Blood type and breed-associated differences in cell marker expression on equine bone marrow-derived mesenchymal stem cells including major histocompatibility complex class II antigen expression

Autoři: J. Lacy Kamm aff001;  Natalie A. Parlane aff003;  Christopher B. Riley aff001;  Erica K. Gee aff001;  Keren E. Dittmer aff001;  C. Wayne McIlwraith aff001
Působiště autorů: Massey University, School of Veterinary Science, Massey University, Palmerston North, New Zealand aff001;  Veterinary Associates, Karaka, Auckland, New Zealand aff002;  AgResearch, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand aff003;  Colorado State University, Orthopaedic Research Center, Fort Collins, Colorado, United States of America aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225161



As the search for an immune privileged allogeneic donor mesenchymal stem cell (MSC) line continues in equine medicine, the characterization of the cells between different sources becomes important. Our research seeks to more clearly define the MSC marker expression of different equine MSC donors.


The bone marrow-derived MSCs from two equine breeds and different blood donor-types were compared over successive culture passages to determine the differential expression of important antigens. Eighteen Thoroughbreds and 18 Standardbreds, including 8 blood donor (erythrocyte Aa, Ca, and Qa antigen negative) horses, were evaluated. Bone marrow was taken from each horse for isolation and culture of MSCs. Samples from passages 2, 4, 6, and 8 were labelled and evaluated by flow cytometry. The cell surface expression of CD11a/18, CD44, CD90 and MHC class II antigens were assessed. Trilineage assays for differentiation into adipogenic, chondrogenic and osteogenic lines were performed to verify characterization of the cells as MSCs.


There were significant differences in mesenchymal stem cell marker expression between breeds and blood antigen-type groups over time. Standardbred horses showed a significantly lower expression of MHC class II than did Thoroughbred horses at passages 2, 4 and 6. CD90 was significantly higher in universal blood donor Standardbreds as compared to non-blood donor Standardbreds over all time points. All MSC samples showed high expression of CD44 and low expression of CD11a/18.


Universal blood donor- type Standardbred MSCs from passages 2–4 show the most ideal antigen expression pattern of the horses and passages that we characterized for use as a single treatment of donor bone marrow-derived MSCs. Further work is needed to determine the significance of this differential expression along with the effect of the expression of MHC I on equine bone marrow-derived MSCs.

Klíčová slova:

Animal husbandry – Blood donors – Cell differentiation – Equines – Horses – Major histocompatibility complex – Mesenchymal stem cells – Red blood cells


1. Richardson SM, Kalamegam G, Pushparaj PN, Matta C, Memic A, Khademhosseini A, et al. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration. Methods (San Diego, Calif). 2016;99:69–80.

2. Joswig AJ, Mitchell A, Cummings KJ, Levine GJ, Gregory CA, Smith R 3rd, et al. Repeated intra-articular injection of allogeneic mesenchymal stem cells causes an adverse response compared to autologous cells in the equine model. Stem cell research & therapy. 2017;8(1):42.

3. McIlwraith CW, Frisbie DD, Rodkey WG, Kisiday JD, Werpy NM, Kawcak CE, et al. Evaluation of intra-articular mesenchymal stem cells to augment healing of microfractured chondral defects. Arthroscopy: the journal of arthroscopic & related surgery: official publication of the Arthroscopy Association of North America and the International Arthroscopy Association. 2011;27(11):1552–61.

4. Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RK. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon. Equine veterinary journal. 2012;44(1):25–32. doi: 10.1111/j.2042-3306.2011.00363.x 21615465

5. Ferris DJ, Frisbie DD, Kisiday JD, McIlwraith CW, Hague BA, Major MD, et al. Clinical outcome after intra-articular administration of bone marrow derived mesenchymal stem cells in 33 horses with stifle injury. Veterinary surgery: VS. 2014;43(3):255–65. doi: 10.1111/j.1532-950X.2014.12100.x 24433318

6. Garvican ER, Cree S, Bull L, Smith RK, Dudhia J. Viability of equine mesenchymal stem cells during transport and implantation. Stem cell research & therapy. 2014;5(4):94.

