Dominance of parental genomes in embryonic stem cell/fibroblast hybrid cells depends on the ploidy of the somatic partner.
Two dozen hybrid clones were produced by fusion of diploid embryonic stem (ES) cells positive for green fluorescent protein (GFP) with tetraploid fibroblasts derived from DD/c and C57BL-I(I)1RK mice. Cytogenetic analysis demonstrated that most cells from these hybrid clones contained near-hexaploid chromosome sets. Additionally, the presence of chromosomes derived from both parental cells was confirmed by polymerase chain reaction (PCR) analysis of polymorphic microsatellites. All hybrid cells were positive for GFP and demonstrated growth characteristics and fibroblast-like morphology. In addition, most hybrid cells were positive for collagen type I, fibronectin, and lamin A/C but were negative for Oct4 and Nanog proteins. Methylation status of the Oct4 and Nanog gene promoters was evaluated by bisulfite genomic sequencing analysis. The methylation sites (CpG-sites) of the Oct4 and Nanog gene promoters were highly methylated in hybrid cells, whereas the CpG-sites were unmethylated in the parental ES cells. Thus, the fibroblast genome dominated the ES genome in the diploid ES cell/tetraploid fibroblast hybrid cells. Immunofluorescent analysis of the pluripotent and fibroblast markers demonstrated that establishment of the fibroblast phenotype occurred shortly after fusion and that the fibroblast phenotype was further maintained in the hybrid cells. Fusion of karyoplasts and cytoplast derived from tetraploid fibroblasts with whole ES cells demonstrated that karyoplasts were able to establish the fibroblast phenotype of the reconstructed cells but not fibroblast cytoplasts. Thus, these data suggest that the dominance of parental genomes in hybrid cells of ES cell/somatic cell type depends on the ploidy of the somatic partner.
Department of Developmental Genetics, Institute of Cytology and Genetics, Academy of Sciences of Russia, Siberian Branch, Novosibirsk, Russia.
Anna A
AA
Kruglova
Natalia M
NM
Matveeva
Maria M
MM
Gridina
Nariman R
NR
Battulin
Anton
A
Karpov
Elena V
EV
Kiseleva
Ksenia N
KN
Morozova
Oleg L
OL
Serov
D000818:Animals; D001483:Base Sequence; D033481:Chromosomes, Mammalian / Q000378:metabolism; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D016678:Genome / Q000235:genetics*; D018398:Homeodomain Proteins / Q000378:metabolism; D006822:Hybrid Cells / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D034904:Lamin Type A / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D011003:Ploidies
D024042:Collagen Type I; D005353:Fibronectins; D018398:Homeodomain Proteins; D034904:Lamin Type A; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3