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Keywords:

  • telocytes;
  • mesenchymal stem cells;
  • fibroblasts;
  • gene expression profile;
  • interstitial cells;
  • stroma;
  • connective tissue;
  • lung

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Telocytes (TCs) are interstitial cells with telopodes – very long prolongations that establish intercellular contacts with various types of cells. Telocytes have been found in many organs and various species and have been characterized ultrastructurally, immunophenotypically and electrophysiologically (www.telocytes.com). Telocytes are distributed through organ stroma forming a three-dimensional network in close contacts with blood vessels, nerve bundles and cells of the local immune system. Moreover, it has been shown that TCs express a broad range of microRNAs, such as pro-angiogenic and stromal-specific miRs. In this study, the gene expression profile of murine lung TCs is compared with other differentiated interstitial cells (fibroblasts) and with stromal stem/progenitor cells. More than 2000 and 4000 genes were found up- or down-regulated, respectively, in TCs as compared with either MSCs or fibroblasts. Several components or regulators of the vascular basement membrane are highly expressed in TCs, such as Nidogen, Collagen type IV and Tissue Inhibitor of Metalloproteinase 3 (TIMP3). Given that TCs locate in close vicinity of small vessels and capillaries, the data suggest the implication of TCs in vascular branching. Telocytes express also matrix metalloproteases Mmp3 and Mmp10, and thus could regulate extracellular matrix during vascular branching and de novo vessel formation. In conclusion, our data show that TCs are not fibroblasts, as the ultrastructure, immunocytochemistry and microRNA assay previously indicated. Gene expression profile demonstrates that TCs are functionally distinct interstitial cells with specific roles in cell signalling, tissue remodelling and angiogenesis.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Recent electron microscopic studies have identified telocytes (TCs), a distinct type of interstitial cells, in many cavitary and non-cavitary organs [1-20]. Telocytes are defined by their very long prolongations – called telopodes (Tps; generally, 2–3/cell; length of up to hundreds of μm) – which emerge from a relatively small cellular body. It has been shown that TCs form a 3D network through the organ interstitium surrounding organ-specific structures, blood capillaries, immune cells and nerve endings. As a specific functional property, TCs are key players in intercellular signalling, at both short and long distance. Thus, the long Tps establish direct contacts (junctions) with neighbouring cells and contribute to the (directional) transport of long-range signals driven by TCs [21]. Local (paracrine) signalling of TCs is achieved by shedding vesicles [8, 20, 22].

The ultrastructural portrait of TCs was recently complemented with the immunophenotypical and electrophysiological characterization and the specific microRNA expression signature [20, 22, 23]. However, the gene expression profile for this type of cells has not been reported yet. Prompted by these studies, we sought to compare murine lung TCs with mesenchymal stem cells (MSCs) and fibroblasts to identify the genes which are specifically regulated in TCs. We choose lung TCs as these are well-characterized ultrastructurally and immunohistochemically in situ and in vitro [4, 5, 11, 16, 17].

Method and Materials

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Cell lines and tissue sampling

Mouse colonies were maintained in Animal Research Center of Fudan University, Shanghai, China. Lung samples were obtained from 20 to 25 g male BABL/c mice, 4–6 weeks of age. The mice were killed with an overdose of anaesthetic and the lung tissues were harvested for the isolation of TCs. The animal study was approved by the Ethic Committee for Animal Care and Use, Fudan University. Mesenchymal stem cells and fibroblast cell lines were obtained from Sciencell Research Laboratories (Cat. no. M7500-57, Carlsbad, CA, USA) and from Chinese Academy of Science (Cat. no. GNM28, Shanghai, China) respectively.

Isolation and primary culture of telocytes from lung tissues

Lung tissues were cut into small pieces and harvested under sterile conditions and collected into sterile tubes containing Dulbecco's Modified Eagle's Medium (DMEM, Gibco, NY, USA), supplemented with 100 UI/ml penicillin and 0.1 mg/ml streptomycin (Sigma Chemical, St. Louis, MO, USA), and the samples were brought to the cell culture room immediately. Samples were further rinsed with sterile DMEM and minced into fragments about 1 mm3, which were then incubated at 37°C for 4 hrs on an orbital shaker, with 1 mg/ml type II collagenase (Sigma-Aldrich, St. Louis, MO, USA) in PBS without Ca2+ and Mg2+. Dispersed cells were separated from non-digested tissue by the filtration through a 40-μm-diameter cell strainer (BD Falcon, Franklin, NJ, USA), harvested by centrifugation, and resuspended in DMEM supplemented with 10% foetal calf serum (Gibco, NY, USA), 100 UI/ml penicillin and 0.1 mg/ml streptomycin. Cell density was counted in a haemocytometer and viability was assessed using the Trypan blue. Cells were distributed in 25 cm2 culture flasks at a density of 1 × 105 cells/cm2 and maintained at 37°C in a humidified atmosphere (5% CO2) until becoming semiconfluent (usually 4 days after plating). Culture medium was changed every 48 hrs. Cultured cells were examined by phase contrast microscope, under an inverted Olympus phase contrast microscope (1 × 51).

