / metabolism
  • Male

Grant Funding

  • K01 OD023219 / NIH HHS

Conflict of Interest Statement

ETHICS APPROVAL AND CONSENT TO PARTICIPATE: All samples were collected after approval by the Ethics Review Committee, University of Georgetown. CONSENT FOR PUBLICATION: Not applicable. COMPETING INTERESTS: The authors declare that they have no competing interests. PUBLISHER’S NOTE: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

This article includes 36 references
  1. Outram AK, Stear NA, Bendrey R, Olsen S, Kasparov A, Zaibert V. The earliest horse harnessing and milking. Science 2009;323:1332–1335.
    doi: 10.1126/science.1168594pubmed: 19265018google scholar: lookup
  2. Ogorev J, Lapanja T, Poklukar K, Tominsek N, Dovc P. Establishment of primary keratinocytes culture from horse tissue biopsates. Acta agriculturae Slovenica 2015;106:2.
  3. Madewell BR. Cancer. UC Davis Book of Horses New York: Harper Collins; 1996. pp. 339–345.
  4. Stannard AA, Barlough J. The skin and various disorders. UC Davis Book of Horses New York: Harper Collins; 1996. pp. 164–167.
  5. Bertone AL. Management of exuberant granulation tissue. Vet Clin North Am Equine Pract 1989;5:551–562.
    pubmed: 2691030
  6. Theoret CL, Barber SM, Moyana TN, Gordon JR. Preliminary observations on expression of transforming growth factors beta1 and beta3 in equine full-thickness skin wounds healing normally or with exuberant granulation tissue. Vet Surg VS Off J Am Coll Vet Surg 2002;31:266–273.
    pubmed: 11994855
  7. Steel CM, Robertson ID, Thomas J, Yovich JV. Effect of topical rh-TGF-beta 1 on second intention wound healing in horses. Aust Vet J 1999;77:734–737.
    doi: 10.1111/j.1751-0813.1999.tb12916.xpubmed: 10685169google scholar: lookup
  8. Dart AJ, Cries L, Jeffcott LB, Hodgson DR, Rose RJ. The effect of equine recombinant growth hormone on second intention wound healing in horses. Vet Surg 2002;31:314–319.
    doi: 10.1053/jvet.2002.33589pubmed: 12094344google scholar: lookup
  9. Cochrane CA. Models in vivo of wound healing in the horse and the role of growth factors. Vet Dermatol 1997;8:259–272.
    doi: 10.1111/j.1365-3164.1997.tb00272.xpubmed: 34645018google scholar: lookup
  10. Cyranoski D. Stem cells boom in vet clinics. Nature 2013;496:148–149.
    doi: 10.1038/496148apubmed: 23579655google scholar: lookup
  11. Guest DJ, Smith MRW, Allen WR. Equine embryonic stem-like cells and mesenchymal stromal cells have different survival rates and migration patterns following their injection into damaged superficial digital flexor tendon. Equine Vet J 2010;42:636–642.
    doi: 10.1111/j.2042-3306.2010.00112.xpubmed: 20840579google scholar: lookup
  12. Becerra P, Valdés Vázquez MA, Dudhia J, Fiske-Jackson AR, Neves F, Hartman NG. Distribution of injected technetium99m-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy. J Orthop Res 2013;31:1096–1102.
    doi: 10.1002/jor.22338pubmed: 23508674google scholar: lookup
  13. Nagy K, Sung HK, Zhang P, Laflamme S, Vincent P, Agha-Mohammadi S. Induced pluripotent stem cell lines derived from equine fibroblasts. Stem Cell Rev 2011;7:693–702.
    doi: 10.1007/s12015-011-9239-5pmc: PMC3137777pubmed: 21347602google scholar: lookup
  14. Breton A, Sharma R, Diaz AC, Parham AG, Graham A, Neil C. Derivation and characterization of induced pluripotent stem cells from equine fibroblasts. Stem Cells Dev 2013;22:611–621.
    doi: 10.1089/scd.2012.0052pmc: PMC3564467pubmed: 22897112google scholar: lookup
  15. Whitworth DJ, D a O, Sun J, Fortuna PRJ, Wolvetang EJ. Generation and characterization of leukemia inhibitory factor-dependent equine induced pluripotent stem cells from adult dermal fibroblasts. Stem Cells Dev 2014;23:1515–1523.
    doi: 10.1089/scd.2013.0461pmc: PMC4066230pubmed: 24555755google scholar: lookup
  16. Aguiar C, Therrien J, Lemire P, Segura M, Smith LC, Theoret CL. Differentiation of equine induced pluripotent stem cells into a keratinocyte lineage. Equine Vet J 2016;48:338–345.
    doi: 10.1111/evj.12438pubmed: 25781637google scholar: lookup
  17. Dahm a M, de Bruin A, Linat A, von Tscharner C, Wyder M, Suter MM. Cultivation and characterisation of primary and subcultured equine keratinocytes. Equine Vet J 2002;34:114–120.
