fibroblasts are one of the most commonly used somatic cells to produce induced pluripotent stem ( ips ) cells [ 1 , 2 ] .
therefore , it is essential to determine the genetic and epigenetic stability of fibroblasts in long - term culture .
it has been shown that long - term in vitro culture of somatic cells increases the risk of cell senescence , chromosomal aneuploidy , as well as multiploidy karyotypes [ 3 , 4 ] .
however , the potential changes in epigenetic gene regulation in long - term cultured human fibroblasts are not well investigated .
dosage compensation of the x chromosome in mammalian female cells is one of classic examples of epigenetic gene regulation .  
it is achieved through an event referred to as x chromosome inactivation ( xci ) in which one of the two x chromosomes is silenced during development [ 57 ] .
xci is initiated in the x - inactivation center of the x chromosome , within which a gene encoding x inactive - specific transcript ( xist ) is expressed solely from the inactivated x chromosome .
xist gene is crucial for xci because its noncoding rna directly interacts with and coats the inactive x chromosome .
after the completion of xci , the two x chromosomes in female cells are distinguished by differential transcription of the xist gene , dna methylation , and histone modifications such as histone h3 lysine 27 trimethylation ( h3k27me3 ) [ 8 , 9 ] . in this study , human dermal fibroblasts ( hdfs )
we then examined the cell - growth rate , the stability of xci in short- and long - term passages .  
we found that early passages of female hdfs retained a normal karyotype with the expected xci pattern while high - passage cells exhibited a higher risk of carrying more than one copy of the inactive x chromosomes due to the presence of three or four copies of x - chromosomes in one nucleus .
this research project was approved and monitored by the ethical committee in the first affiliated hospital of nanjing medical university . written informed consent was obtained from all 8 female patients before collecting their dermal tissue specimens .
tissue specimens were washed with phosphate buffered saline ( pbs ) solution with 100  u / ml penicillin and 100  mg / ml streptomycin .
fat and connective tissues were removed , and the epidermis was scraped off by an operating knife blade .
the remnant tissues were sheared into clumps of approximately 1  mm , which were then planted uniformly onto the bottom of 25  cm flasks .
the flasks were then inverted and placed in a 5% co2    incubator at 37c .
culture medium consists of dulbecco 's modified eagle 's medium ( dmem , pyruvate - free , high - glucose formulation ; invitrogen ) supplemented with 20% fetal bovine serum ( fbs ; invitrogen ) .
when the cells reach 8090% confluence , the culture was split at a ratio of 1  :  2 .
cells from passage 5 through 10 were cultured in a 6-well plate with a density of 4  10 per well .
six days later , the growth curves of 8 hdf cell lines were obtained by counting the number of cells per well every 24 hours .
karyotype analysis on each hdf cell line was carried out via giemsa - banding ( g - banding ) between passages 510 .
when 8090% confluence was reached in the 25  cm flask , culture medium was changed and supplemented with 0.1  g / ml colcemid solution ( diluted from 10  g / ml stock , cat .
, cells were collected and suspended in 5  ml of 0.075  m kcl solution .
. 2  ml of fixation fluid ( methanol : glacial acetic acid at 1  :  3 ) was added and mixed together , and the cells were then collected . after two rounds of fixations , the cell suspension was mounted onto slides and baked at 75c for 3 hours .
cells were plated on either slides or coverslips after being rinsed three times with 1x pbs for 5 minutes and then fixed with 4% paraformaldehyde for 20  minutes at room temperature .
then the cells were washed again with 1x pbs three times and stored in 70% etoh at 20c for subsequent analysis .
all reagents used for rna fish were prepared with diethylpyrocarbonate- ( depc- , sigma ) treated h2o .    
cells were washed three times with 1x pbs and dehydrated with 70% ethanol for 2 minutes and 90% ethanol for 2 minutes .
then the cells were hybridized overnight at 37c in hybridization buffer ( 50% formamide/2x standard saline citrate ( ssc ) using a cy3-hxd1 - 3 probe as previously described in .
the next day , coverslips were rinsed twice with hybridization buffer for 30 minutes each at 37c and then once with 1x pbs .