RNA isolation and preparation

Mouse lung telocytes were isolated after 5 days of culture. Mouse MSCs and fibroblasts were cultured and collected on days 5 and 10 respectively. RNA preparation was performed using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and the RNeasy kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions, including a DNase digestion treatment. The amount and quality of RNA were measured by NanoDrop-1000 spectrophotometer and with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

RNA labelling, array hybridization and DNA microarray

The Mouse 4 × 44K Gene Expression Array (Agilent, Shanghai, China) with about 39,000+ mouse genes and transcripts represented with public domain annotations was applied for the analysis of gene profiles of mouse lung telocytes, MSCs and fibroblasts. Sample labelling and array hybridization were performed according to the protocol of One-Color Microarray-Based Gene Expression Analysis (Agilent Technology). Briefly, 1 μg of total RNA from each sample was linearly amplified and labelled with Cy3-dCTP. The labelled cRNAs were purified by RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labelled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. One microgram of each labelled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25 × Fragmentation Buffer, and heated at 60°C for 30 min. 55 μl 2 × GE Hybridization buffer was added to dilute the labelled cRNA. Hundred microlitre of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hrs at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with the Agilent DNA Microarray Scanner (part number G2505B).

Data analysis

The acquired array images were analysed with Agilent Feature Extraction software (version 10.7.3.1). Quality normalization and subsequent data processing were performed with the GeneSpring GX v11.5.1 software package. The genes detected in all samples were chosen for further data analysis. Differentially expressed genes were identified through Fold Change filtering and hierarchically clustered by the Agilent GeneSpring GX software (version 11.5.1). Gene ontology and String Network analyses were performed with the standard enrichment computation method to study the relation among variant proteins expressed by variant genes. Fisher's exact test was used to find more overlaps between the descriptive list and the GO annotation list than would be expected by chance. The P-value denoted the significance of GO terms enrichment in the descriptive genes.

Results and discussions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

The quality of gene data after filtering and the distribution of data sets were assessed and visualized by Box-Plot. There was no significant difference in distributions of log2 ratios among TCs, MSCs and fibroblasts (Figure S1).

Gene expression analysis

Gene expression array data show that more than 500 genes are at least 10 times higher expressed in TCs comparing with either MSCs or fibroblasts (Table 1). Several genes are found 100 times up-regulated in TCs versus fibroblasts (Cdh2, Cyba, Rnf128, Dpysl3, Fstl1, Rbp1, Gm12892, Cdh2, Aldh1a1, Gm5864) or MSCs (Rbp1 and Glipr1; Table 1A). Additional genes are significantly overexpressed in TCs comparing with MSCs or fibroblasts (Table 1B). Table 2 is a summary of genes found to be down-regulated in TCs. Although many genes are less expressed in TCs comparing with MSCs or fibroblasts, very few are found at least 100 times down-regulated in TCs. Table 2A and B show the genes with known functions that are found at least 30 times down-regulated specifically in TCs comparing with MSCs and fibroblasts.