7. Colbath AC, Dow SW, Phillips JN, McIlwraith CW, Goodrich LR. Autologous and allogeneic equine mesenchymal stem cells exhibit equivalent immunomodulatory properties in vitro. Stem cells and development. 2017;26(7):503–11. doi: 10.1089/scd.2016.0266 27958776

8. Pezzanite LM, Fortier LA, Antczak DF, Cassano JM, Brosnahan MM, Miller D, et al. Equine allogeneic bone marrow-derived mesenchymal stromal cells elicit antibody responses in vivo. Stem cell research & therapy. 2015;6:54.

9. Schnabel LV, Pezzanite LM, Antczak DF, Felippe MJ, Fortier LA. Equine bone marrow-derived mesenchymal stromal cells are heterogeneous in MHC class II expression and capable of inciting an immune response in vitro. Stem cell research & therapy. 2014;5(1):13.

10. Portalska KJ, Groen N, Krenning G, Georgi N, Mentink A, Harmsen MC, et al. The effect of donor variation and senescence on endothelial differentiation of human mesenchymal stromal cells. Tissue engineering Part A. 2013;19(21–22):2318–29. doi: 10.1089/ten.TEA.2012.0646 23676150

11. Griffin MD, Ryan AE, Alagesan S, Lohan P, Treacy O, Ritter T. Anti-donor immune responses elicited by allogeneic mesenchymal stem cells: what have we learned so far? Immunology and cell biology. 2013;91(1):40–51. doi: 10.1038/icb.2012.67 23207278

12. Berglund AK, Fortier LA, Antczak DF, Schnabel LV. Immunoprivileged no more: measuring the immunogenicity of allogeneic adult mesenchymal stem cells. Stem cell research & therapy. 2017;8(1):288.

13. Consentius C, Reinke P, Volk HD. Immunogenicity of allogeneic mesenchymal stromal cells: what has been seen in vitro and in vivo? Regenerative medicine. 2015;10(3):305–15. doi: 10.2217/rme.15.14 25933239

14. Zangi L, Margalit R, Reich-Zeliger S, Bachar-Lustig E, Beilhack A, Negrin R, et al. Direct imaging of immune rejection and memory induction by allogeneic mesenchymal stromal cells. Stem cells (Dayton, Ohio). 2009;27(11):2865–74.

15. Benichou G, Yamada Y, Yun SH, Lin C, Fray M, Tocco G. Immune recognition and rejection of allogeneic skin grafts. Immunotherapy. 2011;3(6):757–70. doi: 10.2217/imt.11.2 21668313

16. Barrachina L, Remacha AR, Romero A, Vazquez FJ, Albareda J, Prades M, et al. Effect of inflammatory environment on equine bone marrow derived mesenchymal stem cells immunogenicity and immunomodulatory properties. Veterinary immunology and immunopathology. 2016;171:57–65. doi: 10.1016/j.vetimm.2016.02.007 26964718

17. Alonso Arias R, Lopez-Vazquez A, Lopez-Larrea C. Immunology and the challenge of transplantation. Advances in experimental medicine and biology. 2012;741:27–43. doi: 10.1007/978-1-4614-2098-9_3 22457101

18. Huang XP, Ludke A, Dhingra S, Guo J, Sun Z, Zhang L, et al. Class II transactivator knockdown limits major histocompatibility complex II expression, diminishes immune rejection, and improves survival of allogeneic bone marrow stem cells in the infarcted heart. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2016;30(9):3069–82.

19. Mei L, Shen B, Ling P, Liu S, Xue J, Liu F, et al. Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model. PLoS One. 2017;12(4):e0176107. doi: 10.1371/journal.pone.0176107 28419155

20. Paebst F, Piehler D, Brehm W, Heller S, Schroeck C, Tarnok A, et al. Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: an approach toward a standardized definition. Cytometry Part A. 2014;85(8):678–87.

