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. Mol. Reprod. Dev. 50:128–138, 1998. © 1998 Wiley‐Liss, Inc.

Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes

“…Most hybrid cells had antigen ECMA-7, a marker typical of early mouse embryos (Pascoe et al 1992), and showed high activity of alkaline phosphatase. A series of experiments in which "injected" chimeras were produced with significant contribution of hybrid cells validated the pluripotency capacities of hybrid cells (Matveeva et al 1998). In fact, analysis of biochemical markers (glucose phosphate isomerase) and coat color demonstrated the presence of the descendants of donor hybrid cells in most chimeric tissues.…”

“…Various in vitro and in vivo tests were used to assess the pluripotency of the hybrid cells. All the clones examined, when kept in suspension, maintained the developmental potential to form embryoid bodies containing the derivatives of all three embryonic germ layers (Matveeva et al 1998). Most hybrid cells had antigen ECMA-7, a marker typical of early mouse embryos (Pascoe et al 1992), and showed high activity of alkaline phosphatase.…”

“…We succeeded in establishing a set of hybrid clones similar to ES cells in morphology (Matveeva et al 1996(Matveeva et al , 1998. Hybrid cell clones were isolated in selective HAT medium, which ensures growth of only HPRT-positive cells.…”

“…Most autosomes in HESS2 and HESS3 clones had only 129/Ola markers of the pluripotent parental ES cell. However, chromosomes 1 and 11 were exceptional: along with markers of ES cells, those of the somatic partner were found to be present in small amounts in the two clones (Matveeva et al 1998(Matveeva et al , 2001). This may reflect incomplete segregation of chromosomes 1 and 11 derived from the somatic partner and is also consistent with cytogenetic data indicating that chromosomes 1 and 11 are trisomic in 30 − 40% of HESS2 and HESS3 cells (Matveeva et al 1998(Matveeva et al , 2001).…”

“…This conclusion follows from analysis of the expression of the Hprt gene, a marker of "the somatic" X-chromosome, in chimeras. The allelic HPRT variant of DD/c mice was identified in all chimeric tissues (Matveeva et al 1998). It is also pertinent to note that, once the hybrid cells happen to be in the blastocoel cavity, they encounter non-selective conditions, and retention of the somatic X in the hybrid cell genome means that it becomes its obligatory component.…”