Table 1. Summary of genes expressed preferentially in TCs, as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
Compared pairs/fold up-regulated>2>10>30>100
TCs vs. MSCs292150017444
TCs vs. Fbs317366129585
(A) Genes up-regulated more than 100-folds in telocytes (TCs) as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
TCs vs. FbsTCs vs. MSCs
GeneFoldsGeneFoldsGeneFolds
Ctgf6151Tm4sf1217Sprr1a2971
Sprr1a2593Sulf1212Cck1242
Myl91668Chi3l3204Wfdc2551
Tagln1545Vopp1198Serinc2527
Cck1206Mfhas1198Chi3l3369
Nid11143Myh14194Glipr1355
Sdpr1004Ogn185Eppk1284
Crlf1942Dsp182Trf259
Anxa8799Mmp10177Myh14246
Cd9718Khdrbs3175Gsta3244
Wfdc2660Atp1b1174Gpr56222
Sox4501Papss2171Cyb561210
Dhcr24496Gprc5c168Gprc5c204
Timp3445Prl2c1165Tjp2202
Trim44410Gas6165Atp1b1194
Serpine1376Rbp1161Lyz1181
Marcksl1356Foxq1156Aldh1a2167
Hs6st2335Cblc149Gpx2152
Gpr56331Aldh1a2149Dsp150
Nrg1327Cdh2136Khdrbs3146
Trf306Crct1133Acp5143
Bmp4298Mmp3131Rbp1141
Cyba293Gpx2126Gprc5c137
Thy1280Gprc5c125Clu131
Lrrc32278Fstl1125Tmc4128
Rab34269Lama2120Acp5114
Dpysl3263Tjp2117Epb4.1l4b114
Decr1256Igsf9116Mfsd6109
Gsta3240Bcr110Cblc107
Evl237Lce1i108Acta1105
Tmem45a233Rnf128107F11r101
Aldh1a1225Klhl13106  
Fzd1223Echdc2103  
Cryab219Trim16101  
Lyz1217    
(B) Genes up-regulated between 30- and 100-folds in telocytes (TCs) as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
TCs vs. FbsTCs vs. MSCs
GeneFoldsGeneFoldsGeneFoldsGeneFolds
Wnt11100Letmd147Pdgfb97Pcgf536
F398Rpgrip146Aldh1a193Fxyd336
Pdgfb97Trp53i1146Itpa90Ctsk35
Fxyd694Hebp246Fxyd687Ctgf35
Fhl294Dkk345Tns179Ckb35
Nox493Cryab45St1478Lama535
Ptprf93Pvrl344Lce1i78Evpl34
Tgfb1i193P2rx244Crip177Col4a634
Ddah192A2bp143S100a1676Chst434
Cd9992Cyba43Klhl1374Apoe33
Irx187Cyr6142Tnk174Pik3r633
Pdlim186Cobl42Mmrn274Panx133
Epb4.1l386Pdlim341Rpgrip172Rnu1b633
Tuft186Map3k941Gsta371Nppb33
Msln83Tlr1341Endod171Sema6a33
Panx183Tjp341Scnn1a69Serpinb6b33
Clic583Grhl241Tacstd269Apoc232
Ggh83Sdcbp241Mboat168Vill32
Bst179Cd1441Gas667Irx131
Mansc179Krt1741Dapk266Isyna130
Slco3a178Loxl240Cpsf3l65Map3k930
Tnfsf1578Cald140Plac964  
Il678Brsk140Krtcap363  
Saa377Ppp1r9a40Mapkapk362  
Fgd377Stxbp239Tbc1d262  
Echdc277Rab2539Tbc1d261  
Mapk1375Stfa339Cytip60  
Tnfrsf11b75Cald139Spint160  
Basp170Brsk139Lcp160  
Slc4a1170Lmo738Grhl259  
Bst169Timp138Wnt1159  
F369Slc35f538Rarb57  
Ubqln269Id138Ctsh57  
Adam868Rnf13037Mansc156  
Parp867Serping137Mmp1056  
Sox467Csf2rb37Ephx155  
Egfl766Olfr138337Coro1a55  
Gsta364Sulf237Rpgrip153  
Tnk164Nhsl137Cd3653  
Fzd264Itm2a37Klf652  
Gpm6b63Slamf937Heph52  
Cgn62Cacnb336Nipsnap150  
Unc13b61Spint136Arhgef1650  
Celsr161Tuba1a36Atp9a50  
Mmrn261Rgs1736Bst149  
Dok261Col4a636Adm49  
Tpm260Tpm136Elovl749  
Ppfibp260Scnn1a35Fcgr2b49  
Npr360Sirpb1a35Tjp348  
Cpsf3l59Clic335Hic148  
Peg1359Klf1335Rab2547  
Arhgef1659Lrrc3335Serpine147  
Lass358Gprc5a35Abcc347  
Dapk258Sgk135Psmg247  
Plac958Ankrd134Col4a446  
Msrb258Mid1ip134Csf2rb245  
Ckb57Coro1a34Tmem8845  
Fam83h57Cd24834Cd9745  
Vcan56Acta134Ppl45  
Acp556Inadl33P2rx244  
Csf1r56Sesn333A2bp143  
Ap1s356Evpl33Akr1c1343  
Pbx356C333St6gal142  
Tmc456Tpm233Efnb141  
Rpgrip155Pilra33Dok241  
Ctsw55H1933Adam841  
Wwc154Pfkfb332Clic541  
Glipr154Zfhx332Sh3bgr40  
Hes654Fcer1g32Fgd339  
Tacstd254Stab 132Csf2rb39  
Nsd154Col1a232Olfr138339  
Cyb56153Igfbp231H1939  
Fcgr2b53Vcam131Sirpb1a39  
Cdc42ep553Chpf231Fcer1g38  
Mdfi52Nppb31Slc39a438  
Galntl452Ccl27a31Fcgr438  
Anxa852Ccl231Sh3bgr38  
Plcg252Tnfaip331Slc22a1838  
Col4a451Fnbp1l31Alcam38  
Acp550Marveld331Stfa338  
Btg349Spint230Ppfibp237  
Ltbp248Sh3bgr30Clic337  
Cd9347Adamts930Csf1r37  
Gadd45b47Abcc330Spint236  
Afap1l247Lcp130Lamc236  
Table 2. Summary of genes less expressed in TCs, as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
Compared pairs/fold down-regulated>2>10>30>100
TCs vs. MSCs4365175325
TCs vs. Fbs54513266316
(A) Genes down-regulated more than 100-folds in telocytes (TCs) as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
TCs vs. FbsTCs vs. MSCs
GeneFoldsGeneFolds
Car6323Ccl5282
Odz4275Hoxc6146
Tenm4269Cdsn159
Pla2g2e253Ifi20363
Cdsn229Gdpd285
Glod5209  
Rarres2180  
Hoxc6152  
Ndufa4l2150  
Hoxc10133  
Rhd122  
Plin4113  
Gm2022105  
Car9102  
(B) Genes down-regulated between 30- and 100-folds in telocytes (TCs) as compared with mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
TCs vs. FbsTCs vs. MSCs
GeneFoldsGeneFoldsGeneFolds
Serpinb9f95Tbx1544Tbx1593
Foxg194Dmrtc1c242Hoxc1092
Mst188Igf2bp341Nkx2-584
Ifi20382Itk41Gbp372
Avil75Paip138Lpar467
Hsd17b1469Rps3a38Hoxb966
Acacb68Slx37Odz458
Angpt167Gchfr35Eif2s158
Csprs67Hc35Pde8b54
Gm495167Ptgir33Ebf346
Mtap1b65Accn232Angpt146
Serpinb9e59Masp232Rsad245
Cox6a259Cbr231Ifi202b45
Matn257Col5a330Fbln137
Pla2g2e54  Ifi20435
Nrxn349  Thbs235
Cbr249  Mx234
Ebf348  Ndufa4l234
Cldn1547  Tgfbr331
Ppargc1a45  Car631

