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Frontiers in Biology

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Front Biol    2010, Vol. 5 Issue (5) : 431-438    https://doi.org/10.1007/s11515-010-0900-1
RESEARCH ARTICLE
Screening for self-renewal factors by a combination of mRNA and CGH microarray in human embryonic stem cells
Lei XIAO1,2(), Lixiazi HE2, Saul J. SHARKIS1()
1. Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; 2. Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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Abstract

Human embryonic stem cells (hESCs) undergo self-renewal while maintaining pluripotency. However, the molecular mechanism that demonstrates how these cells maintain their undifferentiated state and how they self-renew is poorly understood. Here, we characterized an aneuploidy H1 hESC subline (named H1T) using karyotyping and comparative genomic hybridization (CGH) microarray. Because the H1T hESC line displays a self-renewal advantage while maintaining an undifferentiated state, we speculated that the expression patterns of specific genes which are related to pluripotency or differentiation were altered; therefore, we attempted to screen for molecules that are propitious for maintenance of stemness by performing a combination of mRNA and CGH microarray analysis which compared the aneuploidy H1T hESC subline versus the euploid H1 hESC line. It is discovered that some genes are up-regulated in H1T hESC subline such as TBX2 and Wnt3, while some are downregulated, for example, Fbxo7 and HMG2L1. Our findings should fascilitate the study of the complex signaling network which maintains hESC pluripotency and function.
Keywords self-renewal      mRNA microarray      comparative genomic hybridization (CGH) microarray     
Corresponding Author(s): XIAO Lei,Email:leixiao@sibs.ac.cn; SHARKIS Saul J.,Email:ssharkis@jhmi.edu   
Issue Date: 01 October 2010
 Cite this article:   
Lei XIAO,Lixiazi HE,Saul J. SHARKIS. Screening for self-renewal factors by a combination of mRNA and CGH microarray in human embryonic stem cells[J]. Front Biol, 2010, 5(5): 431-438.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-010-0900-1
https://academic.hep.com.cn/fib/EN/Y2010/V5/I5/431
Fig.1  Karyotype of H1T hESC. (a) G-banding analysis shows that chromosome 10 is deleted below band q24.3. (b) Chromosome 17 has a large addition of unidentified material at the tip of the short arm. (c and f) Chromosome 22 appears to be normal. (d and e) SKY analysis demonstrates the deleted chromosome 10; the chromosome 17 with additional material on the short arm which is mostly chromosome 17 material, with a small bit of chromosome 10 on the tip.
Fig.1  Karyotype of H1T hESC. (a) G-banding analysis shows that chromosome 10 is deleted below band q24.3. (b) Chromosome 17 has a large addition of unidentified material at the tip of the short arm. (c and f) Chromosome 22 appears to be normal. (d and e) SKY analysis demonstrates the deleted chromosome 10; the chromosome 17 with additional material on the short arm which is mostly chromosome 17 material, with a small bit of chromosome 10 on the tip.
Fig.2  CGH microarray could not detect the reciprocal translocation between chromosomes 10 and 17(a, b). CGH microarray revealed the addition of chromosome 17q (b), a small deletion of 17p (b), and a small deletion of chromosome 22q(c).
Fig.2  CGH microarray could not detect the reciprocal translocation between chromosomes 10 and 17(a, b). CGH microarray revealed the addition of chromosome 17q (b), a small deletion of 17p (b), and a small deletion of chromosome 22q(c).
