title: Isolation and characterization of residual undifferentiated mouse embryonic stem cells from embryoid body cultures by fluorescence tracking.
abstract: The differentiation of mouse embryonic stem (ES) cells can be induced in vitro after leukemia inhibitory factor (LIF) withdrawal and further enhanced by the formation of "embryoid body" (EB) aggregates. This strategy is being used in order to optimize differentiation protocols that would result in functional cells for experimental cell replacement therapies. However, this study presents the possibility for residual undifferentiated cells to survive after standard in vitro procedures. Mouse ES cells were stably transfected with the enhanced green fluorescent protein (EGFP), under the control of the Oct4 promoter, a transcription factor that is expressed in undifferentiated ES cells but down-regulated on differentiation. Residual fluorescent cells were isolated from EBs that were cultured in standard conditions in absence of LIF. These residual cells displayed recurrent gain of chromosomes 8 and 9. Residual fluorescent cells, further expanded in absence of LIF and cultured as EBs, still displayed a significant Oct4 expression in comparison with parental transfected ES cells. Consequently, these residual cells have an intrinsic resistance to differentiate. The behavior of these cells, observed in vitro, can be overcome in vivo, as they were able to induce teratomas in subcutaneously injected nude mice. Residual undifferentiated cells displayed slight levels of VASA and DAZL expression. These results demonstrate that mouse ES cells cultured in vitro, in standard conditions, can spontaneously acquire recurrent karyotypical changes that may promote an undifferentiated stage, being selected in standard culture conditions in vitro.
authors.affiliation: Instituto de Bioingeniería, Unidad de Bioquímica y Terapia Celular, Universidad Miguel Hernández, Elche, Alicante, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Enseñat-Waser
authors.forename: Alfredo
authors.initials: A
authors.lastname: Santana
authors.forename: Néstor
authors.initials: N
authors.lastname: Vicente-Salar
authors.forename: Juan C
authors.initials: JC
authors.lastname: Cigudosa
authors.forename: Enrique
authors.initials: E
authors.lastname: Roche
authors.forename: Bernat
authors.initials: B
authors.lastname: Soria
authors.forename: Juan A
authors.initials: JA
authors.lastname: Reig
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000235:genetics; D002875:Chromosomes; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000032:analysis; D015850:Interleukin-6 / Q000494:pharmacology; D007621:Karyotyping; D053605:Leukemia Inhibitory Factor; D051379:Mice; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013724:Teratoma / Q000473:pathology
chemical_list: D005456:Fluorescent Dyes; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins