Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells

Abstract

Canine embryonic stem (cES) cell lines were generated to establish a large-animal preclinical model for testing the safety and efficacy of embryonic stem (ES) cell-derived tissue replacement therapy. Putative cES cell lines were initiated from canine blastocysts harvested from natural matings. Times of harvest were estimated as 12-16 days after the presumed surge in circulating levels of luteinizing hormone. Four lines established from blastocysts harvested at days 13-14 postsurge satisfied most of the criteria for embryonic stem cells, whereas lines established after day 14 did not. One line, Fred Hutchinson dog (FHDO)-7, has been maintained through 34 passages and is presented here. FHDO-7 cells are alkaline phosphatase-positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and telomerase but do not express message for Cdx2, which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miRs) (miR-302b, miR-302c, and miR-367) characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders, the cells form embryoid bodies (EBs). Under various culture conditions, the EBs give rise to ectoderm-derived neuronal cells expressing ␥-enolase and ␤3-tubulin; mesoderm-derived cells producing collagen IIA1, cartilage, and bone; and endoderm-derived cells expressing ␣-fetoprotein or Clara cell-specific protein.

Köpek Olfaktorik Mukozasindan Olfaktorik Kök Hücrelerin Izolasyonu ve Karakterizasyonu

“…CD9 belongs to the tetraspanin family and is considered as a pluripotency marker [36] . Afterwards Chaker N. Adra discovered and patented [37] that pluripotent stem cell populations could be obtained from olfactory mucosa and reported that some cells of the pluripotent stem cell population could differentiate into cells of one or more various lineages such as mesenchymal lineages or neuronal lineages or both. Thus, in this study, we also investigated the expression of pluripotent genes such as OCT4, Nanog and Sox2 because canine embryonic stem cells were demonstrated to express Oct4, Nanog, and Sox2 at high levels [38] .…”

Pluripotency of bank vole embryonic cells depends on FGF2 and activin A signaling pathways

“…Obtaining of ES cell lines from species other than mouse and man has proved even more difficult. Protocols optimised for derivation and culture of mouse ES cells were not suitable for establishment of embryonic stem cells from hamster (Doetschman et al, 1988); mink (Sukoyan et al, 1992); dog (Hatoya et al, 2006;Hayes et al, 2008); cat (Yu et al, 2008); rabbit (Honda et al, 2008); horse (Saito et al, 2002); pig (Notarianni et al, 1990;Notarianni et al, 1991;Wheeler, 1994;Li et al, 2003); sheep (Notarianni et al, 1991); buffalo (Verma et al, 2007) and until recently from rat (Iannaccone et al, 1994;Vassilieva et al, 2000;Schulze et al, 2006;Demers et al, 2007). Bank vole seems to be yet another species supplementing the list of ES cells derivation trials.…”

Use of pluripotent stem cells for reproductive medicine: are we there yet?

“…However, the used technology still needs optimization as they undergo spontaneous differentiation over long-term culture, irrespective of the source of derivation, that is, in vivo or in vitro produced blastocysts. Canine ESCs have also been isolated from pre-implantation stage embryos with both in vitro and in vivo differentiation potential (Hatoya et al 2006;Hayes et al 2008;Schneider et al 2008;Vaags et al 2009;Wilcox et al 2009;Schneider et al 2010). These canine ESCs were able to undergo self-renewal for prolonged periods of culture while remaining undifferentiated, and expressed a characteristic pluripotency gene expression profile, such as OCT4, SSEA3 and SOX2.…”