    doi: 10.2746/042516402776767187pubmed: 11902754google scholar: lookup
  18. Visser MB, Pollitt CC. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes. BMC Vet Res 2010;6:16.
    doi: 10.1186/1746-6148-6-16pmc: PMC2847556pubmed: 20230631google scholar: lookup
  19. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 2012;180:599–607.
    doi: 10.1016/j.ajpath.2011.10.036pmc: PMC3349876pubmed: 22189618google scholar: lookup
  20. Liu X, Krawczyk E, Suprynowicz FA, Palechor-Ceron N, Yuan H, Dakic A. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat Protoc 2017;12:439–451.
    doi: 10.1038/nprot.2016.174pmc: PMC6195120pubmed: 28125105google scholar: lookup
  21. Suprynowicz FA, Upadhyay G, Krawczyk E, Kramer SC, Hebert JD, Liu X. Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells. Proc Natl Acad Sci 2012;109:20035–20040.
    doi: 10.1073/pnas.1213241109pmc: PMC3523865pubmed: 23169653google scholar: lookup
  22. Chapman S, Liu X, Meyers C, Schlegel R, McBride AA. Human keratinocytes are efficiently immortalized by a rho kinase inhibitor. J Clin Invest 2010;120:2619–2626.
    doi: 10.1172/JCI42297pmc: PMC2898606pubmed: 20516646google scholar: lookup
  23. Kesmen Z, Sahin F, Yetim H. PCR assay for the identification of animal species in cooked sausages. Meat Sci 2007;77:649–653.
    doi: 10.1016/j.meatsci.2007.05.018pubmed: 22061954google scholar: lookup
  24. Hrdlickova R, Nehyba J, Bose HR. Alternatively spliced telomerase reverse transcriptase variants lacking telomerase activity stimulate cell proliferation. Mol Cell Biol 2012;32:4283–4296.
    doi: 10.1128/MCB.00550-12pmc: PMC3486134pubmed: 22907755google scholar: lookup
  25. Raudsepp T, Chowdhary BP. FISH for mapping single copy genes. Methods Mol Biol 2008;422:31–49.
    doi: 10.1007/978-1-59745-581-7_3pubmed: 18629659google scholar: lookup
  26. Arrighi FE, Hsu TC. Localization of heterochromatin in human chromosomes. Cytogenet Genome Res 1971;10:81–86.
    doi: 10.1159/000130130pubmed: 4106483google scholar: lookup
  27. Seabright M. A rapid banding technique for human chromosomes. Lancet 1971;298:971–972.
    doi: 10.1016/S0140-6736(71)90287-Xpubmed: 4107917google scholar: lookup
  28. Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331–344.
    doi: 10.1016/S0092-8674(75)80001-8pubmed: 1052771google scholar: lookup
  29. Shamir ER, Ewald AJ. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat Rev Mol Cell Biol 2014;15:647–664.
    doi: 10.1038/nrm3873pmc: PMC4352326pubmed: 25237826google scholar: lookup
  30. Knottenbelt DC. Equine wound management: are there significant differences in healing at different sites on the body?. Vet Dermatol 1997;8:273–290.
    doi: 10.1111/j.1365-3164.1997.tb00273.xpubmed: 34645017google scholar: lookup
  31. Sharma R, Barakzai SZ, Taylor SE, Donadeu FX. Epidermal-like architecture obtained from equine keratinocytes in three-dimensional cultures. J Tissue Eng Regen Med 2016;10:627–636.
    doi: 10.1002/term.1788pubmed: 23897780google scholar: lookup
  32. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 2007;25:681–686.
    doi: 10.1038/nbt1310pubmed: 17529971google scholar: lookup
  33. Terunuma A, Limgala RP, Park CJ, Choudhary I, Vogel JC. Efficient procurement of epithelial stem cells from human tissue specimens using a rho-associated protein kinase inhibitor Y-27632. Tissue Eng Part A 2010;16:1363–1368.
    doi: 10.1089/ten.tea.2009.0339pmc: PMC2862604pubmed: 19912046google scholar: lookup
  34. Dakic A, DiVito K, Fang S, Suprynowicz F, Gaur A, Li X. ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes. Oncotarget 2016;7:66740–66753.
    doi: 10.18632/oncotarget.11458pmc: PMC5341834pubmed: 27556514google scholar: lookup
  35. Staiger EA, Al Abri MA, Pflug KM, Kalla SE, Ainsworth DM, Miller D. Skeletal variation in Tennessee walking horses maps to the LCORL/NCAPG gene region. Physiol Genomics 2016;48:325–335.
    doi: 10.1152/physiolgenomics.00100.2015pubmed: 26931356google scholar: lookup
  36. Ghosh S, Qu Z, Das PJ, Fang E, Juras R, Cothran EG. Copy number variation in the horse genome. PLoS Genet 2014;10:e1004712.
    doi: 10.1371/journal.pgen.1004712pmc: PMC4207638pubmed: 25340504google scholar: lookup

