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本帖最后由 bioon 于 2015-2-28 11:52 编辑 " _' e3 W6 ~! U+ ?% N
$ m; u' v3 I, z; mIncreased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions7 q1 @3 N/ D9 K* @ ~1 J
http://journals.plos.org/plosone ... ournal.pone.0118307
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The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.
2 K3 G& _: U# Q1 j) U' @4 eMaterials and Methods: D, P8 S0 H& m
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Cell Culture& I. e; \+ \+ c3 D" w8 u
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The HDF51iPS1, HDF51iPS7, and HDF51iPS11 lines were three independent hiPSC clones reprogrammed from human fetal dermal fibroblasts using the standard reprogramming factors (OCT4/POU5F1, SOX2, KLF4, and MYC) carried by retroviral vectors [3,12]. WA09 is an hESC line derived by the Thomson laboratory [13], and was obtained directly from WiCell Research Institute. WA09, HDF51iPS1, HDF51iPS7, and HDF51iPS11 cells were cultured at 37°C and 5% CO2 on extracellular matrix (Geltrex; Life Technologies) or irradiated mouse embryonic fibroblasts (MEFs). Cells on feeder layers were cultured in standard hPSC medium, consisting of Dulbecco’s modified eagle medium DMEM/F12 (Life Technologies) with 20% Knockout Serum Replacement (Life Technologies), 1 mM GlutaMAX (Life Technologies), 0.1 mM non-essential amino acids (NEAA, Life Technologies), and 12 ng/ml basic FGF (Life Technologies) and passaged mechanically (MefMech) by cutting colonies into small pieces with a 18G needle or enzymatically dissociated (MefEnz) with Accutase (Life Technologies). Cells cultured on Geltrex in StemPro hESC SFM (Life Technologies) with 0.1 mM β-mercaptoethanol (Life Technologies) and 12 ng/ml basic FGF (Life Technologies), were passaged mechanically (EcmMech) or enzymatically (EcmEnz) with Accutase. Cells on feeder layers were passaged once a week and feeder-free cultures were passaged every three to four days. At each passage, 150,000 cells were seeded per well of a 6-well plate (equivalent to a cell plating density of ~15,600 cells/cm2). To accurately count the number of cells in mechanically passaged cultures, the clumps of cells were collected and gently triturated, and an aliquot was removed and completely dissociated to single cell suspension using Accutase. The medium was changed daily., |, O) u7 D+ g. Z
, ~: t& {' C# p W$ y3 dIn transitioning the original cell cultures from the MefMech condition into the other conditions, we started counting the passages in the new condition as soon as the process of transition started. For transitioning from mechanical to enzymatic passaging, we simply started using Accutase (Life Technologies). For transitioning from the Mef condition (which included culture on mouse embryonic fibroblast feeder layers with the standard hPSC medium) to the Ecm condition (which included culture on Geltrex (Life Technologies) with StemPro hESC SFM (Life Technologies)), we initially passaged onto Geltrex (Life Technologies) using a 1:1 mixture of standard hPSC medium:StemPro hESC SFM for 24 hours, followed by a 1:3 mixture of standard hPSC medium:StemPro hESC SFM for 24 hours, and then 100% StemPro hESC SFM thereafter.5 B! Z2 X4 E) `! [ a0 D+ p) d
1 B1 {4 ?, |) ^; vTeratoma formation, histopathologic evaluation and immunohistochemistry- M- A3 a+ Q7 }4 x
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All procedures were done following the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Scripps Research Institute and UCSD Institutional Animal Care and Use Committees (IACUCs). For the teratoma assays, we injected two mice per replicate (12 mice per culture condition) at two sites per mouse (1 testis capsule and 1 leg injection). WA09 hESCs were harvested by Accutase (Life Technologies), washed with PBS (Life Technologies), re-suspended in 30 μl of a 1:1 mixture of DMEM/F12 (Life Technologies) and Matrigel (BD Biosciences), and placed on ice. One million cells were injected per site into the right testis capsule and left lower leg muscle of CB-17-Prkdcscid mice (Charles River) with a 1cc syringe and a 27G, ½ inch needle. The injection site was monitored for tumor growth weekly and six to eight weeks after injection the mice were euthanized and the tumors removed. The tumors were measured, weighed, and fixed with PBS containing 4% paraformaldehyde (Sigma-Aldrich). The specimens were sectioned and stained with hematoxylin and eosin and evaluated microscopically by a board-certified anatomic pathologist (MP) to detect derivatives of the three germ layers. Selected sections were stained with a rabbit anti-OCT4 primary antibody (Abcam, Cat # ab19857) at 1:400, using a Ventana Discovery Ultra automated immunostainer (Ventana Medical Systems) with standard antigen retrieval and reagents per the manufacturer’s protocol.
