Animal Reproduction (AR)
Animal Reproduction (AR)

Characterization of hematopoietic stem cells from the canine yolk sac

Bárbara Rossi de Sousa; Vanessa Cristina de Oliveira; Alessandra Oliveira Pinheiro; Carlos Eduardo Ambrósio

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The characterization of hematopoietic stem cells (HSC) from the canine yolk sac (cYS) can contribute to future gene therapies because it is possible to obtain information about the beginning of the development of the circulatory system through the characterization. The cYS is a likely source of HSC, which is a source of blood cell development in mammals. Studies in this field have been conducted for decades; however, interest in cellular therapy is currently at its peak with greater visibility, and these cells are a promising therapeutic tool for the treatment of diseases related to animals and humans. The aim of this study was to isolate and characterize HSC from the cYS embryos at 30 to 45 days of gestational age. Our results showed that the cYS was macroscopically located in the ventral region with a central portion and extremities. The cells in culture presented a circular morphology and cell clusters. The average cell viability was 22.55% dead cells out of 6.5 × 104 total cells. The cells were also able to form colonies on methylcellulose. Flow cytometry analysis revealed the expression of CD34, CD117, and CD45. Our results suggest that the cYS can be used as a source of hematopoietic cells, and this study is very important to understand the mechanism and development of the hematopoietic system in dogs.


yolk sac, canine, hematopoietic, stem cell and embryo


Alves PMM, Carrondo MJT, Cruz P. Introdução à tecnologia de cultivo de células animais. In: Morais AM, Augusto EFP, Castilho LR. Tecnologia do cultivo de células animais: de biofármacos à terapia gênica. São Paulo: Roca; 2007. p. 2-14.

Ambrósio CE, Wenceslau CV, Nogueira JL, Abreu DK, Rodrigues EAF, Lessa TB, Martin DS, Bertolini LR, Miglino MA. Fetal membranes stem cells applications in pets. Acta Sci Vet. 2011;39:97-101.

Auerbach R, Huang H, Lu L. Hematopoietic stem cells in the mouse embryonic yolk sac. Stem Cells. 1996;14(3):269-80. PMid:8724693.

Bian Z, Gong Y, Huang T, Lee CZW, Bian L, Bai Z, Shi H, Zeng Y, Liu C, He J, Zhou J, Li X, Li Z, Ni Y, Ma C, Cui L, Zhang R, Chan JKY, Ng LG, Lan Y, Ginhoux F, Liu B. Deciphering human macrophage development at single-cell resolution. Nature. 2020;582(7813):571-6. PMid:32499656.

Bjorkman N, Dantzer V, Leiser R. Comparative placentation in laboratory animals: a review. Scand J Lab Anim Sci. 1989;16:129-58.

Bruno B, Goerner MA, Nash RA, Storb R, Kiem HP, McSweeney PA. Purified canine CD34+Lin- marrow cells transduced with retroviral vectors give rise to long-term multi-lineage hematopoiesis. Biol Blood Marrow Transplant. 2001;7(10):543-51. PMid:11760086.

Bruno B, Nash RA, Wallace PM, Gass MJ, Thompson J, Storb R, McSweeney PA. CD34+ selected bone marrow grafts are radioprotective and establish mixed chimerism in dogs given high dose total body irradiation. Transplantation. 1999;68(3):338-44. PMid:10459536.

Cardoso MT, Pinheiro AO, Vidane AS, Casals JB, de Oliveira VC, Gonçalves N, Martins DS, Ambrósio CE. Characterization of teratogenic potential and gene expression in canine and feline amniotic membrane-derived stem cells. Reprod Domest Anim. 2017;52(Suppl 2):58-64. PMid:27774699.

Cumano A, Ferraz JC, Klaine M, Di Santo JP, Godin I. Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution. Immunity. 2001;15(3):477-85. PMid:11567637.

De Vita B, Loreta LC, Listoni AJ, Maia L, Sudano LJ, Curcio BR, Ladim-Alvarenga FC, Prestes N. Isolamento, caracterização e diferenciação de células mesenquimais do liquido amniótico equino obtido em diferentes idades gestacionais. Pesq Vet Bras. 2013;33(4):535-42.

Ferkowicz MJ, Yoder M. Blood island formation: longstanding observations and modern interpretations. Exp Hematol. 2005;33(9):1041-7. PMid:16140152.

Herzog EL, Chai L, Krause DS. Plasticity of marrow-derived stem cells. Blood. 2003;102(10):3483-93. PMid:12893756.

Hyttel P, Sinowatz F, Vejlsted M, Betteridge K. Essentials of domestic animal embryology. London: Elsevier Health Sciences; 2009.

Jaffredo T, Bollerot K, Sugiyama D, Gautier R, Drevon C. Tracing the hemangioblast during embryogenesis: developmental relationships between endothelial and hematopoietic cells. Int J Dev Biol. 2005;49(2-3):269-77. PMid:15906241.

Jindal N, Minhas G, Prabhakar S, Anand A. Characterization of Lin-ve CD34 and CD117 cell population reveals an increased expression in bone marrow derived stem cells. Curr Neurovasc Res. 2014;11(1):68-74. PMid:24321025.

Jones CJ, Jauniaux E. Ultrastructure of the materno-embryonic interface in the first trimester of pregnancy. Micron. 1995;26(2):145-73. PMid:7767634.

Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA. 2001;98(19):10716-21. PMid:11535826.

Kirkness EF, Bafna V, Halpern AL, Levy S, Remington K, Rusch DB, Delcher AL, Pop M, Wang W, Fraser CM, Venter JC. The dog genome: survey sequencing and comparative analysis. Science. 2003;301(5641):1898-903.