addition and upFold change
WNT3NM_030753wingless-type MMTV integration site family, member 3 (WNT3), mRNA4.51
GOSR2NM_004287golgi SNAP receptor complex member 2 (GOSR2), transcript variant A,1.89
ATP5G1NM_005175ATP synthase, H+ transporting, mitochondrial F0 complex,2.02
FLJ13855NM_023079Homo sapiens hypothetical protein FLJ13855 (FLJ13855), mRNA1.69
IMP-1NM_006546Homo sapiens IGF-II mRNA-binding protein 1 (IMP-1), mRNA1.89
PHBNM_002634Homo sapiens prohibitin (PHB), mRNA2.25
SPOPNM_003563Homo sapiens speckle-type POZ protein (SPOP), mRNA1.61
SLC35B1NM_005827Homo sapiens solute carrier family 35, member B1 (SLC35B1), mRNA1.70
MYST2NM_007067Homo sapiens MYST histone acetyltransferase 2 (MYST2), mRNA1.68
COL1A1NM_000088Homo sapiens collagen, type I, alpha 1 (COL1A1), mRNA2.94
PRO1855NM_018509Homo sapiens hypothetical protein PRO1855 (PRO1855), mRNA1.82
NME1NM_000269non-metastatic cells 1, protein (NM23A) expressed in (NME1), mRNA1.89
NME2NM_002512non-metastatic cells 2, protein (NM23B) expressed in (NME2), nuclear gene encoding mitochondrial protein, mRNA1.74
CGI-48NM_016001CGI-48 protein (CGI-48), mRNA1.77
MRPS23NM_016070mitochondrial ribosomal protein S232.72
FLJ20345NM_017777Homo sapiens hypothetical protein FLJ20345 (FLJ20345), mRNA1.73
TEX14NM_031272Homo sapiens testis expressed sequence 14 (TEX14), mRNA6.77
RAD51CNM_002876RAD51 homolog C (S. cerevisiae) (RAD51C), transcript variant 2, mRNA2.57
TBX2NM_005994Homo sapiens T-box 2 (TBX2), mRNA3.29
CYB561NM_001915cytochrome b-561 (CYB561), mRNA1.81
LOC51204NM_016360clone HQ0477 PRO0477p (LOC51204), mRNA2.31
FTSJ3NM_017647Homo sapiens FtsJ homolog 3 (E. coli) (FTSJ3), mRNA1.95
PSMC5NM_002805proteasome (prosome, macropain) 26S subunit, ATPase, 5 (PSMC5)2.07
SMARCD2NM_003077SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 2 (SMARCD2), mRNA1.77
HT008NM_018469uncharacterized hypothalamus protein HT008 (HT008), mRNA1.97
LOC90799NM_138363Homo sapiens hypothetical protein BC009518 (LOC90799), mRNA1.68
FLJ14775NM_032837Homo sapiens hypothetical protein FLJ14775 (FLJ14775), mRNA1.73
AK027166AK027166Homo sapiens cDNA: FLJ23513 fis, clone LNG038691.55
RPL38NM_000999Homo sapiens ribosomal protein L38 (RPL38), mRNA1.64
SLC9A3R1NM_004252solute carrier family 9, isoform 3 regulatory factor 1 (SLC9A3R1), mRNA1.56
ICT1NM_001545Homo sapiens immature colon carcinoma transcript 1 (ICT1), mRNA1.95
SLC25A19NM_021734solute carrier family 25, member 19 (SLC25A19), mRNA2.03
HCNGPNM_013260transcriptional regulator protein (HCNGP), mRNA1.71
MRPL38NM_032478mitochondrial ribosomal protein L381.94
SRP68NM_014230signal recognition particle 68kDa (SRP68), mRNA1.59
PTDSRNM_015167phosphatidylserine receptor (PTDSR), mRNA1.96
TIMP2NM_003255Homo sapiens tissue inhibitor of metalloproteinase 2 (TIMP2), mRNA2.81
MGC25062NM_019020Homo sapiens hypothetical protein FLJ20748 (FLJ20748), mRNA1.66
raptorNM_020761Homo sapiens raptor (raptor), mRNA1.71
BAIAP2NM_017450BAI1-associated protein 2 (BAIAP2), transcript variant 1, mRNA2.45
HGSNM_004712hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), mRNA1.80
P4HBNM_000918procollagen-proline, 2-oxoglutarate 4-dioxygenase, beta polypeptide2.40
THOC4NM_005782Homo sapiens THO complex 4 (THOC4), mRNA3.02
TIMP2NM_016476Homo sapiens tissue inhibitor of metalloproteinase 2 (TIMP2), mRNA2.81
SIRT7NM_016538sirtuin 71.89
RAC3NM_005052ras-related C3 botulinum toxin substrate 3 rho family, small GTP binding protein Rac32.35
PP3111NM_022156Homo sapiens PP3111 protein (PP3111), mRNA2.03
HOXB7Homo sapiens homeo box B7, mRNA (cDNA clone MGC:21362 IMAGE:4413080), complete cds
GRB2Homo sapiens growth factor receptor-bound protein 2 (GRB2), mRNA
survivinHomo sapiens baculoviral IAP repeat-containing 5 (survivin), mRNA (cDNA clone IMAGE:3506845), partial cds
Tab.1  Genes effective in anti-apoptosis and pluripotency maintenance
Deletion and downfold down
MYH9NM_002473myosin, heavy polypeptide 9, non-muscle (MYH9), mRNA1.93
RAC2NM_002872rho family, small GTP binding protein Rac22.08
TSTNM_003312thiosulfate sulfurtransferase, nuclear gene encoding mitochondrial protein,3.24
SYN3NM_003490synapsin III (SYN3), transcript variant IIIa, mRNA4.07
