Isolation and characterization of embryonic stem-like cells from canine blastocysts

Abstract

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.

Canine Embryo‐Derived Stem Cells—Toward Clinically Relevant Animal Models for Evaluating Efficacy and Safety of Cell Therapies

“…Sequencing confirmed that the obtained polymerase chain reaction (PCR) products correspond to canine sequences. Furthermore, in agreement with the report by Hatoya et al [9], the canine ES-like cells showed expression of SSEA-1 but were negative for SSEA-4 (data not shown).…”

“…In a recent report, Hatoya and colleagues [9] describe the isolation of two ES-like cell lines from canine blastocysts. The cell lines were shown to exhibit characteristic ES-like morphology and expression of pluripotency markers.…”

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

“…In the case of antibodies specific for SSEA-1, also known as CD15, which is expressed on human granulocytes as well as human ES cells, there was no specific labeling of dog granulocytes or ES cells (data not shown). This differed from the two previous reports on dog ES cells [15,16]; however, in the first study, no specificity controls for staining were included, and in the second, the data were not shown and the methods were not discussed.…”

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

“…Corresponding results were reported by [30], in which cat ES-like cells expressed the "stemness" transcription factors and the levels of expression of OCT-4, NANOG and C-MYC were reduced in cat ES-like colonies as compared to that seen in blastocysts. In other study, expression of transcription factors like Oct 4 and Nanog were used as molecular markers for ES cells in various species [16,17,[41][42][43]. Moreover, when undifferentiated primary colonies were analyzed for the expression of pluripotent transcription factors, Gómez et al [30] found that OCT-4 was down regulated regardless of the expression of NANOG.…”