the cells prepared as above were rinsed and incubated sequentially by the following procedures : cells were rinsed with 1x pbs three times , incubated in 1x pbs plus 0.4% triton x-100 ( sigma ) for 20 minutes and rinsed again with 1x pbs three times at room temperature .
blocking of nonspecific staining was conducted with 10% normalgoat serum in 1x pbs for 1 hour at room temperature , after which the cells were incubated overnight with a rabbit anti - h3k27me3 antibody ( 1  :  1000 , from update biotechnology , usa ) at 4c in a humidified chamber .
the cells were subsequently rinsed in 1x pbs three times and incubated for 1  hour in the dark with fitc - conjugated secondary antibody ( 1  :  200 , beijing zhong shan golden bridge biotechnology co. ltd , china ) .
the cells were then rinsed with 1x pbs three times , counterstained with 4,6-diamidino-2-phenylindole ( dapi , vysis , inc . , downers grove , il , usa ) , and mounted onto slides after 5 minutes .  
slides were immersed in 2x ssc solution jar , shook for 5 seconds , and removed after 10 minutes .
hybridization areas were made , and the temperature of the denaturing solution ( 70% formamide/2x ssc ) was maintained at 73c .  
after about 5 minutes , slides were dehydrated for 1 minute in 70% etoh , followed by 1 minute in 85% etoh , and then 1 minute in 100% etoh .
concurrently , the probe mixture was prepared and placed in a 73c water bath for 5 minutes
. 10  l of probe mixture was then added to one target area after which the coverslip was immediately applied .
the next day , the coverslip was removed from one slide and the slides were immediately immersed in the 0.4x ssc solution for 2 minutes at 70c .
finally , the slides were air - dried in the dark and 10  l of counterstains were applied to the target area of the slides and the coverslips were added . a commercially available probe ( vysis , inc .
, downers grove , il , usa ) targeting the centromeric region of x chromosome ( green fluorescence ) was used to evaluate the number of x chromosomes of the cells .
two hundred cells were examined randomly from each culture , and the numbers of signals in each cell nucleus were counted .
after the first 24-hour inoculation , dermal tissues were completely adhered to the bottom of the flasks and hdfs began growing out in the following days .  
when hdfs reached 8090% confluence , they were passaged as described in materials and methods .   within 10 passages , few of the cells exhibited senescence including intumescences , arborescence , and cell death .
the growth curves showed that the number of hdf slowly increased in the first 24 hours after passage . from 24 to 96 hours , hdf proliferated exponentially .
hdf cells entered stationary phase from 96 to 144 hours after passage ( figure 1 ) .  
g - banding was conducted to determine the karyotypes of 8 hdf cell lines , and the results demonstrated that each culture exhibited normal karyotype in twenty metaphase spreads examined within the first 10 passages ( data not shown ) .    
one intense punctate signal of h3k27me3 was found in most hdf nuclei within passages 1 and 5 ( figure 2 ) .  
occasionally , we also found cells that exhibit either zero or two punctuate staining signals .
after randomly selecting and counting the number of nuclei with 0 , 1 , and 2 h3k27me3 signals , we found that the number of cells without h3k27me3 punctate signals appeared to increase at a higher passage number ( table 1 ) .
the xist rna fish analysis yielded the same results as h3k27me3 immunostaining ( figure 3 ) .
interestingly , in four out of total eight cell lines ( # 1 , 2 , 3 , and 8) , our counting data showed that two positive signals of xist rna coating or h3k27me3 staining of inactive x chromosomes were present in approximately 8.5 ~ 18.5% hdf cells at passage 10 ( figures 2 and 4 ) .
consistent with the increased number of inactive x - chromosome , a few cells showed three or four copies of x chromosome signals from dna fish analysis of total number of x chromosomes .  
our counting data showed that approximately 6% of the cells exhibited three copies of x chromosomes and 16% with four copies of x - chromosomes ( figure 4 ) .
pluripotent cells are considered a major resource for regenerative medicine , and fibroblasts are the most commonly used cell type to produce ips cells by reprogramming [ 1 , 2 ] . in order to produce quality ips cells , the biological properties of fibroblasts must first be characterized prior to reprogramming .
these include cell growth state , stability of cytogenetic and epigenetic status such as x chromosome inactivation . as a pioneer on cell senescence study ,
hayflick was the first to establish a reliable protocol to maintain fibroblast growth in vitro .