EIF3S7NM_003753eukaryotic translation initiation factor 3, subunit 7 zeta, 66/67kDa (EIF3S7),1.72
LARGENM_004737Homo sapiens like-glycosyltransferase (LARGE), transcript variant 1, mRNA2.40
HMG2L1NM_005487Homo sapiens high-mobility group protein 2-like 1 (HMG2L1), mRNA2.18
YWHAENM_006761tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (YWHAE),1.59
RABL4NM_006860RAB, member of RAS oncogene family-like 4 (RABL4)2.32
FBXO7NM_012179Homo sapiens F-box only protein 7 (FBXO7), mRNA1.73
HSPC117NM_014306Homo sapiens hypothetical protein HSPC117 (HSPC117), mRNA1.68
ZNF278NM_014323Homo sapiens zinc finger protein 278 (ZNF278), transcript variant 11.81
GEMIN4NM_015721Homo sapiens gem (nuclear organelle) associated protein 4 (GEMIN4)1.69
CGI-150NM_016080Homo sapiens CGI-150 protein (CGI-150), mRNA1.73
FLJ10581NM_018146Homo sapiens putative RNA methyltransferase (FLJ10581), mRNA2.37
EIF4ENIF1NM_019843eukaryotic translation initiation factor 4E nuclear import factor 1 (EIF4ENIF1),2.49
MPSTNM_021126Homo sapiens mercaptopyruvate sulfurtransferase (MPST), mRNA2.02
CT120NM_024792membrane protein expressed in epithelial-like lung adenocarcinoma (CT120)1.78
Tab.2  Genes having effects on facilitating differentiation or inhibiting self-renewal
1 Ambrosini G, Adida C, Altieri D C (1997). A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med , 3(8): 917–921
doi: 10.1038/nm0897-917
2 Amit M, Shariki, C. Margulets V, Itskovitz-Eldor J (2004). Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod , 70(3): 837–845
doi: 10.1095/biolreprod.103.021147
3 Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying Q L, Smith A (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell , 135(7): 1287–1298
4 Chang Y F, Cheng C M, Chang L K, Jong Y J, Yuo C Y (2006). The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination. Biochem Biophys Res Commun , 342(4): 1022–1026
doi: 10.1016/j.bbrc.2006.02.061
5 Chen G, Liu D, Tadokoro M, Hirochika R, Ohgushi H, Tanaka J, Tateishi T (2004). Chondrogenic differentiation of human mesenchymal stem cells cultured in a cobweb-like biodegradable scaffold. Biochem Biophys Res Commun , 322(1): 50–55
doi: 10.1016/j.bbrc.2004.07.071
6 Coumoul X, Li W, Wang R H, Deng C (2004). Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system. Nucleic Acids Res , 32(10): e85
doi: 10.1093/nar/gnh083
7 Draper J S, Smith K, Gokhale P, Moore H D, Maltby E, Johnson J, Meisner L, Zwaka T P, Thomson J A, Andrews P W (2004). Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol , 22(1): 53–54
doi: 10.1038/nbt922
8 Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X, Cheng L (2005). Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells , 23(10): 1489–1501
doi: 10.1634/stemcells.2005-0034
9 Guo Y, Mantel C, Hromas R A, Broxmeyer H E (2008). Oct-4 is critical for survival/antiapoptosis of murine embryonic stem cells subjected to stress: effects associated with Stat3/survivin. Stem Cells , 26(1): 30–34
doi: 10.1634/stemcells.2007-0401
10 He Z, Feng L, Zhang X, Geng Y, Parodi D A, Suarez-Quian C, Dym M (2005). Expression of Col1a1, Col1a2 and procollagen I in germ cells of immature and adult mouse testis. Reproduction , 130(3): 333–341
doi: 10.1530/rep.1.00694
11 Ikeda M, Hikita T, Taya S, Uraguchi-Asaki J, Toyo-oka K, Wynshaw-Boris A, Ujike H, Inada T, Takao K, Miyakawa T, Ozaki N, Kaibuchi K, Iwata N (2008). Identification of YWHAE, a gene encoding 14-3-3epsilon, as a possible susceptibility gene for schizophrenia. Hum Mol Genet , 17(20): 3212–3222
doi: 10.1093/hmg/ddn217
12 Jacobs J J, Keblusek P, Robanus-Maandag E, Kristel P, Lingbeek M, Nederlof P M, van Welsem T, van de Vijver M J, Koh E Y, Daley G Q, van Lohuizen M (2000). Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers. Nat Genet , 26(3): 291–299
doi: 10.1038/81583
13 Lee S K, Park S O, Joe C O, Kim Y S (2007). Interaction of HCV core protein with 14-3-3epsilon protein releases Bax to activate apoptosis. Biochem Biophys Res Commun , 352(3): 756–762
doi: 10.1016/j.bbrc.2006.11.098
14 Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson R E, Schulze E N, Song H, Hsieh C L, Pera M F, Ying Q L (2008). Germline competent embryonic stem cells derived from rat blastocysts. Cell , 135(7): 1299–1310