21. De Schauwer C, Piepers S, Van de Walle GR, Demeyere K, Hoogewijs MK, Govaere JL, et al. In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry. Cytometry Part A. 2012;81(4):312–23.

22. Zahedi M, Parham A, Dehghani H, Mehrjerdi HK. Stemness Signature of Equine Marrow-derived Mesenchymal Stem Cells. International journal of stem cells. 2017;10(1):93–102. doi: 10.15283/ijsc16036 28222255

23. Radcliffe CH, Flaminio MJ, Fortier LA. Temporal analysis of equine bone marrow aspirate during establishment of putative mesenchymal progenitor cell populations. Stem cells and development. 2010;19(2):269–82. doi: 10.1089/scd.2009.0091 19604071

24. Song X, Hong C, Zheng Q, Zhao H, Song K, Liu Z, et al. Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro. In vitro cellular & developmental biology Animal. 2017;53(1):77–82.

25. Zhao YF, Xiong W, Wu XL. Mesenchymal stem cell-based developmental endothelial locus-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice. Molecular medicine reports. 2014;9(5):1583–9. doi: 10.3892/mmr.2014.1988 24573341

26. Dvorakova J, Hruba A, Velebny V, Kubala L. Isolation and characterization of mesenchymal stem cell population entrapped in bone marrow collection sets. Cell biology international. 2008;32(9):1116–25. doi: 10.1016/j.cellbi.2008.04.024 18562221

27. Becht JL, Semrad SD. Hematology, blood typing, and immunology of the neonatal foal. The Veterinary clinics of North America Equine practice. 1985;1(1):91–116. doi: 10.1016/s0749-0739(17)30771-x 3907769

28. Angelos J, Oppenheim Y, Rebhun W, Mohammed H, Antczak DF. Evaluation of breed as a risk factor for sarcoid and uveitis in horses. Animal genetics. 1988;19(4):417–25. doi: 10.1111/j.1365-2052.1988.tb00833.x 3232865

29. Tomlinson JE, Taberner E, Boston RC, Owens SD, Nolen-Walston RD. Survival Time of Cross-Match Incompatible Red Blood Cells in Adult Horses. Journal of veterinary internal medicine. 2015;29(6):1683–8. doi: 10.1111/jvim.13627 26478135

30. Ogawa M, Larue AC, Watson PM, Watson DK. Hematopoietic stem cell origin of mesenchymal cells: opportunity for novel therapeutic approaches. International journal of hematology. 2010;91(3):353–9. doi: 10.1007/s12185-010-0554-4 20336396

31. Waselau M, Sutter WW, Genovese RL, Bertone AL. Intralesional injection of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses. Journal of the American Veterinary Medical Association. 2008;232(10):1515–20. doi: 10.2460/javma.232.10.1515 18479242

32. Lacitignola L, Crovace A, Rossi G, Francioso E. Cell therapy for tendinitis, experimental and clinical report. Veterinary research communications. 2008;32 Suppl 1:S33–8.

33. Kisiday JD, Kopesky PW, Evans CH, Grodzinsky AJ, McIlwraith CW, Frisbie DD. Evaluation of adult equine bone marrow- and adipose-derived progenitor cell chondrogenesis in hydrogel cultures. Journal of orthopaedic research: official publication of the Orthopaedic Research Society. 2008;26(3):322–31.

34. Gibco. StemPro Differentiation Kit: Product use manual. In: Scientific TF, editor. Waltham, MA, USA2014.

35. Snyder L. Blood Typing. In: Wilson D, editor. Clinical Veterinary Advisor: The Horse. Pennsylvania, USA Elsevier Saunders; 2012.

