Characterization of Canine Embryonic Stem Cell Lines Derived From Different Niche Microenvironments

Abstract

Embryo-derived stem cells hold enormous potential for producing cell-based transplantation therapies, allowing high-throughput drug screening and delineating early embryonic development. However, potential clinical applications must first be tested for safety and efficacy in preclinical animal models. Due to physiological and genetic parity to humans, the domestic dog is widely used as a clinically relevant animal model for cardiovascular, neurodegenerative, orthopedic, and oncologic diseases. Therefore, we established numerous putative canine embryonic stem cell (cESC) lines by immunodissection of the inner cell mass (ICM), which we termed OVC.ID.1-23, and by explant outgrowths from whole canine blastocysts, named OVC.EX.1-16. All characterized lines were immunopositive for OCT4, SOX2, NANOG, SSEA-3, and SSEA-4; displayed high telomerase and alkaline phosphatase (ALP) activities; and were maintained in this state up to 37 passages ( approximately 160 days). Colonies from OVC.EX lines showed classic domed hESC-like morphology surrounded by a ring of fibroblast-like cells, whereas all OVC.ID lines exhibited a mixed cell colony of tightly packed cESCs surrounded by a GATA6+/CDX2- hypoblast-derived support layer. Spontaneous serum-only differentiation without feeder layers demonstrated a strong lineage selection associated with the colony niche type, and not the isolation method. Upon differentiation, cESC lines formed embryoid bodies (EB) comprised of cells representative of all germinal layers, and differentiated into cell types of each layer. Canine ESC lines such as these have the potential to identify differences between embryonic stem cell line derivations, and to develop or to test cell-based transplantation therapies in the dog before attempting human clinical trials.

Preclinical Derivation and Imaging of Autologously Transplanted Canine Induced Pluripotent Stem Cells

“…Our culture system for ciPSCs may provide additional clues to help establishing other large ani- mal ESC lines like pig and sheep for which culture conditions have yet to be determined. Unlike the pig and sheep for which ESC lines are not available, isolation and culture of cESCs have been documented (7)(8)(9), thus allowing us to verify pluripotency and self-renewal properties of ciPSCs derived in this study. Importantly, we have used a noninvasive multimodality imaging approach to monitor autologous iPSC transplantation in a large animal model of cardiac delivery.…”

“…Among large animals, the canine model is especially well suited for translational studies of iPSC-based therapies. Unlike the pig, canine embryonic stem cells (cESCs) have already been isolated and characterized and thus provide criteria for pluripotent canine cells (7)(8)(9). Although both monkey ESCs and iPSCs have been previously derived, the use of primates remains controversial (7,10).…”

“…Unlike the pig, canine embryonic stem cells (cESCs) have already been isolated and characterized and thus provide criteria for pluripotent canine cells (7)(8)(9). Although both monkey ESCs and iPSCs have been previously derived, the use of primates remains controversial (7,10). In this study, we generated ciPSCs resembling cESCs from the somatic skin and fat of adult mongrel dogs.…”

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

“…For the derivation of bovine ES cells several different cell types have been used as feeder layers, including bovine fetal fibroblasts, bovine uterus epithelial cells, mouse fetal fibroblasts, STO cells and human lung fibroblasts but none of these cell types has proven feasible to sustain prolonged proliferation of bovine ES cell-like cells [31], whereas mixed fibroblast feeder cells were more efficient for longterm culture of bovine ES cell lines than using only mouse or bovine fibroblasts [32]. On the other hand, the use of homologous or heterologous feeder cells (mouse) equally supported the generation of canine ES cell lines, but the use of heterologous mouse feeder cells was required to continue canine ES cell propagation [33]. This further complicates the use of either homologous or heterologous feeder layer suggesting the species specific differences in the use of feeder layers for ES cell derivation and culture.…”

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.…”