Hierarchical cluster and gene ontology analyses

The hierarchical cluster of the genes with more than twofold changes among telocytes, MSCs and fibroblasts is shown in Figure 1. Remarkably, the MCSs and fibroblast gene expression profiles relate each other to higher extent than to TCs supporting the view that TCs have a distinct gene expression pattern. In fact this is an important additional proof that TCs and fibroblasts are different cells. The GO analysis indicates that the genes differentially expressed in TCs are mainly involved in development, in tissue and organ morphogenesis and in transport and maintenance of a biological compound to a specific location (Fig. 2A). In addition, many of the differentially expressed genes likely function in extracellular compartments (Fig. 2B) and may play roles in cell survival, growth and differentiation through autocrine and paracrine activity (Fig. 2C). The relationships, including direct (physical) and indirect (functional) associations, of those genes were analysed by String Network analysis (www.string-db.org). Among the 156 co-expressed genes, 46 genes were found to have certain interactions (Fig. 3).

image

Figure 1. Hierarchical cluster analysis of the differentially expressed genes among telocytes (TCs), mesenchymal stem cells (MSCs) and fibroblasts (Fbs).

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image

Figure 2. Gene ontology of the genes with at least twofolds difference among telocytes (TCs), mesenchymal stem cells (MSCs) and fibroblast (Fbs), analysed under following categories: Biological Processes (A), Cellular Components (B) and Molecular Function (C). (P ≤ 0.01).

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image

Figure 3. String Network of the proteins that are differentially expressed among telocytes (TCs), mesenchymal stem cells (MSCs) and fibroblast (Fbs). A group of 46 genes are found connected functionally. Strong associations are represented by thick lines.

