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. 2015 May 3;5:3–6. doi: 10.1016/j.gdata.2015.04.021

Genome-wide copy number profiling of mouse neural stem cells during differentiation

U Fischer a,⁎, N Ludwig a, A Keller b, C Backes b, E Meese a
PMCID: PMC4583622  PMID: 26484209

Abstract

There is growing evidence that gene amplifications were present in neural stem and progenitor cells during differentiation. We used array-CGH to discover copy number changes including gene amplifications and deletions during differentiation of mouse neural stem cells using TGF-ß and FCS for differentiation induction. Array data were deposited in GEO (Gene Expression Omnibus, NCBI) under accession number GSE35523. Here, we describe in detail the cell culture features and our TaqMan qPCR-experiments to validate the array-CGH analysis. Interpretation of array-CGH experiments regarding gene amplifications in mouse and further detailed analysis of amplified chromosome regions associated with these experiments were published by Fischer and colleagues in Oncotarget (Fischer et al., 2015). We provide additional information on deleted chromosome regions during differentiation and give an impressive overview on copy number changes during differentiation induction at a time line.

Keywords: Gene amplification, Neural stem cell differentiation, Array-CGH, qPCR

Specifications
Organism/cell line/tissue Mus musculus
Sex n.d.
Sequencer or array type NimbleGen 720K mouse whole genome tiling arrays.
Data format Raw data: PAIR file, analyzed data: txt file
Experimental factors SFME cells vs normal mouse genomic DNA, SFME cells grown as spheres and after differentiation induction using TGF-ß or FCS
Experimental features SFME cells were grown as spheres for undifferentiated state. Differentiation was induced by withdrawal of EGF and addition of TGF-ß or FCS. Array-CGH experiments were done with undifferentiated cells, 24 h-TGF-ß differentiation induced cells and 12 h-FCS differentiation induced cells.
Consent n/a
Sample source location SFME cells (CRL-9392™) from ATCC
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1. Direct link to deposited data

Deposited data can be found here: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35523.

2. Experimental design, materials and methods

2.1. Cell culture and differentiation

SFME cells cultured in the absence of fibronectin formed spheres and served as non-differentiated controls. SFME cells were seeded on fibronectin-coated cultureware and allowed to grow for 18 h prior to differentiation induction with TGF-β or FCS. SFME cells were differentiation induced using above supplemented ATCC DMEM:F12 Medium containing TGF-β (10 ng/ml) for 8 h, 12 h and 24 h or DMEM:F12 supplemented with FCS for 8 h, 12 h and 24 h.

Cells were harvested and cell pellet was frozen before proceeding to DNA extraction as described previously (Fischer et al., 2014 genomics data) [1].

2.2. Array-CGH data analysis

Array data were deposited in GEO under accession number GSE35523.

Signal intensity data were extracted from scanned images of each array using Roche NimbleGen NimbleScan v2.6 software. After spatial correction, the Cy3 and Cy5 signal intensities were normalized using qspline normalization. Following normalization a 10 × window–averaging step is applied. For amplification and deletion detection we used the dynamic segMNT algorithm that identifies segments by minimizing the squared error relative to the segment means. To detect representative alterations and to minimize the identification of random alterations, we extracted segments with segment means greater than 0.1 threshold and a size greater than 250 kb. Deletions detected in undifferentiated, TGF-ß differentiation induced and FCS differentiation induced cells were summarized in Table 1.

Table 1.

Overview of deleted chromosome regions.

Start and end points of deleted chromosome regions are according to NCBI37/mm9. Size is displayed in kb.