early studies showed that hdfs can reach over 50 population doublings before senescence sets in , but its proliferative capacity depends on the age of the donor and the biopsy site . in our cultures of eight female hdf lines , cells with fusiform shape and senescence phenotype
nevertheless , in vitro cultures may still affect genetic and epigenetic stability of the cultured hdfs . by counting the cell numbers after passaging , we plotted the cell growth curves which demonstrated that cells were in delitescence in the first 24 hours after cell passage .
the cells then proliferated rapidly in exponential growth phase from 24 to 96 hours and entered stationary phase from 96 to 144 hours ( figure 1 ) .
previous in vitro culture studies report spontaneously mutated clones in most species , especially in rodents , usually after 57 passages .
these mutated clones have abnormal karyotypes including chromosomal aneuploidy and multi - ploidy karyotypes . in our results
, we did not find any definitively abnormal karyotypes through the standard g - banding karyotyping .  
however , in a detailed analysis of x - inactivation status , we found that a portion of cells ( 8.518.5% ) in half of our cultures ( four out of eight hdf lines ) exhibited more than two copies of x - chromosomes .
our data suggest that identification of the number of inactive x chromosomes by xist rna fish or the total number of x chromosomes by dna fish analysis is a more sensitive method to detect chromosomal variations than the standard g - banding karyotyping .
xci is a fundamental epigenetic mechanism in female somatic cells , involving the dosage compensation of x - linkage genes .
any improper changes in the dosage of x - linkage gene would impair the normal function of somatic cells .
research on human female embryonic stem cell ( esc ) lines indicated that xci patterns of these lines could change during passaging .
xist gene expression in cultured hesc lines was unstable and subjected to epigenetic silencing by dna methylation . in this study , xist gene rna fish and h3k27me3 immunofluorescence
were performed to detect the xci status in passages 1 , 5 , and 10 of hdf cells .
the results indicated that in most cell nucleus only one xci signal ( green spot for h3k27me3 or red spot for xist rna ) was observed , and few of the cells had no xci markers ( figures 2 and 3 ) .
cell numbers without xci signals tended to increase along with hdf population doublings ( table ) , which could be dna methylation on the xist promoter to silence its expression .
additionally , two xist rna signals in coupling with 3 - 4 signals of x chromosome can be observed in the same nucleus in a few cells at passage 10 ( figure 4 ) .
the presence of two xist rna signals indicate two copies of inactive x chromosomes , consistent with the increased number of total x - chromosomes . in summary
, our present study demonstrates that early passages of female hdf cultures are genetically more stable than cultures after 10 passages . by monitoring xci status and the number of inactive x chromosome , we can effectively determine whether the chromosome ploidy of human female somatic cells changes over the course of cell culture .  
the information of genome stability is important for evaluating the suitability of these cells for other applications such as the derivation of ips cells in regenerative medicine .
 human primary fibroblasts are a popular type of somatic cells for the production of induced pluripotent stem ( ips ) cells .   here 
 we characterized biological properties of primary fibroblasts in terms of cell - growth rate , cytogenetic stability , and the number of inactive x chromosomes during long - term passaging . 
 we produced eight lines of female human dermal fibroblasts ( hdfs ) and found normal karyotype and expected pattern of x chromosome inactivation ( xci ) at low passages ( passage p1 - 5 ) . 
 however , four out of the eight hdf lines at high passage numbers (  p10 ) exhibited duplicated hallmarks of inactive x chromosome including two punctuate signals of histone h3 lysine 27 trimethylation ( h3k27me3 ) and x inactive - specific transcript ( xist ) rna signals in approximately 8.518.5% of the cells . 
 our data suggest that the copy number of inactive x chromosomes in a subset of female hdf is increased by a two - fold . 
 consistently , dna fluorescent in situ hybridization ( fish ) identified 3 - 4 copies of x chromosomes in one nucleus in this subset of cells with two inactive xs .   
 we conclude that female hdf cultures exhibit a higher risk of genetic anomalies such as carrying an increased number of x chromosomes including both active and inactive x chromosomes at a high passage (  p10 ) . 
 
1. Introduction
2. Material and Method
3. Results
4. Discussion