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TCs are potentially involved in tissue remodelling and basement membrane homeostasis

A set of genes are specifically up- or down-regulated in TCs comparing with both fibroblasts and MSC (Table 3). As last two cell types are developmentally and functionally quite different, one being progenitors and the other differentiated, specialized cells, this set of genes should connect to the specific biological activities of TCs among the other stromal cells. Thus, we have found that several genes with roles in tissue remodelling and repair are significantly up-regulated in TCs (Tables 1A and 3): connective tissue growth factor (CTGF) [24, 25], Transgelin (Tagln) [26], Nidogen 1 (Nid1) [27, 28], tissue inhibitor of metalloproteinase 3 (TIMP3) [29], collagen type IV, alpha (Col4a4, Col4a6, Col4a5) [28, 30], Matrix Metallopeptidase 10 (Mmp10) [31-33], Matrix Metallopeptidase 3 (Mmp3) [31-33] and Retinol-binding protein 1 (RBP1). RBP1 (also known as CRABP-I, CRBP, CRBP1, CRBPI, RBPC) is required in tissue remodelling [34]. Regarding the molecular mechanisms, RBP1 delivers vitamin A to other cells through the plasma membrane protein STRA6 involved in JAK/STAT signalling and the intracellular metabolism of the vitamin [35]. Remarkably, two main components of basement membrane, Collagen type IV and Nidogen 1 are up-regulated in the cultured TCs comparing with both MSCs and fibroblasts. Moreover, TIMP3 is an extracellular matrix-anchored metalloproteinase inhibitor that acts specifically to increase vascular (endothelial) basement membrane stability [36, 37]. As TCs express Matrix Metalloproteases Mmp3 and Mmp10 also, it is likely that TCs are involved in both basement membrane assembly (stability) and surrounding extracellular matrix remodelling.

Table 3. Genes up- or down-regulated in telocytes (TCs) relative to both mesenchymal stem cells (MSCs) and fibroblasts (Fbs)
Gene nameTCs vs. FbsTCs vs. MSCs
Fold changeRegFold changeReg
Ctgf6150Up35Up
Mmp10177Up56Up
Mmp3131Up25Up
Col4a446Up51Up
Col4a634Up36Up
Col4a58Up32Up
Unc13b61Up7Up
Mapk1375Up13Up
Igsf9115Up3Up
Glipr154Up355Up
Clic583Up41Up
Myh14194Up245Up
Aldh1a1225Up92Up
Aldh1a2148Up167Up
Rbp1161Up141Up
Gprc5c125Up136Up
Gsta364Up70Up
Plac957Up63Up
Fgd377Up39Up
Dok260Up41Up
Scnn1a35Up68Up
Car6323Down31Down
Odz4275Down59Down
Oz/ten-m269Down56Down
Cdsn229Down153Down
Hoxc6152Down207Down
Ifi20382Down150Down

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Overall, the data indicate that TCs are clearly distinct from both MSCs and fibroblasts, and the gene signature of TCs suggests specific biological functions in (a) development and tissue morphogenesis, (b) biological compound transport and (c) extracellular matrix remodelling. It has been proposed that TCs play essential roles in angiogenesis given that TCs are frequently found in close vicinity of small vessels and express angiogenesis-related factors (VEGF, NO) and pro-angiogenic microRNAs [22]. The data presented here bring additional support to this view suggesting that TCs may also regulate vascular basement membrane remodelling as key step in vascular branching and de novo vessel formation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

The authors would like to thank Hongjian Gao, Department of Electronic Microscopy, Shanghai Medical College, Fudan University, for the technical assistance in TEM; Biomedical Research Center of Fudan University Zhongshan Hospital for technical supports and facility supplies. The work was supported by Shanghai Leading Academic Discipline Project (Project Number: B115), Fudan University (Distinguished Professor Grant), Shanghai Science & Technology Committee Grants for International Collaboration (11410708600), Project of Science and Technology Innovation Plan in Biomedicine, National Natural Science foundation of China (H0108) and National Natural Key Science foundation of China: ‘Lung injury of ischemic reperfusion’ (30930090). This study is partially supported by the Sectorial Operational Programme Human Resources Development (SOPHRD), financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/64153 (to V.B.C) and by grant 350/2012 PN-II-ID-PCE-2011-3-0134 of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI (to L.M.P).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Method and Materials
  5. Results and discussions
  6. Concluding remarks
  7. Acknowledgements
  8. Conflict of interest
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
jcmm12052-sup-0001-FigS1.pptxapplication/mspowerpoint104KFigure S1 Box-Plot of Quality assessment of gene data after filtering. After normalization, the distributions of log2 ratios among all samples are nearly the same.

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