Nuclear cloning of embryonal carcinoma cells

Abstract

Embryonal carcinoma (EC) cells have served as a model to study the relationship between cancer and cellular differentiation given their potential to produce tumors and, to varying degrees, participate in embryonic development. Here, nuclear transplantation was used to assess the extent to which the tumorigenic and developmental potential of EC cells is governed by epigenetic as opposed to genetic alterations. Nuclei from three independent mouse EC cell lines (F9, P19, and METT-1) with differing developmental and tumorigenic potentials all were able to direct early embryo development, producing morphologically normal blastocysts that gave rise to nuclear transfer (NT)-derived embryonic stem (ES) cell lines at a high efficiency. However, when tested for tumor or chimera formation, the resulting NT ES cells displayed an identical potential as their respective donor EC cells, in stark contrast to previously reported NT ES cells derived from transfer of untransformed cells. Consistent with this finding, comparative genomic hybridization identified previously undescribed genetic lesions in the EC cell lines. Therefore, nonreprogrammable genetic modifications within EC nuclei define the developmental and tumorigenic potential of resulting NT ES cells. Our findings support the notion that cancer results from the deregulation of stem cells and further suggest that the genetics of ECs will reveal genes involved in stem cell self-renewal and pluripotency.

Fig. 1.

Teratoma analysis of EC and corresponding NT ES cell lines. Teratomas from EC (A, C, and E) and NT ES (B, D, and F) cell lines. (A and B) F9ECandNT ES teratomas remain completely undifferentiated (*). Host dermal and epidermal tissue is seen in bottom and lower left of A and D, respectively. (C and D) P19 EC and NT ES cells differentiate into immature neuroepithelium distinguished by rosette formation (arrow). (E and F) METT-1 EC and NT ES cells differentiate into a variety of tissues, including mature neural and glandular tissue (asterisk and arrow, respectively).

Fig. 2.

Chimera analysis of EC and corresponding NT ES cell lines. (A–D) Midgestation (embryonic day 14.5) chimeras. (A and C) P19 EC and NT ES cells contribute to developing bone (best seen in ribcage) and brain. Head and spinal morphogenesis is abnormal. (B and D) METT-1 EC and NT ES cells show a broad contribution to embryos, including bone, soft tissue, and skin, as seen here. (E–H) P19 EC and NT ES chimeras are born with head and neck embryocarcinomas. Tumors are obvious on external evaluation of P19EC (E) and P19 NT ES (F) pups (arrows). Hematoxylin/eosin staining (G) and GFP immunostaining (H) of a representative P19 NT ES chimera confirm tumors are ECs of donor cell origin.

Fig. 3.

CGH analysis of EC and corresponding NT ES cell lines. CGH profiles of F9 (A), P19 (B), and METT-1 (C) EC and NT ES cell lines. The x axis represents chromosomes 1–19 (sex chromosomes are not included). The y axis represents relative copy numbers of genetic material along chromosomes. (B Lower) SKY image of chromosomes 6 and 14 in a P19 EC cell, showing two normal chromosomes 6, one normal chromosome 14, and a nonreciprocal translocation of tip of chromosome 6 to chromosome 14, resulting in gain of 6q and loss of 14q. (Left) SKY images. (Center) Reverse 4′,6-diamidino-2-phenylindole images. (Right) Computer-generated representations of SKY images.