Citations

This article has been cited 15 times.
  1. Krawczyk E. Conditionally Reprogrammed Cells as Preclinical Model for Rare Cancers. Cancers (Basel) 2025 Aug 29;17(17).
    doi: 10.3390/cancers17172834pubmed: 40940935google scholar: lookup
  2. Santiviparat S, Swangchan-Uthai T, Stout TAE, Buranapraditkun S, Setthawong P, Taephatthanasagon T, Rodprasert W, Sawangmake C, Tharasanit T. De novo reconstruction of a functional in vivo-like equine endometrium using collagen-based tissue engineering. Sci Rep 2024 Apr 19;14(1):9012.
    doi: 10.1038/s41598-024-59471-zpubmed: 38641671google scholar: lookup
  3. Baumbach CM, Anantama NA, Savkovic V, Mülling CKW, Schinköthe J, Michler JK. 3D Approaches to Culturing Bovine Skin: Explant Culture versus Organotypic Skin Model. Cells Tissues Organs 2024;213(5):424-438.
    doi: 10.1159/000538438pubmed: 38508156google scholar: lookup
  4. Dhamija B, Marathe S, Sawant V, Basu M, Attrish D, Mukherjee D, Kumar S, Pai MGJ, Wad S, Sawant A, Nayak C, Venkatesh KV, Srivastava S, Barthel SR, Purwar R. IL-17A Orchestrates Reactive Oxygen Species/HIF1α-Mediated Metabolic Reprogramming in Psoriasis. J Immunol 2024 Jan 15;212(2):302-316.
    doi: 10.4049/jimmunol.2300319pubmed: 38019129google scholar: lookup
  5. Alkhilaiwi F. Conditionally Reprogrammed Cells and Robotic High-Throughput Screening for Precision Cancer Therapy. Front Oncol 2021;11:761986.
    doi: 10.3389/fonc.2021.761986pubmed: 34737964google scholar: lookup
  6. Marx C, Gardner S, Harman RM, Wagner B, Van de Walle GR. Mesenchymal stromal cell-secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes. Stem Cells Transl Med 2021 Dec;10(12):1666-1679.
    doi: 10.1002/sctm.21-0058pubmed: 34528765google scholar: lookup
  7. Ramsauer AS, Wachoski-Dark GL, Fraefel C, Ackermann M, Brandt S, Grest P, Knight CG, Favrot C, Tobler K. Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection. Viruses 2021 Jul 19;13(7).
    doi: 10.3390/v13071404pubmed: 34372610google scholar: lookup
  8. Chen Z, He W, Leung TCN, Chung HY. Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632. Int J Mol Sci 2021 Jun 24;22(13).
    doi: 10.3390/ijms22136782pubmed: 34202585google scholar: lookup
  9. Souci L, Denesvre C. 3D skin models in domestic animals. Vet Res 2021 Feb 15;52(1):21.
    doi: 10.1186/s13567-020-00888-5pubmed: 33588939google scholar: lookup
  10. Wu X, Wang S, Li M, Li J, Shen J, Zhao Y, Pang J, Wen Q, Chen M, Wei B, Kaboli PJ, Du F, Zhao Q, Cho CH, Wang Y, Xiao Z, Wu X. Conditional reprogramming: next generation cell culture. Acta Pharm Sin B 2020 Aug;10(8):1360-1381.
    doi: 10.1016/j.apsb.2020.01.011pubmed: 32963937google scholar: lookup
  11. Xia S, Wu M, Chen S, Zhang T, Ye L, Liu J, Li H. Long Term Culture of Human Kidney Proximal Tubule Epithelial Cells Maintains Lineage Functions and Serves as an Ex vivo Model for Coronavirus Associated Kidney Injury. Virol Sin 2020 Jun;35(3):311-320.
    doi: 10.1007/s12250-020-00253-ypubmed: 32602046google scholar: lookup
  12. Liu X, Wu Y, Rong L. Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses. Virol Sin 2020 Jun;35(3):280-289.
    doi: 10.1007/s12250-020-00244-zpubmed: 32557270google scholar: lookup
  13. Liu X, Mondal AM. Conditional cell reprogramming for modeling host-virus interactions and human viral diseases. J Med Virol 2020 Nov;92(11):2440-2452.
    doi: 10.1002/jmv.26093pubmed: 32478897google scholar: lookup
  14. Palechor-Ceron N, Krawczyk E, Dakic A, Simic V, Yuan H, Blancato J, Wang W, Hubbard F, Zheng YL, Dan H, Strome S, Cullen K, Davidson B, Deeken JF, Choudhury S, Ahn PH, Agarwal S, Zhou X, Schlegel R, Furth PA, Pan CX, Liu X. Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks. Cells 2019 Oct 27;8(11).
    doi: 10.3390/cells8111327pubmed: 31717887google scholar: lookup
  15. Yang Q, Lopez MJ. Ultrastructural morphology is distinct among primary progenitor cell isolates from normal, inflamed, and cryopreserved equine hoof tissue and CD105(+)K14(+) progenitor cells. In Vitro Cell Dev Biol Anim 2019 Sep;55(8):641-655.
    doi: 10.1007/s11626-019-00380-1pubmed: 31297697google scholar: lookup
Subscribe to our newsletter
Join 99,579 horse owners like you who receive our email updates on horse health and nutrition.