Animal Reproduction (AR)
https://animal-reproduction.org/article/doi/10.1590/1984-3143-AR2022-0063
Animal Reproduction (AR)
ORIGINAL ARTICLE

Secretion pattern of canine amniotic stem cells derived extracellular vesicles

Rafael Garcia Karam; Lina Castelo Branco Motta; Matheus Ferreira de Almeida; Alessandra Bridi; Juliano Coelho da Silveira; Carlos Eduardo Ambrósio

Downloads: 1
Views: 675

Abstract

Extracellular vesicles (EVs) derived from stem cells (SCs) have regenerative potential and the possibility of being used in treating chronic diseases. EVs present lower risk of tumorigenicity and easily to isolation and storage. Therefore, this research aims to compare the morphological characteristics of the EVs (up to 150nm) derived from stem cells obtained from canine amniotic membranes in different passages during the in vitro culture. For this, cells from the amniotic membranes were isolated, cultured, and characterized. In order to answer our aim, the number of cells was normalized at each passage to generate conditioned media for EVs separation. The cells were differentiated into adipogenic, chondrogenic, and osteogenic tissue, to characterize these cells as mesenchymal stem cells (MSC). Moreover, flow cytometry analysis was performed and showed that the MSC were positive for CD90, CD105 and negative for CD34, CD45, mesenchymal and hematopoietic markers, respectively. For EVs analysis, MSC in different passages (P0-P2) were culture until 80% of confluence, then the medium was replaced by EVs depleted medium. After 48h, culture medium was collected and centrifuged to separate EVs, followed by nanoparticle tracking analysis. The EVs were also characterized by western blot and transmission electron microscopy (TEM). EVs were positive for Alix and negative for Cytochrome C as well as presented the traditional cup-shape by transmission electronic microscopy. Our results demonstrated that the concentration in the different passages was increased in P0 compared to P1 and P2 (p<0.05). No differences were found in EVs size (P0=132nm, P1=130nm and P2=120nm). Together, these results demonstrate that P0 of MSC is enriched of EVs when compared to later passages, suggesting that this passage would be the best to be applied in pre-clinical tests. Despite that, more studies are necessary to identify the EVs content and how the cells will respond to treatment with them.

Keywords

extracellular vesicles, canine, mesenchymal

References

Bonafede R, Scambi I, Peroni D, Potrich V, Boschi F, Benati D, Bonetti B, Mariotti R. Exosome derived from murine adipose-derived stromal cells: neuroprotective effect on in vitro model of amyotrophic lateral sclerosis. Exp Cell Res. 2016;340(1):150-8. http://dx.doi.org/10.1016/j.yexcr.2015.12.009. PMid:26708289.

Borghesi J, Mario LC, Carreira AC, Miglino MA, Favaron PO. Phenotype and multipotency of rabbit (Oryctolagus cuniculus) amniotic stem cells. Stem Cell Res Ther. 2017;8(1):1-14. http://dx.doi.org/10.1186/s13287-016-0468-z. PMid:28173846.

Cardoso M, 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. http://dx.doi.org/10.1111/rda.12832. PMid:27774699.

Connolly KD, Guschina IA, Yeung V, Clayton A, Draman MS, Von Ruhland C, Ludgate M, James PE, Rees DA. Characterisation of adipocyte-derived extracellular vesicles released pre-and post-adipogenesis. J Extracell Vesicles. 2015;4(1):29159. http://dx.doi.org/10.3402/jev.v4.29159. PMid:26609807.

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-7. http://dx.doi.org/10.1080/14653240600855905. PMid:16923606.

Evans HE, Sack WO. Prenatal development of domestic and laboratory mammals: growth curves, external features and selected references. Zentralbl Veterinärmed C. 1973;2(1):11-45. http://dx.doi.org/10.1111/j.1439-0264.1973.tb00253.x. PMid:4745140.

