title: In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes.
abstract: Hypoxanthine phosphoribosyltransferase-deficient (HPRT-) mouse embryonic stem (ES) cells, HM-1 cells (genotype XY), were fused with adult female DD/c mouse spleen cells. As a result, a set of HAT-resistant clones was isolated. Four hybrid clones most similar in morphology and growth characteristics to the HM-1 cells were studied in detail with respect to their pluripotency. Of these, three clones contained 41-43 chromosomes, and one clone was nearly tetraploid. All the clones had the XXY set of sex chromosomes and expressed the HPRT of the somatic partner only. The hybrid clones shared features with the HM-1 cells, indicating that they retained their pluripotent properties: (1) embryonic ECMA-7 antigen, not TROMA-1 antigen, was present in most cells; (2) the hybrid cells showed high activity of endogenous alkaline phosphatase (AP); (3) all the hybrid clones were able to form complex embryoid bodies containing derivatives of all the embryonic germinal layers; (4) the hybrid cells contained synchronously replicating X chromosomes, indicating that they were in an active state; and (5) a set of chimeric animals was generated by injecting hybrid cells into BALB/c and C57BL/6J mouse blastocysts. Evidence for chimerism was provided by the spotted coat derived from 129/Ola mice and identification of 129/Ola glucose phosphate isomerase (GPI) in many organs. Thus the results obtained demonstrated that the hybrid cells retain their high pluripotency level despite the close contact of the "pluripotent" HM-1 genome with the "somatic" spleen cell genome during hybrid cell formation and the presence of the "somatic" X chromosome during many cell generations. The presence of HPRT of the somatic partner in many organs and tissues, including the testes in chimeric animals, shows that the "somatic" X chromosome segregates weakly, if at all, during development of the chimeras. There were no individuals with the 129/Ola genotype among the more than 50 offspring from chimeric mice. The lack of the 129/Ola genotype is explained by the imbalance of the sex chromosomes in the hybrid cells rendering the passage of hybrid cell descendants through meiosis in chimeras impossible. As a result, chimeras become unable to produce gametes of the hybrid cell genotype.
authors.affiliation: Institute of Cytology and Genetics, Academy of Sciences of Russia, Siberian Department, Novosibirsk.
authors.forename: N M
authors.initials: NM
authors.lastname: Matveeva
authors.forename: A G
authors.initials: AG
authors.lastname: Shilov
authors.forename: E M
authors.initials: EM
authors.lastname: Kaftanovskaya
authors.forename: L P
authors.initials: LP
authors.lastname: Maximovsky
authors.forename: A I
authors.initials: AI
authors.lastname: Zhelezova
authors.forename: A N
authors.initials: AN
authors.lastname: Golubitsa
authors.forename: S I
authors.initials: SI
authors.lastname: Bayborodin
authors.forename: M M
authors.initials: MM
authors.lastname: Fokina
authors.forename: O L
authors.initials: OL
authors.lastname: Serov
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015415:Biomarkers; D002454:Cell Differentiation; D002460:Cell Line; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D006822:Hybrid Cells; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D013154:Spleen; D013234:Stem Cells / Q000166:cytology; D014960:X Chromosome
chemical_list: D015415:Biomarkers; D016326:Extracellular Matrix Proteins; D007381:Intermediate Filament Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D000469:Alkaline Phosphatase