Isolation and culture of primary bovine embryonic stem cell colonies by a novel method

Abstract

Authentic bovine embryonic stem (ES) cell lines have not been established despite progress made for more than two decades. Isolation and culture of primary ES cell colonies are the first critical step towards establishment of stable ES cell lines. Here we report a novel method designated as "Separate and Seed" that contributes remarkably to efficient derivation of bovine primary ES-like cell colonies from blastocysts. These primary cultured bovine ES-like cells exhibit morphology typical of ES cells and express pluripotent molecular markers including Oct4, Nanog and alkaline phosphatase. Interestingly, bovine primary ES-like cell colonies distinctively express both stage-specific embryonic antigens 1 and 4 (SSEA1 and SSEA4), unlike mouse and human ES cells. These pluripotent markers may be used for characterization of authentic bovine ES cell lines in later studies. In contrast, whole embryos or inner cell mass (ICM) used for primary culture by conventional methods fails to produce primary bovine ES cell colonies that express all pluripotent stem cell markers shown above. Furthermore, bFGF improves growth and maintained undifferentiated state of bovine ES-like cells for several passages, whereas LIF and ERK inhibitor PD98059 known to promote pluripotency of mouse ES cells are unable to sustain bovine ES-like cells. Although continued efforts are required for improving long-term culture of bovine ES cells, this novel "Separate and Seed" method provides an initial effective step that may eventually lead to derivation of authentic bovine ES cell lines.

The use of parthenotegenetic and IVF bovine blastocysts as a model for the creation of human embryonic stem cells under defined conditions

“…From the adhered ICMs we obtained 39 % and 33 % of expanded colonies, respectively, suggesting that cells interacted satisfactory with fibronectin and maintained their proliferative capacity forming typical primary stem cell-like colonies. These results contrast with previously reported data, where 15 mechanically dissected bovine ICMs plated onto mouse embryonic feeder cells did not adhere to the substrate and disappeared in few days [3]. In addition, assays for pluripotency markers presented very clear results indicating that the parthenogenetic and IVF colonies created by these methodologies were primary bESCs.…”

Derivation and Characterization of Bovine Induced Pluripotent Stem Cells by Transposon-Mediated Reprogramming

“…Numerous attempts to establish bovine ESC lines have been undertaken; however, they have had limited success (Gong et al, 2010;Jin et al, 2012;Maruotti et al, 2012;Saito et al, 1992;Saito et al, 2003;Solter et al, 2000). Most ESC-like cultures proliferated slowly, lost pluripotency markers, and ceased to divide at early passages (Cao et al, 2009;Malaver-Ortega et al, 2012;Maruotti et al, 2012;Munoz et al, 2008;Saito et al, 1992).…”

Embryonic stem-like cells derived from in vitro produced bovine blastocysts

“…The maintenance of a bovine ES cell lineage seemed to depend less on the technical procedures than the knowledge of the mechanisms that promote or influence cell pluripotency. This is not an easy task, as recently reported by Cao et al (2009), the pathways needed to maintain the undifferentiated state of bovine ES cells seem to be different from the already known pathways found in mice and humans. In summary, we were able to isolate bovine ICMderived ES-like cells by mechanical procedures.…”

“…Conflicting results have been reported in relation to AP staining in the bovine embryonic stem cells. Cao et al (2009) detected a strong AP activity in both, the primary bovine ES-like colony and trophoctoderm cells. Yadav et al (2005) observed a weak or absent AP activity in the bovine embryonic ES-like cells.…”

“…Usually, a micromanipulation chamber over the stage of an inverted microscope are used, equipment not frequently disposable to cell culturists that aims to isolate the bovine ES. Furthermore, until now, no authentic bovine ES cells have been obtained despite the use of these more sophisticated techniques (Talbot and Blombergle, 2008;Cao et al, 2009). The aim of this study was to establish and characterize the embryonic stem cells from fresh in vitro produced bovine blastocysts (IVP), using the mechanical isolation of ICM and mechanical propagation of the colonies.…”