Ercolin ACM, Roballo KC, Casals JB, Pieri NC, Souza AF, Barreto RS, Bressan FF, Feitosa ML, Miglino MA, Meirelles FV, Ambrósio CE. Rabbit olfactory stem cells. Isolation protocol and characterization. Acta Cir Bras. 2016;31(1):59-66. http://dx.doi.org/10.1590/S0102-865020160010000009. PMid:26840357.

Filioli Uranio M, Valentini L, Lange-Consiglio A, Caira M, Guaricci AC, L’Abbate A, Catacchio CR, Ventura M, Cremonesi F, Dell’Aquila ME. Isolation, proliferation, cytogenetic, and molecular characterization and in vitro differentiation potency of canine stem cells from foetal adnexa: a comparative study of amniotic fluid, amnion, and umbilical cord matrix. Mol Reprod Dev. 2011;78(5):361-73. http://dx.doi.org/10.1002/mrd.21311. PMid:21491540.

Huang C-C, Narayanan R, Alapati S, Ravindran S. Exosomes as biomimetic tools for stem cell differentiation: applications in dental pulp tissue regeneration. Biomaterials. 2016;111:103-15. http://dx.doi.org/10.1016/j.biomaterials.2016.09.029. PMid:27728810.

Hung ME, Leonard JN. A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery. J Extracell Vesicles. 2016;5(1):31027. http://dx.doi.org/10.3402/jev.v5.31027. PMid:27189348.

Lange-Consiglio UM, Corradetti B, Bizzaro D, Magatti M, Ressel L, Tassan S, Parolini O, Cremonesi F. Characterization and potential applications of progenitor-like cells isolated from horse amniotic membrane. J Tissue Eng Regen Med. 2012;6(8):622-35. http://dx.doi.org/10.1002/term.465. PMid:21948689.

Nardi NB, Meirelles LS. Mesenchymal stem cells: Isolation, in vitro expansion and characterization. Handb Exp Pharmacol. 2006;174(174):249-82. http://dx.doi.org/10.1007/3-540-31265-X_11. PMid:16370331.

Park S-B, Seo MS, Kim HS, Kang KS. Isolation and characterization of canine amniotic membrane derived multipotent stem cells. PLoS One. 2012;7(9):e44693. http://dx.doi.org/10.1371/journal.pone.0044693. PMid:23024756.

Pieri N, Souza AF, Casals JB, Roballo K, Ambrósio CE, Martins DS. Comparative development of embryonic age by organogenesis in domestic dogs and cats. Reprod Domest Anim. 2015;50(4):625-31. http://dx.doi.org/10.1111/rda.12539. PMid:25990819.

Russell KA, Chow NH, Dukoff D, Gibson TW, LaMarre J, Betts DH, Koch TG. Characterization and immunomodulatory effects of canine adipose tissue and bone marrow-derived mesenchymal stromal cells. PLoS One. 2016;11(12):e0167442. http://dx.doi.org/10.1371/journal.pone.0167442. PMid:27907211.

Saulnier N, Loriau J, Febre M, Robert C, Rakic R, Bonte T, Buff S, Maddens S. Canine placenta: a promising potential source of highly proliferative and immunomodulatory mesenchymal stromal cells? Vet Immunol Immunopathol. 2016;171:47-55. http://dx.doi.org/10.1016/j.vetimm.2016.02.005. PMid:26964717.