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Karyotyping
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Karyotyping was performed only on selected MefEnz cultures. Cell Line Genetics performed G-banded karyotyping on the hESC cultures. 30 spreads were counted per culture.2 ~& A& }8 Z0 Z( z3 B2 K& }9 Y, S
1 ?4 g3 |& x; n! `3 CMycoplasma Testing& S8 e! A! k9 b9 [5 R, U8 C
3 D9 x* q; N+ JSupernatant of the cultures was collected and mycoplasma was tested monthly using the Lonza Luminescence test (Lonza) and VenorGeM PRC Test (Sigma-Aldrich) according to the manufacturers’ protocols./ P G5 X7 y8 f, l: T0 f* ^/ E, c
3 S7 I! L3 M9 pIn Vitro Differentiation
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2 v7 N: Q) f8 A% q/ d7 p) |0 Y# X! iFor embryoid body (EB) formation, WA09 hESCs were harvested manually and transferred to low attachment plates in Dulbecco’s modified eagle medium DMEM/F12 with 20% Knockout Serum Replacement, 1 mM Glutamax, 0.1 mM non-essential amino acids (NEAA), 0.1 mM β-mercaptoethanol. The cells were cultured in suspension for 7 days to allow for EB formation and the media was changed every other day. On the eighth day, the EBs were transferred to gelatin (Life Technologies) coated coverslips with DMEM (Life Technologies), 2 mM Glutamax, 20% fetal bovine serum (Life Technologies), and 0.1 mM NEAA, and cultured seven more days changing the medium every other day. EBs were then washed with PBS and fixed for 15 minutes with PBS containing 4% paraformaldehyde.1 _$ U9 z1 x+ D# u- Y5 X2 e: e, p) n9 P
- x9 J9 O o; Q% aImmunocytochemistry
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Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After washing with PBS, the cells were treated with blocking solution (2% BSA (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich), and 2% low fat milk in PBS) for one hour at room temperature. Primary antibodies include OCT4 (1:100, Santa Cruz); SSEA-4 (1:200, R&D system); SOX2 (1:100, Millipore); TUJ1 (1:15,000, Millipore); Brachyury (1:300, Santa Cruz); Smooth Muscle Actin (SMA) (1:2000, Millipore); alpha-fetoprotein (AFP) (1:400, Millipore). Fluorescent dye-labeled goat anti mouse, goat anti rat or donkey anti mouse was used as the secondary antibody (Life Technology, Molecular Probes).
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' _# m9 u9 Z# a9 E+ T; nEDU Incorporation Assay
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Cells were incubated with 20 μM EdU (Life Technologies) for 1 hour at 37°C, harvested, washed with 1% BSA in PBS, and fixed in suspension for 15 minutes at room temperature with 4% paraformaldehyde. Cells were washed again, permeabilized with Triton X-100 for 30 minutes at room temperature, washed again, and incubated with the Click-iT EDU CellCycle 633-red (Life Technologies) for 30 minutes at room temperature and protected from light. The samples were then analyzed by flow cytometry.
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MTT Assay
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: L4 I+ d+ r" E7 J5 I( VCells cultured on a 96-well plate were incubated in medium containing 0.4 mg/mL 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-2H-tetrazoliumbromide (MTT; TCI America) at 37°C for 1 h. Reduced MTT was solubilized in DMSO (Sigma-Aldrich) for determination of absorbance at 570 nm.