Sphere
 24 h TGF-ß
 12 h FCS
Start End log2 Size Start End log2 Size Start End log2 Size
chr1 3019999 9259999 − 0.11308 6240
chr1 10419999 12539999 − 0.16881 2120
chr1 21459999 33339999 − 0.12453 11,880
chr1 47059999 51179999 − 0.20953 4120
chr1 67859999 68979999 − 0.20877 1120
chr1 95859999 106179999 − 0.11556 10,320
chr1 108899999 120099999 − 0.13136 11,200
chr1 110459999 112459999 − 0.15437 2000
chr1 125099999 125779999 − 0.1133 680
chr1 141739999 151499999 − 0.12807 9760
chr1 157499999 166019999 − 0.11672 8520 chr1 157699999 164339999 − 0.11251 6640
chr1 179699999 180099999 − 0.17032 400
chr2 39299999 49379999 − 0.11759 10,080
chr2 80899999 83139999 − 0.16313 2240
chr2 85619999 89979999 − 0.1151 4360 chr2 85539999 89979999 − 0.19832 4440
chr2 94819999 101179999 − 0.10144 6360 chr2 94419999 101179999 − 0.14893 6760
chr2 140259999 140739999 − 0.10916 480
chr2 174619999 176979999 − 0.10819 2360 chr2 174539999 176979999 − 0.10409 2440
chr3 3179999 7819999 − 0.16459 4640
chr3 10779999 15219999 − 0.10217 4440 chr3 10699999 14179999 − 0.21013 3480
chr3 15259999 15819999 − 0.25966 560 chr3 15339999 18379999 − 0.18784 3040 chr3 15459999 15779999 − 0.2969 320
chr3 23219999 26019999 − 0.12403 2800
chr3 41659999 48699999 − 0.20394 7040
chr3 47419999 48019999 − 0.20803 600 chr3 48739999 50819999 − 0.10307 2080
chr3 66659999 67219999 − 0.14445 560
chr3 69859999 71299999 − 0.14 1440
chr3 71339999 73539999 − 0.1502 2200 chr3 71339999 72859999 − 0.24188 1520
chr3 72899999 75019999 − 0.12964 2120
chr3 76179999 78579999 − 0.10363 2400
chr3 80059999 81099999 − 0.14237 1040 chr3 80659999 81059999 − 0.20763 400
chr3 93699999 94059999 − 0.18368 360 chr3 93699999 94059999 − 0.16031 360
chr3 110219999 115099999 − 0.16927 4880
chr3 116859999 120779999 − 0.1109 3920
chr3 123499999 125779999 − 0.2668 2280 chr3 123339999 125779999 − 0.35956 2440 chr3 123059999 127819999 − 0.16916 4760
chr3 125819999 127739999 − 0.16144 1920 chr3 125819999 128939999 − 0.16294 3120
chr3 131419999 132379999 − 0.13963 960
chr3 140139999 140899999 − 0.18969 760
chr3 149859999 151299999 − 0.18356 1440
chr3 154499999 159578619 − 0.10621 5079
chr4 12379999 32099999 − 0.13785 19,720
chr4 35779999 39859999 − 0.20166 4080
chr4 64579999 75579999 − 0.11545 11,000
chr4 75659999 80779999 − 0.2069 5120 chr4 75619999 80779999 − 0.2917 5160 chr4 75579999 78339999 − 0.19677 2760
chr4 89259999 94339999 − 0.13013 5080
chr5 5939999 7859999 − 0.15729 1920
chr5 11859999 19539999 − 0.12821 7680
chr5 54859999 61859999 − 0.20136 7000
chr5 67699999 72699999 − 0.13854 5000
chr5 78299999 91059999 − 0.10839 12,760 chr5 81459999 81739999 − 0.14919 280
chr5 146259999 146579999 − 0.10129 320
chr6 41499999 47339999 − 0.10686 5840 chr6 41539999 47099999 − 0.16256 5560
chr6 55739999 66419999 − 0.10699 10,680
chr6 73379999 81459999 − 0.11413 8080
chr6 103779999 112019999 − 0.1065 8240
chr6 138299999 140059999 − 0.14334 1760 chr6 138379999 140059999 − 0.22473 1680
chr7 56899999 70659999 − 0.10699 13,760
chr7 75539999 79379999 − 0.11891 3840
chr7 91779999 103339999 − 0.10018 11,560
chr7 10699999 12139999 − 0.24888 1440
chr7 110659999 111699999 − 0.23809 1040 chr7 110619999 111379999 − 0.20134 760
chr8 4579999 9659999 − 0.11752 5080
chr8 99539999 106019999 − 0.10809 6480
chr8 29979999 34539999 − 0.13338 4560
chr8 49459999 55619999 − 0.16084 6160
chr8 98739999 106419999 − 0.17182 7680
chr9 3139999 7299999 − 0.10507 4160
chr9 10419999 12939999 − 0.15382 2520
chr9 16739999 20259999 − 0.10813 3520
chr9 33219999 34019999 − 0.11795 800