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, Antoniou A, Arab T, Archer F, Atkin-Smith GK, Ayre DC, Bach JM, Bachurski D, Baharvand H, Balaj L, Baldacchino S, Bauer NN, Baxter AA, Bebawy M, Beckham C, Bedina Zavec A, Benmoussa A, Berardi AC, Bergese P, Bielska E, Blenkiron C, Bobis-Wozowicz S, Boilard E, Boireau W, Bongiovanni A, Borràs FE, Bosch S, Boulanger CM, Breakefield X, Breglio AM, Brennan MÁ, Brigstock DR, Brisson A, Broekman ML, Bromberg JF, Bryl-Górecka P, Buch S, Buck AH, Burger D, Busatto S, Buschmann D, Bussolati B, Buzás EI, Byrd JB, Camussi G, Carter DR, Caruso S, Chamley LW, Chang YT, Chen C, Chen S, Cheng L, Chin AR, Clayton A, Clerici SP, Cocks A, Cocucci E, Coffey RJ, Cordeiro-da-Silva A, Couch Y, Coumans FA, Coyle B, Crescitelli R, Criado MF, D’Souza-Schorey C, Das S, Datta Chaudhuri A, de Candia P, De Santana EF, De Wever O, Del Portillo HA, Demaret T, Deville S, Devitt A, Dhondt B, Di Vizio D, Dieterich LC, Dolo V, Dominguez Rubio AP, Dominici M, Dourado MR, Driedonks TA, Duarte FV, Duncan HM, Eichenberger RM, Ekström K, El Andaloussi S, Elie-Caille C, Erdbrügger U, Falcón-Pérez JM, Fatima F, Fish JE, Flores-Bellver M, Försönits A, Frelet-Barrand A, Fricke F, Fuhrmann G, Gabrielsson S, Gámez-Valero A, Gardiner C, Gärtner K, Gaudin R, Gho YS, Giebel B, Gilbert C, Gimona M, Giusti I, Goberdhan DC, Görgens A, Gorski SM, Greening DW, Gross JC, Gualerzi A, Gupta GN, Gustafson D, Handberg A, Haraszti RA, Harrison P, Hegyesi H, Hendrix A, Hill AF, Hochberg FH, Hoffmann KF, Holder B, Holthofer H, Hosseinkhani B, Hu G, Huang Y, Huber V, Hunt S, Ibrahim AG, Ikezu T, Inal JM, Isin M, Ivanova A, Jackson HK, Jacobsen S, Jay SM, Jayachandran M, Jenster G, Jiang L, Johnson SM, Jones JC, Jong A, Jovanovic-Talisman T, Jung S, Kalluri R, Kano SI, Kaur S, Kawamura Y, Keller ET, Khamari D, Khomyakova E, Khvorova A, Kierulf P, Kim KP, Kislinger T, Klingeborn M, Klinke DJ 2nd, Kornek M, Kosanović MM, Kovács ÁF, Krämer-Albers EM, Krasemann S, Krause M, Kurochkin IV, Kusuma GD, Kuypers S, Laitinen S, Langevin SM, Languino LR, Lannigan J, Lässer C, Laurent LC, Lavieu G, Lázaro-Ibáñez E, Le Lay S, Lee MS, Lee YXF, Lemos DS, Lenassi M, Leszczynska A, Li IT, Liao K, Libregts SF, Ligeti E, Lim R, Lim SK, Linē A, Linnemannstöns K, Llorente A, Lombard CA, Lorenowicz MJ, Lörincz ÁM, Lötvall J, Lovett J, Lowry MC, Loyer X, Lu Q, Lukomska B, Lunavat TR, Maas SL, Malhi H, Marcilla A, Mariani J, Mariscal J, Martens-Uzunova ES, Martin-Jaular L, Martinez MC, Martins VR, Mathieu M, Mathivanan S, Maugeri M, McGinnis LK, McVey MJ, Meckes DG Jr, Meehan KL, Mertens I, Minciacchi VR, Möller A, Møller Jørgensen M, Morales-Kastresana A, Morhayim J, Mullier F, Muraca M, Musante L, Mussack V, Muth DC, Myburgh KH, Najrana T, Nawaz M, Nazarenko I, Nejsum P, Neri C, Neri T, Nieuwland R, Nimrichter L, Nolan JP, Nolte-’t Hoen EN, Noren Hooten N, O’Driscoll L, O’Grady T, O’Loghlen A, Ochiya T, Olivier M, Ortiz A, Ortiz LA, Osteikoetxea X, Østergaard O, Ostrowski M, Park J, Pegtel DM, Peinado H, Perut F, Pfaffl MW, Phinney DG, Pieters BC, Pink RC, Pisetsky DS, Pogge von Strandmann E, Polakovicova I, Poon IK, Powell BH, Prada I, Pulliam L, Quesenberry P, Radeghieri A, Raffai RL, Raimondo S, Rak J, Ramirez MI, Raposo G, Rayyan MS, Regev-Rudzki N, Ricklefs FL, Robbins PD, Roberts DD, Rodrigues SC, Rohde E, Rome S, Rouschop KM, Rughetti A, Russell AE, Saá P, Sahoo S, Salas-Huenuleo E, Sánchez C, Saugstad JA, Saul MJ, Schiffelers RM, Schneider R, Schøyen TH, Scott A, Shahaj E, Sharma S, Shatnyeva O, Shekari F, Shelke GV, Shetty AK, Shiba K, Siljander PR, Silva AM, Skowronek A, Snyder OL 2nd, Soares RP, Sódar BW, Soekmadji C, Sotillo J, Stahl PD, Stoorvogel W, Stott SL, Strasser EF, Swift S, Tahara H, Tewari M, Timms K, Tiwari S, Tixeira R, Tkach M, Toh WS, Tomasini R, Torrecilhas AC, Tosar JP, Toxavidis V, Urbanelli L, Vader P, van Balkom BW, van der Grein SG, Van Deun J, van Herwijnen MJ, Van Keuren-Jensen K, van Niel G, van Royen ME, van Wijnen AJ, Vasconcelos MH, Vechetti IJ Jr, Veit TD, Vella LJ, Velot É, Verweij FJ, Vestad B, Viñas JL, Visnovitz T, Vukman KV, Wahlgren J, Watson DC, Wauben MH, Weaver A, Webber JP, Weber V, Wehman AM, Weiss DJ, Welsh JA, Wendt S, Wheelock AM, Wiener Z, Witte L, Wolfram J, Xagorari A, Xander P, Xu J, Yan X, Yáñez-Mó M, Yin H, Yuana Y, Zappulli V, Zarubova J, Žėkas V, Zhang JY, Zhao Z, Zheng L, Zheutlin AR, Zickler AM, Zimmermann P, Zivkovic AM, Zocco D, Zuba-Surma EK. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. http://dx.doi.org/10.1080/20013078.2018.1535750. PMid:30637094.

