Chromosomal spread preparation of human embryonic stem cells for karyotyping.
Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used, while manual dissection of colonies for passaging retains a stable karyotype. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis. Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies.
Campos
Priscila B
PB
Institute of Biomedical Sciences, Federal University of Rio De Janeiro-UFRJ.
Sartore
Rafaela C
RC
Abdalla
Stacie N
SN
Rehen
Stevens K
SK
D002478:Cells, Cultured; D002869:Chromosome Aberrations; D002877:Chromosomes, Human; D053595:Embryonic Stem Cells / Q000166:cytology / Q000648:ultrastructure*; D006801:Humans; D007621:Karyotyping / Q000379:methods*