chr9 35659999 35939999 − 0.21324 280 chr9 35659999 36299999 − 0.16454 640 chr9 35659999 36059999 − 0.16676 400
chr9 37699999 38899999 − 0.10023 1200 chr9 37419999 39979999 − 0.10149 2560
chr9 71699999 72019999 − 0.1087 320
chr9 115059999 115619999 − 0.10853 560
chr10 15059999 16899999 − 0.1919 1840
chr10 35579999 35939999 − 0.17695 360 chr10 26739999 38059999 − 0.11928 11,320
chr10 45899999 51019999 − 0.12617 5120 chr10 45899999 49499999 − 0.20934 3600 chr10 47739999 49099999 − 0.13582 1360
chr10 63059999 65899999 − 0.11266 2840
chr10 71099999 74299999 − 0.10106 3200
chr10 100819999 105099999 − 0.16547 4280 chr10 100819999 105099999 − 0.22175 4280 chr10 101059999 104739999 − 0.11145 3680
chr10 111619999 114339999 − 0.19567 2720
chr10 122579999 126139999 − 0.11643 3560
chr10 128539999 129975647 − 0.10464 1436 chr10 128539999 129975647 − 0.14084 1436
chr11 8979999 18619999 − 0.11164 9640 chr11 17579999 18619999 − 0.10657 1040
chr11 36019999 42459999 − 0.11313 6440 chr11 36459999 42459999 − 0.19152 6000
chr11 90499999 93299999 − 0.14477 2800
chr12 89939999 99619999 − 0.12512 9680
chr12 114979999 116219999 − 0.10697 1240
chr13 76539999 90659999 − 0.12234 14,120
chr13 115939999 120282113 − 0.12049 4342
chr14 49819999 53379999 − 0.12933 3560 chr14 50499999 52099999 − 0.12137 1600
chr14 52379999 53419999 − 0.10299 1040
chr14 76859999 78259999 − 0.10653 1400 chr14 76939999 125175837 − 0.15227 48,236
chr14 88619999 95699999 − 0.17732 7080 chr14 80339999 98619999 − 0.31002 18,280
chr14 98659999 106899999 − 0.14224 8240
chr14 106939999 118059999 − 0.26608 11,120
chr14 118099999 125175837 − 0.13545 7076
chr15 9419999 10299999 − 0.13815 880
chr15 13459999 23819999 − 0.1377 10,360 chr15 13339999 24459999 − 0.20317 11,120
6360 chr15 13499999 14979999 − 0.10807 1480
chr15 46139999 47259999 − 0.15127 1120 chr15 44659999 51019999 − 0.17027 chr15 19699999 22619999 − 0.10222 2920
chr15 47419999 51059999 − 0.12808 3640
chr15 89459999 90219999 − 0.12766 760
chr16 59059999 89699999 − 0.13622 30,640
chr17 17499999 22579999 − 0.11022 5080 chr17 18299999 20899999 − 0.2772 2600
chr17 37259999 42659999 − 0.17895 5400
chr17 50899999 56019999 − 0.11015 5120
chr17 57539999 62859999 − 0.14589 5320
chr17 76139999 78259999 − 0.13494 2120 chr17 76139999 78299999 − 0.17557 2160
chr17 81499999 83219999 − 0.10341 1720
chr17 89499999 95255954 − 0.10669 5756 chr17 89259999 95255954 − 0.15303 5996
chr18 16939999 19899999 − 0.11972 2960 chr18 16979999 19779999 − 0.1813 2800 chr18 7899999 8339999 − 0.13658 440
chr18 26139999 31459999 − 0.11968 5320 chr18 26219999 31379999 − 0.21126 5160
chr18 51059999 52299999 − 0.14649 1240 chr18 50739999 52659999 − 0.19077 1920 chr18 51659999 52099999 − 0.12795 440
chr18 70659999 73179999 − 0.1304 2520
chr18 75979999 76259999 − 0.16726 280 chr18 75779999 76499999 − 0.11312 720
chr18 85659999 90459999 − 0.1178 4800 chr18 85299999 90765552 − 0.13801 5466
chr19 47779999 52739999 − 0.12561 4960
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We used this low threshold of 0.1 for amplification and deletion detection because we were using a mixture of cells. Fluorescence in situ hybridization experiments at a single cell level had shown that gene amplifications were present at various percentages of the cells and in various copy numbers per single cell [2]. For further confirmation of the usefulness of a low threshold we did TaqMan copy number assay for two amplified genes namely GFAP and FZR1. GFAP revealed a 0.347 log2 ratio and FZR1 a 0.2 log2 ratio.