Vidane AS, Souza AF, Sampaio RV, Bressan FF, Pieri NC, Martins DS, Meirelles FV, Miglino MA, Ambrósio CE. Cat amniotic membrane multipotent cells are nontumorigenic and are safe for use in cell transplantation. Stem Cells Cloning. 2014;7:71-8. PMid:25249758.

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. http://dx.doi.org/10.1089/ten.tea.2010.0678. PMid:21529262.

Yamahara K, Harada K, Ohshima M, Ishikane S, Ohnishi S, Tsuda H, Otani K, Taguchi A, Soma T, Ogawa H, Katsuragi S, Yoshimatsu J, Harada-Shiba M, Kangawa K, Ikeda T. Comparison of angiogenic, cytoprotective, and immunosuppressive properties of human amnion- and chorion-derived mesenchymal stem cells. PLoS One. 2014;9(2):e88319. http://dx.doi.org/10.1371/journal.pone.0088319. PMid:24551087.

Zhao B, Zhang Y, Han S, Zhang W, Zhou Q, Guan H, Liu J, Shi J, Su L, Hu D. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation. J Mol Histol. 2017;48(2):121-32. http://dx.doi.org/10.1007/s10735-017-9711-x. PMid:28229263.

Zhang J, Li S, Li L, Li M, Guo C, Yao J, Mi S. Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics Proteomics Bioinformatics. 2015;13(1):17-24. http://dx.doi.org/10.1016/j.gpb.2015.02.001. PMid:25724326.
 


Submitted date:
06/15/2022

Accepted date:
10/25/2022

636ba94fa953956606786575 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections