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Abstract
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Introduction: The formation of chimeras is an important technique for evaluating the developmental potential of human pluripotent stem cells (hPSCs). Our previous research indicated that the chicken embryo is a suitable host due to its developmental similarity to the human embryo at the primitive streak stage. However, we encountered challenges associated with the lengthy process of transferring the fertilized egg to the surrogate eggshell, leading to increased embryo mortality. To address this issue, we suggest a new approach to enhance human-chicken chimera formation.
Methods: We injected primed Royan H6 cells at a rate of 3000-5000 cells in 3 microliters into HH4 stage embryos. Instead of using the previous method, where a surrogate eggshell was used to inject the cells from the wide top, fertilized eggs were placed on their side for at least 1 hour and injected the cells directly into that side. After 7 days, we examined the morphology and survival of the embryos, as well as the presence of human cells using the STEM121 antibody.
Results: After comparing the old and new methods, we found that increased efficiency in chimera formation reduced fetal mortality during cell injection into the primitive streak after 7 days and decreased the number of consumed chick embryos. Although we observed abnormalities in the morphology of some chick chimera, staining with STEM121 antibody revealed the presence of hPSCs.
Conclusion: Our approach enhances precision, simplifies handling, and speeds up chimera generation. Furthermore, we validated the reproducibility of chimera formation in chick embryos using hPSCs.
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