RESUMEN
To study the genomic plasticity of somatic cells without ectopic genetic manipulation, we cultured mouse fibroblasts with ovarian cells, embryonic fibroblasts of different strains, and parthenogenetic embryonic stem cells (ESCs). Of 41 trials, cell aggregation resembling nascent ESC colony from inner cell mass was detected in 9 cases (22%), and 6 cases (67%) yielded fibroblast-derived colonies with ESC morphology. Cells used in coculture provided the critical (P=0.0061) inducing factor for the aggregation. These colony-forming fibroblasts (CFFs) showed similar characteristics to those in ESCs and induced pluripotent stem cells (iPSCs), including pluripotency gene expression, in vitro differentiation, and teratoma formation. Furthermore, CFFs produced somatic chimera, although none showed germline chimerism. CFFs had a tetraploid-like karyotype, and their imprinting patterns differed from parthenogenetic ESCs, thereby confirming their nongermline transmissibility. We observed dysregulation of cell cycle-related proteins, as well as both homologous and heterologous recombination of genomic single-nucleotide polymorphisms in CFFs. Our observations provide information on somatic cell plasticity, resulting in stemness or tumorigenesis, regardless of colony-forming cell progenitors in the fibroblast population. The plasticity of somatic genomes under environmental influences, as well as acquisition of pluripotency by cell fusion, is also implicated.
Asunto(s)
Desdiferenciación Celular , Fibroblastos/citología , Nicho de Células Madre , Células Madre/citología , Animales , Agregación Celular , Fusión Celular , Células Cultivadas , Aberraciones Cromosómicas , Técnicas de Cocultivo , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Femenino , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Perfilación de la Expresión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/ultraestructura , Cariotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Microscopía Electrónica de Transmisión , Análisis de Secuencia por Matrices de Oligonucleótidos , Ovario/citología , Especificidad de la Especie , Células Madre/metabolismo , Células Madre/ultraestructuraRESUMEN
OBJECTIVE: To suggest an alternative strategy for deriving histocompatible stems cells without undertaking genetic manipulation. DESIGN: Prospective approach using an animal model. SETTING: Stem cell and bioevaluation laboratory, Seoul National University. ANIMAL(S): F1 (C57BL6 X DBA2) and outbred (ICR) mice. INTERVENTION(S): Ovarian stroma cells of less than 40 mum in diameter were subcultured with fibroblast monolayer, and colony-forming cells were characterized. MAIN OUTCOME MEASURE(S): Stemness, genotype, and imprinted gene methylation. RESULT(S): Two-lines of colony-forming cells were established, which expressed markers specific for embryonic stem cells (ESC) and formed embryoid bodies and teratomas. Complete matching of microsatellite markers with the cell donor strain confirmed their establishment from ovarian tissue, and identification of both homozygotic and heterozygotic chromosomes raised the possibility of their derivation from parthenogenetic oocytes. However, the use of cells smaller than mature oocytes for primary culture, the difference in imprinted gene methylation compared with parthenogenetic ESCs, and failure to establish the ESC-like cells by primary follicle culture collectively suggested the irrelevancy to gametes. CONCLUSION(S): Coculture of adult ovarian cells with somatic fibroblasts can yield colony-forming cells having ESC-like activity, which may provide an alternative for establishing autologous stem cells from adults that can be obtained without genetic manipulation.