Lee SY, Anderson JW, Scott GL, Mossman HW. Ultrastructure of the placenta and fetal membranes of the dog: II. The yolk sac. Am J Anat. 1983;166(3):313-27. PMid:6846208.

Li W, Johnson SA, Shelley WC, Ferkowicz M, Morrison P, Li Y, Yoder MC. Primary endothelial cells isolated from the yolk sac and para-aortic splanchnopleura support the expansion of adult marrow stem cells in vitro. Blood. 2003;102(13):4345-53. PMid:12893772.

Lim R. Concise review: fetal membranes in regenerative medicine: new tricks from an old dog. Stem Cells Transl Med. 2017;6(9):1767-76. PMid:28834402.

Medvinsky A, Taoudi S, Mendes S, Dzierzak E. Analysis and manipulation of hematopoietic progenitor and stem cells from murine embryonic tissues. Curr. Protoc. Stem Cell Biol. 2008;Chapter 2:Unit 2A.6.

Miettinen M, Lasota J. KIT (CD117): a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol AIMM. 2005;13(3):205-20. PMid:16082245.

Mikkola HKA, Orkin SH. The journey of developing hematopoietic stem cells. Develop. 2006;133(19):3733-44.

Moore KL, Persaud T. Embriologia básica. 6. ed. São Paulo: Elsevier; 2004.

Nakage APM, Santana AE. Células-tronco hematopoéticas em cães. Cienc Rural. 2006;36(1):325-9.

Noden DM, De Lahunta A, Ferrol N. Embriologia de los animales domésticos: mecanismos de desarrollo y malformaciones. Espana: Zaragoza; 1990. 399 p.

Oliveira VC, Mançanares CAF, Oliveira LJ, Gonçalves NJN, Miglino MA, Perecin F, Meirelles FV, Piedrahita J, Ambrósio CE. Characterization of putative haematopoietic cells from bovine yolk sac. J Tissue Eng Regen Med. 2017;11(4):1132-40. PMid:25712733.

Palis J, Yoder MC. Yolk-sac hematopoiesis: the first blood cells of mouse and man. Exp Hematol. 2001;29(8):927-36. PMid:11495698.

Pessolato AGT, Martins DS, Galdos AR, Fontes AM, Ambrósio CE, Rici REG, Magalhães DAR, Castilho-Fernandes A, Covas DT, Miglino MA. Microscopic aspects of the yolk sac hematopoiesis from ovine embryos. In: Méndez-Vilas A, editor. Current microscopy contributions to advances in science and technology. Badajoz: Formatex Research Center; 2012. p. 610-6.

Riveros A. Analise Prôteomica do saco vitelino de bovinos [dissertação]. São Paulo: Universidade de São Paulo; 2009.

Robb L, Lyons I, Li R, Hartley L, Köntgen F, Harvey RP, Metcalf D, Begley CG. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci USA. 1995;92(15):7075-9. PMid:7624372.

Rüsse I, Sinowatz F, Richter L, Lehmann M, Schallenberger E. Die Entwicklung des Dottersackes beim Wiederkäuer (Schaf und Rind). ANHE. 1992;21(4):324-47.

Santos HSL, Azoubel R. Embriologia comparada. Jaboticabal: FUNEP; 1996.

Sasaki T, Sasaki-Irie J, Penninger JM. New insights into the transmembrane protein tyrosine phosphatase CD45. Int. J. Biochem.Cell Biol. 2001;33:1041-46.

Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature. 1995;373(6513):432-4. PMid:7830794.

Sutherland DR, Stewart AK, Keating A. CD34 antigen: molecular features and potential clinical applications. Stem Cells. 1993;11(Suppl 3):50-7. PMid:7507757.

Tárnok A, Ulrich H, Bocsi J. Phenotypes of stem cells from diverse origin. Cytometry A. 2010;77A:6-10.

Vidane AS, Pinheiro A, Casals J, Passarelli D, Hage M, Bueno R, Martins DS, Ambrósio CE. Transplantation of amniotic membrane-derived multipotent cells ameliorates and delays the progression of chronic kidney disease in cats. Reprod Domest Anim. 2017;52(Suppl 2):316-26. PMid:27774657.

Wang F, Du L, Ge S. PTH/SDF-1α cotherapy induces CD90+CD34− stromal cells migration and promotes tissue regeneration in a rat periodontal defect model. Sci Rep. 2016;6(1):30403. PMid:27480134.

Wenceslau CV, Miglino MA, Martins DS, Ambrósio CE, Lizier NF, Pignatari GC, Kerkis I. Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow. Tissue Eng Part A. 2011;17(17-18):2165-76. PMid:21529262.

Wilpshaar J, Bhatia M, Kanhai HHH, Breese R, Heilman DK, Johnson CS, Falkenburg JHF, Srour EF. Engraftment potential of human fetal hematopoietic cells in NOD/SCID mice is not restricted to mitotically quiescent cells. Blood. 2002;100(1):120-7. PMid:12070016.

Wolf E, Arnold GJ, Bauersachs S, Beier HM, Blum H, Einspanier R, Fröhlich T, Herrler A, Hiendleder S, Kölle S, Prelle K, Reichenbach HD, Stojkovic M, Wenigerkind H, Sinowatz F. Embryo-maternal communication in bovine - strategies for deciphering a complex cross-talk. Reprod Domest Anim. 2003;38(4):276-89. PMid:12887567.

Yokomizo T, Ogawa M, Osato M, Kanno T, Yoshida H, Fujimoto T, Fraser S, Nishikawa S, Okada H, Satake M, Noda T, Nishikawa S, Ito Y. Requirement of Runx1/AML1/PEBP2αB for the generation of haematopoietic cells from endothelial cells. Genes Cells. 2001;6(1):13-23. PMid:11168593.

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