Clonal isolation and characterization of myoblast-like reconstituted cells formed by fusion of karyoplasts from mouse teratocarcinoma cells with rat myoblast cytoplasts.
To examine the roles of the cytoplasms of differentiated somatic cells on nuclear gene expression, reconstituted cells (RC-cells) were isolated clonally by fusing karyoplasts (isolated nuclei) from neomycin-resistant mouse teratocarcinoma PCC4-neor cells with cytoplasts (isolated cytoplasms) of chloramphenicol (CAP)-resistant rat myoblasts L6TG.CAPr cells, and after double selection in the medium containing 400 micrograms/ml of neomycin and 100 micrograms/ml of CAP (G418 plus CAP medium). The RC-cells were characterized by the presence of two genetic markers, neomycin- and CAP-resistance, by the absence of latex beads which had incorporated into karyoplast donor PCC4-neor cells as a cytoplasmic physical marker, and by the similar karyotypes as that of parental PCC4-neor cells. In contrast to the teratocarcinoma cybrids previously isolated, all the isolated RC-clones expressed myoblast-like morphologies of three types. The phenotypic expression of these RC-cells was compared with that of PCD-1 cells, a teratocarcinoma-derived myoblast line. RC-cells and PCD-1 cells did not express alkaline phosphatase (ALPase) activity while parental PCC4-neor expressed it strongly. After induction of myogenic differentiation by treatments with excess thymidine and conditioned medium, two clones were capable of forming short multinucleated cells. The protein synthetic patterns of RC-cells analysed by two-dimensional polyacrylamide gel were different from PCC4-neor cells, and quite resembled those of PCD-1 cells. Particularly, multinucleated RC-clones expressed alpha-tropomyosin, and contained 10 nm filaments, characteristic markers of early myogenic cells. These results suggest that the RC-cells are myoblast-like cells, that a few of them maturate to partially differentiated myogenic cells, that the rat myoblast cytoplasm contains regulatory factor(s) able to determine the myogenic cell lineage of the undifferentiated stem cells, and that this factor is continuously expressed in these myoblasts.
Tosu
M
M
Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan.
Terasaki
T
T
Iwakura
Y
Y
Yoshida
M
M
Sekiguchi
T
T
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002459:Cell Fusion; D002467:Cell Nucleus / Q000502:physiology; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced; D002999:Clone Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000502:physiology; D005819:Genetic Markers; D007621:Karyotyping; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000196:embryology*; D010641:Phenotype; D051381:Rats; D013724:Teratoma / Q000473:pathology*; D013936:Thymidine / Q000494:pharmacology
D005819:Genetic Markers; D000469:Alkaline Phosphatase; D013936:Thymidine