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$ e* Q3 `( v8 m& u s% D: LApoptosis Assay
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% Y& }" H+ ?& K2 KApoptosis rate was measured using the In Situ Cell Death Detection Kit (Roche). Cells were harvested and a cell suspension of 106 cells/ 100 μl was fixed with 4% paraformaldehyde for 60 min at room temperature with shaking, washed with PBS, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (Sigma-Aldrich) for 2 minutes on ice. The labeling of DNA strand breaks was performed for 60 minutes at 37°C by Terminal deoxynucleotidyltransferase (TdT), which catalyzes polymerization of fluorescein labeled dUTP to free 3’-OH DNA ends in a template independent manner, also known as TUNEL reaction. The cells were then washed with PBS and analyzed by flow cytometry at an excitation wavelength of 488 nm and detection at 515–565 nm.* `# D& H1 c- L2 I2 o1 K
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Telomere Length Assay# a* p4 X/ U! j7 }
/ e: L7 P) I) \! TTelomere length was measured using TeloTAGGG Telomere length assay (Roche). Cells were harvested and genomic DNA was isolated with DNeasy Blood & Tissue kit (Qiagen). The concentration of genomic DNA was measured with the Qubit Quantitation Platform (Invitrogen), the DNA concentration for each sample was normalized, and 1.5 μg of genomic DNA from the sample or control DNA was digested with 40 units of Hinf I (New England Biolabs) and Rsa I (New England Biolabs) for 2 hours at 37°C. The digestion was stopped with gel electrophoresis loading buffer and the digested DNA was separated by 0.8% agarose gel electrophoresis. For Southern blots, the digested DNA was transferred to a positively charged nylon membrane (Roche) using capillary transfer. The DNA was fixed on the wet blotting membrane by UV-crosslinking at 120 mJ. The blotted DNA fragments were then hybridized to a digoxigenin (DIG)-labeled probe specific for telomeric repeats for 3 hours at 42°C, washed several times, and incubated for 30 minutes at room temperature with a DIG-specific antibody covalently coupled to alkaline phosphatase. The membrane was incubated with the chemiluminescent substrate CDP-Star and imaged (Gel Doc XR System, Bio-Rad). The mean terminal restriction fragment (TRF) length, which comprises the variable terminal array and the subtelomeric region, was calculated by measuring the chemiluminescent signal and the length of each TRF.& Y% \/ T: f! @ Y ^# {* g
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Telomerase Activity Assay
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Telomerase activity was measured using TRAPeze RT Telomerase Detection kit (Millipore). Cells were harvested, washed with PBS, resuspended in 200 μl of CHAPS lysis buffer (provided in TRAPeze kit)/106 cells, and incubated on ice for 30 minutes. The samples were spun down at 12,000 x g for 20 minutes at 4°C, 160 μl of supernatant was collected, protein concentration determined with the BCA Protein Assay (Pierce), and the concentration of total protein was normalized for each sample. The reaction mixture contained 5 μl 5X TRAPeze RT Reaction Mix, 1 μl Taq polymerase (Titanium Taq, Clontech #639220), 17 μl nuclease free water, and 2 μl of TSR8 dilution (for standard curve), positive extract control, telomerase negative control, no template control, experimental samples, or heat treated experimental samples. The parameters for the qRT-PCR were 30 minutes at 30°C (1 cycle), 2 minutes at 95°C (1 cycle), and then15 s at 94°C, 60 s at 59°C, 10 s at 45°C for 45 cycles using the primers are supplied by the manufacturer.( t, T3 h/ D5 \. q/ j& \0 J* ]
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SNP Genotyping2 v/ n, q2 Q- h' ^
+ V, F( s; t7 b: _) tSNP Genotyping was performed on the OmniQuad version 1 (Illumina, Inc.), which interrogates 1,140,419 SNPs across the human genome. One μg input genomic DNA purified with the DNeasy Blood & Tissue Kit (Qiagen) (the yield from approximately 200,000 cells) was amplified and labeled according to the manufacturer’s instructions. DNA was quantified using the PicoGreen reagent (Life Technologies). Labeled product was then hybridized to the array and scanned on a BeadArray Reader (Illumina, Inc.). Genotyping calls were made using BeadStudio (Illumina, Inc.), using the standard cluster files provided by the manufacturer. The GenCall threshold was set to 0.15, and the call rates were between 0.9911858 (EcmMech P147 D) and 0.9981838 (Control).- Y. _) d; {+ l& q' F" G3 Y# j
1 j i" D6 |; k5 x# k# @& _0 MCopy Number Variation Assessment
