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1Institute of Biomedical Sciences, Federal University of Rio De Janeiro-UFRJ
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Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.
Campos, P. B., Sartore, R. C., Abdalla, S. N., Rehen, S. K. Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping. J. Vis. Exp. (31), e1512, doi:10.3791/1512 (2009).
Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, 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 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. 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 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.
Treating the cells with colcemid
In this procedure we used the hESC line H9 cultured onto mouse embryonic fibroblasts (mEF) inactivated with mitomycin C (Sigma) and maintained in DMEM/F12 (Invitrogen) supplemented with 20% knockout serum replacement (KSR, Gibco) and 8ng/mL of fibroblast growth factor (FGF-2, R&D).
In order to obtain a large number of metaphases spreads, it is recommended to use cultures with high mitotic index in which there are many dividing cells.
Fixing the cells
CLEANING THE GLASS SLIDES
Before starting making your chromosome spreads make sure that your slides are properly cleaned or you may lose some nuclei and also metaphases. This step is as well important if you want to try any hybridization technique 9.
NOTE: For in situ hybridization methods, the slides cannot be stored for more than 2 days into 96% ethanol
Washing the cells
Preparing the slides
How choose an adequate metaphase to be analyzed
Choosing round and isolated metaphases (figure 2D), you may avoid considering false aneuploidies as the gain of chromosomes generated by two or more spreads mixed together or the loss of chromosomes generated by a launched chromosome. So, avoid choosing metaphases near nuclei because some chromosomes may be hidden by them (figure 2A and 2B). Besides, look for round metaphases and be sure that any chromosome is separated from the metaphase (figure 2C).
Feeder cells and the hESC karyotype
For G-banding and SKY analysis, the presence of feeder cells in hESC culture does not interfere with the results, because feeder cells are not under a dividing state (inactivated by mitomycin C or irradiation) and will not be part of the metaphase population. However, for the application of FISH technique in interphase nuclei, it is preferred to separate the hESC population from feeder cells by harvesting the colonies manually before trypsinization for FISH analysis.
Figure 1: How know the appropriate cell density in the slides. Images of phase contrast microscope in 10x objective. (A) The cell density is too high. The arrows point to metaphase spreads too close of nuclei. (B) A slide with good cell density. The circles show metaphases spreads isolated from nuclei or other metaphases. Please click here to see a larger version of figure 1.
Figure 2: How to choose an adequate metaphase to be analysed. Chromosomes are stained with DAPI and the images obtained by a fluorescent microscope in 100x objective. (A) (B) Avoid metaphases near nuclei (indicated by the arrows) (C) and metaphases not round that present chromosomes separated (circle). (D) A round metaphase is a good metaphase to be analysed. Please click here to see a larger version of figure 2.
The preparation of chromosome spreads is a critical step for a successful analysis of the genetic status of embryonic stem cells by routinely techniques as G-banding, and more sophisticated techniques such as FISH, SKY and CGH.
This procedure can be applied for many cell types by varying the period of colcemid incubation, which depends on the cell cycle length. For colonies of embryonic stem cells, we wait 3 hours while for embryoid bodies (EB) this time can be up to 6 hours.
In the case of working with cell aggregates (like embryoid bodies) you will need to dissociate very well in order to obtain a single cell solution. In this case, after trypsin and mechanical dissociation, you can make use of a 40μm nylon cell strainer (BD Biosciences) and then give sequence to the procedure (from step 4 of the procedures topic).
In the procedure here described it is worth noting that the cell suspension is dropped "closely" onto the slides in order to avoid excessive spreading of the chromosomes and consequently generation of artifacts.
We believe that this protocol is an useful and simple tool to be routinely applied not only by laboratories dealing with embryonic stem cells but by other investigators interested in studying chromosomal instability in other stem cells.
The authors have nothing to disclose.
|15 ml centrifuge tube||Techno Plastic Products||91015|
|Colcemid Karyo MAX||GIBCO, by Life Technologies||15212-012|
|Glacial acetic acid||Isofar||100|
|Potassium Chloride (KCl)||Merck & Co., Inc.||104.931.000|
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