2.3. qPCR analysis

TaqMan Copy Number Assays for genes GFAP and FZR1 were performed following manufacturer's instructions. We used the TERT TaqMan Copy Number reference assay for relative quantitation of copy number of target genes. Mouse genomic DNA (Clontech) was used as control standard for normal diploid copy number. TaqMan assays were run in two independent experiments, each in four technical replicates and results were analyzed using StepOne™ Software v2.0 and copy numbers were analyzed using CopyCaller™ software. Mean results of four technical replicates were summarized in Fig. 1a (GFAP) and b (FZR1). The copy number calculated by Software Copy Caller™ revealed an increased copy number 3-fold of GFAP for SFME cell differentiation induced by TGFß for 8 h, 12 h and 24 h. In SFME cell differentiation induced by FCS for 8 h, 12 h and 24 h, the copy number was 2.5, 3 and 2.5-fold respectively. The software also identified an increased copy number of 2.5-fold for FZR1 for SFME cell differentiation induced by TGFß for 8 h, 12 h and 24 h. Likewise we found an increased copy number of 2.5-fold for SFME cell differentiation induced by FCS for 24 h. These results confirmed our previous array-CGH analysis and FISH experiments. Interestingly the higher log2 ratio values for GFAP in array-CGH experiments corresponded to an elevated copy number value in TaqMan qPCR experiments.

Fig. 1.

Fig. 1

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Amplification analysis using q-PCR.

Amplification of GFAP and FZR1 was analyzed by qPCR using the TaqMan copy number assays. SFME cells grown as spheres served as undifferentiated controls. SFME cells were investigated at three different time points with TGF-ß and FCS differentiation induction. Mouse genomic DNA served as standard for normal diploid copy number. The average copy number was 3 of GFAP in SFME cell differentiation induced by TGF-ß for 8 h, 12 h and 24 h. In SFME cell differentiation induced by FCS for 8 h, 12 h and 24 h, the average copy number was 2.5, 3 and 2.5 respectively. The average copy number was 2.5 for FZR1 in SFME cell differentiation induced by TGF-ß for 8 h, 12 h and 24 h, induced by FCS for 24 h. There was no copy number gain for FZR1 detectable in SFME cell differentiation induced by FCS for 8 h and 12 h.

3. Discussion

Here we report detailed information on threshold choice for detection of gene amplification using NimbleGen 730K mouse whole genome array and correlation between log2 ratio values and copy number values from TaqMan qPCR experiments. Here and in our previous report we detected a complex pattern of amplifications and deletions. Both amplifications and deletions were only detectable after a low threshold setting. Threshold settings of 0.8 used in many studies were very likely to miss alterations that were present in a subpopulation of the investigated cells. Our confirmation using qPCR strongly argues for a low threshold setting. This dataset is an additional step towards uncovering copy number changes upon differentiation in mammalian stem cells.

Acknowledgements

The “Deutsche Forschungsgemeinschaft” funded this study (Fi644/2-1; Fi644/2-2).

References

  • 1.Fischer U., Keller A., Backes C., Meese E. Genome-wide copy number profiling to detect gene amplifications in neural progenitor cells. Genomics Data. 2014;2:162–165. doi: 10.1016/j.gdata.2014.06.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Fischer U. Gene amplification during differentiation of mammalian neural stem cells in vitro and in vivo. Oncotarget. 2015;6(9):7023–7039. doi: 10.18632/oncotarget.3248. [DOI] [PMC free article] [PubMed] [Google Scholar]

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