36. Hardy J. Venous and arterial catheterization and fluid therapy. In: JAE MWaH, editor. Equine Anesthesia: Monitoring and Emergency Therapy 2nd ed. Missouri, USA Mosby Elsevier; 2009.

37. Whittington NC, Wray S. Suppression of red blood cell autofluorescence for immunohistochemistry on fixed embryonic mouse tissue. Curr Protoc Neurosci:81:2.28.2–12.

38. Maecker HT, Frey T, Nomura LE, Trotter J. Selecting fluorochrome conjugates for maximum sensitivity. Cytometry Part A: the journal of the International Society for Analytical Cytology. 2004;62(2):169–73.

39. Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B. Aging of mesenchymal stem cell in vitro. BMC cell biology. 2006;7:14. doi: 10.1186/1471-2121-7-14 16529651

40. Cribari-Neto F ZA. Beta Regression in R. Journal of Statistical Software 2010;34(2):1–24.

41. He X, Houde ALS, Pitcher TE, Heath DD. Genetic architecture of gene transcription in two Atlantic salmon (Salmo salar) populations. Heredity. 2017;119(2):117–24. doi: 10.1038/hdy.2017.24 28467401

42. Talbot B, Chen TW, Zimmerman S, Joost S, Eckert AJ, Crow TM, et al. Combining Genotype, Phenotype, and Environment to Infer Potential Candidate Genes. The Journal of heredity. 2017;108(2):207–16. doi: 10.1093/jhered/esw077 28003371

43. Kundrotas G, Gasperskaja E, Slapsyte G, Gudleviciene Z, Krasko J, Stumbryte A, et al. Identity, proliferation capacity, genomic stability and novel senescence markers of mesenchymal stem cells isolated from low volume of human bone marrow. Oncotarget. 2016;7(10):10788–802. doi: 10.18632/oncotarget.7456 26910916

44. Esteves CL, Sheldrake TA, Dawson L, Menghini T, Rink BE, Amilon K, et al. Equine Mesenchymal Stromal Cells Retain a Pericyte-Like Phenotype. Stem cells and development. 2017;26(13):964–72. doi: 10.1089/scd.2017.0017 28376684

45. Ranera B, Lyahyai J, Romero A, Vazquez FJ, Remacha AR, Bernal ML, et al. Immunophenotype and gene expression profiles of cell surface markers of mesenchymal stem cells derived from equine bone marrow and adipose tissue. Veterinary immunology and immunopathology. 2011;144(1–2):147–54. doi: 10.1016/j.vetimm.2011.06.033 21782255

46. Campioni D, Lanza F, Moretti S, Ferrari L, Cuneo A. Loss of Thy-1 (CD90) antigen expression on mesenchymal stromal cells from hematologic malignancies is induced by in vitro angiogenic stimuli and is associated with peculiar functional and phenotypic characteristics. Cytotherapy. 2008;10(1):69–82. doi: 10.1080/14653240701762364 18202976

47. Moraes DA, Sibov TT, Pavon LF, Alvim PQ, Bonadio RS, Da Silva JR, et al. A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells. Stem cell research & therapy. 2016;7(1):97.

48. Favi PM, Benson RS, Neilsen NR, Hammonds RL, Bates CC, Stephens CP, et al. Cell proliferation, viability, and in vitro differentiation of equine mesenchymal stem cells seeded on bacterial cellulose hydrogel scaffolds. Materials science & engineering C, Materials for biological applications. 2013;33(4):1935–44.

49. van Kooyk Y, Weder P, Heije K, de Waal Malefijt R, Figdor CG. Role of intracellular Ca2+ levels in the regulation of CD11a/CD18 mediated cell adhesion. Cell adhesion and communication. 1993;1(1):21–32. doi: 10.3109/15419069309095679 7915956

50. Kisselbach L, Merges M, Bossie A, Boyd A. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures. Cytotechnology. 2009;59(1):31–44. doi: 10.1007/s10616-009-9190-3 19296231

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