15 Maitra A, Arking D E, Shivapurkar N, Ikeda M, Stastny V, Kassauei K, Sui G, Cutler D J, Liu Y, Brimble S N, Noaksson K, Hyllner J, Schulz T C, Zeng X, Freed W J, Crook J, Abraham S, Colman A, Sartipy P, Matsui S, Carpenter M, Gazdar A F, Rao M, Chakravarti A (2005). Genomic alterations in cultured human embryonic stem cells. Nat Genet , 37(10): 1099–1103
doi: 10.1038/ng1631
16 Mitalipova M M, Rao R R, Hoyer D M, Johnson J A, Meisner L F, Jones K L, Dalton S, Stice S L (2005). Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol , 23(1): 19–20
doi: 10.1038/nbt0105-19
17 Mitelman F (1995). An International System for Human Cytogenetic Nomenclature in ISCN. Basel, Karger AG
18 O’Connor D S, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio P C, Altieri D C (2000). Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci U S A , 97(24): 13103–13107
doi: 10.1073/pnas.240390697
19 Pease S, Braghetta P, Gearing D., Grail D, Williams R L (1990). Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF). Dev Biol , 141(2): 344–352
doi: 10.1016/0012-1606(90)90390-5
20 Prakash O, Yunis J J (1984). High resolution chromosomes of the t(9;22) positive leukemias. Cancer Genet Cytogenet , 11(4): 361–367
doi: 10.1016/0165-4608(84)90015-3
21 Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou A H (2004). Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med , 10(1): 55–63
doi: 10.1038/nm979
22 Schr?ck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith M A, Ning Y, Ledbetter D H, Bar-Am I, Soenksen D, Garini Y, Ried T (1996). Multicolor spectral karyotyping of human chromosomes. Science , 273(5274): 494–497
doi: 10.1126/science.273.5274.494
23 Shtivelman E, Lifshitz B, Gale R P, Canaani E (1985). Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature , 315(6020): 550–554
doi: 10.1038/315550a0
24 Silva C, Wood J R, Salvador L, Zhang Z, Kostetskii I, Williams C J, Strauss J F 3rd (2009). Expression profile of male germ cell-associated genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone. Mol Reprod Dev , 76(1): 11–21
doi: 10.1002/mrd.20925
25 Thomson J A, Itskovitz-Eldor J, Shapiro S S, Waknitz M A, Swiergiel J J, Marshall V S, Jones J M (1998). Embryonic stem cell lines derived from human blastocysts. Science , 282(5391): 1145–1147
doi: 10.1126/science.282.5391.1145
26 Vance K W, Carreira S, Brosch G, Goding C R (2005). Tbx2 is overexpressed and plays an important role in maintaining proliferation and suppression of senescence in melanomas. Cancer Res , 65(6): 2260–2268
doi: 10.1158/0008-5472.CAN-04-3045
27 Wang Y, Hai T, Liu Z, Zhou S, Lv Z, Ding C, Liu L, Niu Y, Zhao X, Tong M, Wang L, Jouneau A, Zhang X, Ji W, Zhou Q (2010). HSPC117 deficiency in cloned embryos causes placental abnormality and fetal death. Biochem Biophys Res Commun , 397(3): 407–412
doi: 10.1016/j.bbrc.2010.05.105
28 Xiao L, Yuan X, Sharkis S J (2006). Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells , 24(6): 1476–1486
doi: 10.1634/stemcells.2005-0299
29 Xu C, Inokuma M S, Denham J, Golds K, Kundu P, Gold J D, Carpenter M K (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol , 19(10): 971–974
doi: 10.1038/nbt1001-971
30 Yamada M, Ohkawara B, Ichimura N, Hyodo-Miura J, Urushiyama S, Shirakabe K., Shibuya H (2003). Negative regulation of Wnt signalling by HMG2L1, a novel NLK-binding protein. Genes Cells , 8(8): 677–684
doi: 10.1046/j.1365-2443.2003.00666.x
31 Ying Q L, Nichols J, Chambers I, Smith A (2003). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell , 115(3): 281–292
32 Zeng X, Chen J, Liu Y, Luo Y, Schulz T C, Robins A J, Rao M S, Freed W J (2004). BG01V: a variant human embryonic stem cell line which exhibits rapid growth after passaging and reliable dopaminergic differentiation. Restor Neurol Neurosci , 22(6): 421–428
33 Zhou J, Hu G, Wang X (2010). Repression of smooth muscle differentiation by a novel high mobility group box-containing protein, HMG2L1. J Biol Chem , 285(30): 23177–23185
doi: 10.1074/jbc.M110.109868
[1] Jin He. Function of Polycomb repressive complexes in stem cells[J]. Front. Biol., 2016, 11(2): 65-74.
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