Abstract
Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cell embryos with embryonic stem cells. The fully agouti colored chimeras were generated from both injection and coculture of 8-cell embryos with embryonic stem cells. Additionally, microsatellite DNA screening showed that the fully agouti colored chimeras were fully embryonic stem cell derived mice. Unlike embryonic stem cells, the mouse chimeras were only generated from injection of 8-cell embryos with induced pluripotent stem cells and none of these showed germline transmission. The results indicated that injection of 8-cell embryos is the most efficient method for assessing stem cell pluripotency and generating induced pluripotent stem cell chimeras, embryonic stem cell chimeras with germline transmission, and fully mouse embryonic stem cell derived mice.
Highlights
Chimeric mice are important tools for investigating embryonic development as they can provide insights into the function of a specific gene, they can trace the origin of the cell lineage, and they can assess the potential of cells
While embryonic stem cells (ESCs) are recognized as pluripotent stem cells, we tested the chimeric contributions of induced pluripotent stem cells (iPSCs) on chimera generation by microinjection and coculture in the well-of-the-well (WOW) system
When ESCs at passage 20–22 were used for chimera production (Figure 1), 29.1% (25/86), 18.1% (23/127), and 38.4% (28/73) of the pups derived from, respectively, 8-cell embryo coculture (Figure 2), 8-cell embryo injection (Figures 3(a) and 3(b)), and blastocyst injection showed coat color chimerism (Table 2)
Summary
Chimeric mice are important tools for investigating embryonic development as they can provide insights into the function of a specific gene, they can trace the origin of the cell lineage, and they can assess the potential of cells. Microinjection will produce good germline transmitted chimeras, specialized equipment is needed For these reasons, the coculture method was developed to produce chimeric mice [3, 4, 19] even though it is far less efficient than the microinjection and well sandwich aggregation techniques. Stem Cells International a high degree of chimerism and germline transmission which utilized coculture of denuded mouse embryos and ES cells in Eppendorf tubes was reported [20] Such aggregation in Eppendorf tubes can cause embryo adhesion and form 2- or 3-embryo clusters mixed with ESCs. Recently, 8-cell stage embryos were used to generate fully ESC-derived mice in a laser- [21] and piezo-assisted micromanipulation system [22]. While ESCs are recognized as pluripotent stem cells, we tested the chimeric contributions of iPSCs on chimera generation by microinjection and coculture in the well-of-the-well (WOW) system
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