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Title: Isolation and characterization of residual undifferentiated mouse embryonic stem cells from embryoid body cultures by fluorescence tracking
Authors: Enseñat-Waser, Roberto
Santana, Alfredo 
Vicente-Salar, Néstor
Cigudosa, Juan C.
Roche, Enrique
Soria, Bernat
Reig, Juan A.
UNESCO Clasification: 3109 Ciencias veterinarias
Keywords: Cell differentiation
Cell culture
Flow cytometry
Oct4, et al
Issue Date: 2006
Journal: In Vitro Cellular and Developmental Biology - Animal 
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.
ISSN: 1071-2690
DOI: 10.1290/0509063.1
Source: In Vitro Cellular and Developmental Biology - Animal [ISSN 1071-2690], v. 42, p. 115-123
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