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For the SNP Genotyping data, data preprocessing was performed in BeadScan (Illumina, Inc.). Data cleaning, SNP calling, and replicate error identification was performed in GenomeStudio (Illumina, Inc.). CNVPartition 3.2.0 [14] was used as the CNV-calling algorithm for calling duplications, with a CNV score threshold set at 50, and a minimum number of 10 SNPs per CNV region. CNV locations are displayed according to the hg19/NCBI36 version of the human genome. We have previously shown that approximately 80% of the duplications called by this method are validated by qPCR [3]. Since we previously found that only 40% of the regions of deletion identified by CNVPartition were validated by qPCR [3], we required that regions of deletion be identified both as regions of deletion by CNVPartition and as new regions of LOH by replicate error analysis. All CNV calls were manually curated, and regions that were identified manually are indicated with a CNV Confidence Value designated as “NA”. Manual curation was also used to combine adjacent areas called by CNVPartition into larger regions of CNV; in these cases, the CNV confidence interval is indicated as the range of CNV confidence intervals calculated by CNVPartition for the encompassed subregions. During manual curation, we identified some regions of CNV that were present in only a subpopulation of cells, which we termed “partial” duplications and deletions. We also identified some large regions of partial CNV for which we could not determine with certainty whether they were partial duplications or partial deletions, and these we termed “Complex CNV.”8 s0 }" s% M0 ^. o) u7 [7 q9 t
# w# K5 D0 \ f7 z h6 qCNV-RT-PCR1 b3 J, t& c. L' T
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Validation of the CNV values, obtained from CNVPartition 3.2.0., was done using TaqMan Copy Number Assays (Life Technologies). The DNA used for validation was the same on which SNP genotyping analysis was performed. The DNA was diluted in order to obtain a final concentration of 5 ng/μl. 20 ng of DNA were loaded in the plate. Following the manufacturer’s protocol, we prepared the reaction mixture (10 μl of TaqMan Genotyping Master Mix (Life Technologies), 1 μl of probe of choice (Life Technologies), 1 μl of TaqMan Copy Number Reference Assay (Life Technologies), and 4 μl of nuclease free water) and added to each sample. qPCR was performed using these cycling conditions: 10 minutes at 95°C (1 cycle), and then 15 s at 95°C, 60 s at 60°C, for 40 cycles. Six probes (three for the short arm and three for the long arm of chromosome 12, Life Technologies)) were used to validate the CNV values of the samples: Hs03295596_cn (12p11.22b chr12:29111191), Hs04404518_cn (12p11.21b ch12:31250144), Hs03812366_cn (12p11.21b chr12:31944796), Hs03841181_cn (12q23.1b chr12:979244490), Hs06947059_cn (12q24.11d chr12:111495082), and Hs06363301_cn (12q24.23a chr12:118819770). The results were analyzed using the ΔΔCt method, using RNaseP as the control.
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. H: \$ D: O1 Y3 k1 q/ rGene Expression Microarray" m$ r# O$ U) c
8 k1 B) \' m$ f/ G( g8 J& FGene expression profiling was performed using HumanHT-12 v3 Expression BeadChips according to the manufacturer’s instructions (Illumina, Inc.), with the exception of the definitive mesoderm sample, for which gene expression profiling was performed using HumanHT-12 v4 BeadChips (Illumina, Inc.). Samples were prepared using the TotalPrep kit (Ambion, Inc.) according to the manufacturer’s instructions. Probes were filtered with a detection-P value cut-off of 0.05, and normalized using the LUMI package in R with the RSN (Robust spline normalization) method.! ?' P1 s8 G7 b2 x! I$ m) x
' d* g6 h5 K% d W3 {Multivariate analysis was performed on all the undifferentiated samples using R. Different variables were compared: time in culture (early vs. late), passage method (Enz vs. Mech), and substrate (Mef vs. Ecm). The output was filtered with a p-value cutoff of <0.01 and a fold-change cutoff of >1.8.; Q" l# i' |; r" ?
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Hierarchical clustering analysis was performed to determine the relative expression levels of the pluripotency-associated genes using Cluster 3.0. The dendrograms and heatmaps were generated using Java Treeview [15].
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Pathways analysis was performed with DAVID Bioinformatics Resources 6.7 [16].
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DNA Methylation Microarray
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* y( l4 X* B R, ^6 c3 cDNA was extracted from 106 cells (Qiagen DNeasy kit) according to the manufacturer’s protocol and quantified using the Picogreen reagent (Life Technologies). Bisulfite conversion was performed using the Bisulfite Conversion Kit from Zymo Research. Bisulfite converted DNA was labeled and hybridized to Infinium HumanMethylation450 BeadChips (Illumina, Inc.), scanned with an Illumina iScan BeadArray Scanner and quality controlled in GenomeStudio (Illumina, Inc.). Pathway analysis was performed using GREAT [17], and visualized using REVIGO [18].
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1 Y: t/ ]( n2 Y0 i0 s5 W7 i: dAccession Numbers
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The hESC DNA methylation, gene expression, and SNP genotyping array data are available at the NCBI GEO database under the accession designation GSE34982. The hiPSC SNP genotyping data are available under accession number GSE56834. The hESC and hiPSC DNA methylation data are also available under SuperSeries GSE56851. |
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发表于 2015-2-28 11:36
