ABSTRACT
Embryonic stem cells (ESCs) are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, we derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.
Subject(s)
Blastocyst/physiology , Cattle/embryology , Embryonic Stem Cells/physiology , Pluripotent Stem Cells/physiology , Animals , Biomarkers , Cell Culture Techniques/veterinary , Cell Differentiation , Cells, Cultured , Cloning, Organism , Embryo Culture Techniques/veterinary , Epigenesis, Genetic , Gene Expression Regulation, Developmental/physiology , Nuclear Transfer Techniques/veterinaryABSTRACT
Genome editing using programmable nucleases has revolutionized biomedical research. CRISPR-Cas9 mediated zygote genome editing enables high efficient production of knockout animals suitable for studying development and relevant human diseases. Here we report efficient disabling pancreatogenesis in pig embryos via zygotic co-delivery of Cas9 mRNA and dual sgRNAs targeting the PDX1 gene, which when combined with chimeric-competent human pluriopotent stem cells may serve as a suitable platform for the xeno-generation of human tissues and organs in pigs.
Subject(s)
CRISPR-Cas Systems , Genetic Therapy/veterinary , Organogenesis/genetics , Pancreas/metabolism , Swine/genetics , Animals , Genetic Therapy/methods , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Pancreas/embryology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/transplantation , Stem Cell Transplantation/methods , Stem Cell Transplantation/veterinary , Swine/embryology , Tissue and Organ Harvesting/methods , Tissue and Organ Harvesting/veterinary , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
