{"paper_id":"b4b6ee16-d6fa-4045-bede-fb09fa1e4653","body_text":"392 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009\nINTRODUCTION\nHuman endometrium undergoes cycli-\ncal processes of growth, differentiation,\nshedding, and regeneration as part of the\nmenstrual cycle during the reproductive\nlife of women (1).\nEndometriosis is a multifactorial\n estrogen-dependent disease that affects\n5% to 10% of women of reproductive\nage in the Western countries. Its defining\nfeature is the presence of endometrium-\nlike tissue in sites outside the uterine cav-\nity, primarily on the pelvic peritoneum\nand ovaries (2).\nEndometriosis can originate from\nanatomical or biochemical aberrations of\nuterine function. Theories on the histo-\ngenesis of endometriosis belong to five\ncategories: coelomic metaplasia, retro-\ngrade menstruation, embryonic cell rest,\ninduction, and lymphatic and vascular\ndissemination (3).\nMany studies thus far have focused on\nthe biomolecular and cellular characteris-\ntics of endometriosis compared to endo -\nmetrium and with the possible molecular\nmechanisms at the basis of the develop-\nment of endometriotic lesions. Among\nthese investigations, of particular interest\nis a recent analysis revealing a list of 22\nmicroRNAs differentially expressed in\npaired ectopic and eutopic endometrial\ntissues, which could contribute to en-\ndometriosis progression through their\ncognate target mRNAs (4). Other studies\nhighlighted a differential expression of\nthe genes SF1 and estrogen receptor beta\nin endometriotic tissue compared with\nendometrium. Results indicated that ex-\npression was primarily controlled by a\nmethylation-dependent epigenetic mech-\nanism (5,6). In addition, various chromo-\nsomal aberrations have been reported in\nendometriotic samples and in ovarian\ncarcinoma (7).\nDifferences in stromal cell migration, in-\nflammatory markers, and other pathways\nbetween eutopic and ectopic endometrial\ntissues have been also highlighted (8).\nIt should also be mentioned that en-\ndometriosis may have a genetic basis, be-\ncause its incidence in relatives of affected\nwomen is much higher than the incidence\nin women without a family history (9).\nStem cells are increasingly becoming\nthe focus of many areas of biomedical re-\nsearch. Stem cells are rare undifferentiated\ncells present in virtually all adult tissues\nand organs. These cells retain high prolif-\nerative, self-renewal, and differentiation\npotential. The number of stem cells in\nadult tissues is actively regulated through\na strict balance between cell proliferation,\ncell differentiation, and cell death (10). Re-\ncent studies revealed the presence of\nExpression Pattern of Stemness-Related Genes in Human\nEndometrial and Endometriotic Tissues\nAmalia Forte,1 Maria Teresa Schettino,2 Mauro Finicelli,1 Marilena Cipollaro,1 Nicola Colacurci,2\nLuigi Cobellis,2 and Umberto Galderisi1\nDepartments of 1Experimental Medicine and 2Gynaecology, Obstetrics and Reproductive Medicine, Second University of Naples, Italy\nEndometriosis is a chronic disease characterized by the presence of ectopic endometrial tissue outside of the uterus with mixed\ntraits of benign and malignant pathology. In this study we analyzed in endometrial and endometriotic tissues the differential e x-\npression of a panel of genes that are involved in preservation of stemness status and consequently considered as markers of stem\ncell presence. The expression profiles of a panel of 13 genes ( SOX2, SOX15, ERAS, SALL4, OCT4, NANOG, UTF1, DPPA2, BMI1, GDF3,\nZFP42, KLF4, TCL1) were analyzed by reverse transcription–polymerase chain reaction in human endometriotic (n = 12) and en-\ndometrial samples (n = 14). The expression of SALL4 and OCT4 was further analyzed by immunohistochemical methods. Genes\nUTF1, TCL1, and ZFP42 showed a trend for higher frequency of expression in endometriosis than in endometrium (P < 0.05 for UTF1),\nwhereas GDF3 showed a higher frequency of expression in endometrial samples. Immunohistochemical analysis revealed that\nSALL4 was expressed in endometriotic samples but not in endometrium samples, despite the expression of the corresponding\nmRNA in both the sample groups. This study highlights a differential expression of stemness-related genes in ectopic and eutopic\nendometrium and suggests a possible role of SALL4-positive cells in the pathogenesis of endometriosis.\n© 2009 The Feinstein Institute for Medical Research, www.feinsteininstitute.org\nOnline address: http://www.molmed.org\ndoi: 10.2119/molmed.2009.00068\nAddress correspondence and reprint requests to Luigi Cobellis, Department of Gynae-\ncology, Obstetrics and Reproductive Medicine, Second University of Naples, Largo\nMadonna delle Grazie, 1-80138 Naples, Italy. Phone: +39-081-5665608; Fax +39-081-\n5665610; E-mail: luigi.cobellis@unina2.it.\nSubmitted May 27, 2009; Accepted for publication August 10, 2009; Epub\n(www.molmed.org) ahead of print August 10, 2009.\n\nRESEARCH ARTICLE\nMOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 393\nadult stem cells in endometrium. In par-\nticular, work by Chan et al. (11) revealed\nclonogenic stromal and epithelial cells in\nhuman endometrium, possibly indicating\nthe presence of stem cells.\nResults of two studies (12,13) using the\nlabel-retaining cell approach suggested\nthe presence of stem cells in murine en-\ndometrium. Other studies, also con-\nducted in murine models, demonstrated\nthat stem cells in endometrium derive\nfrom bone marrow (14).\nThe presence of stem cells in en-\ndometrium has been demonstrated\nmainly through the analysis of their sur-\nface markers, their clonogenic properties,\nand their differentiation ability.\nEndometriosis can evolve into ovarian\ncancer (3,15) and other malignant dis-\neases in which stem cells could play a\nrole, as recently demonstrated (16). The\nrelationship of endometriosis and ovar-\nian cancer has been demonstrated both\nby epidemiological studies and by com-\nmon genetic alterations (3).\nStudies on transcriptional profiling of\nstem cells allowed a preliminary identifi-\ncation of stemness-related genes actively\ninvolved in the control of stem cell prop-\nerties, such as self-renewal ability and re-\ntention of an uncommitted state. Initially,\ngenes that control stemness were identi-\nfied in embryonic stem cells (17,18). In\nadult stem cells, some embryonal stem-\nness genes are not expressed.\nIn this study we aimed to detect the\nexpression of a panel of 13 genes consid-\nered as stem cell markers in eutopic en-\ndometrium and in endometriotic tissue,\nthrough analysis at the mRNA level for\nall the 13 genes and verification of the\ndata at the protein level for 2 of them.\nThe 13 genes were selected on the basis\nof data reported in the currently avail-\nable literature.\nAmong these genes, BMI1 (BMI1 poly-\ncomb ring finger oncogene) plays a central\nrole in the inheritance of stemness. BMI1\nbelongs to the polycomb group (PcG)\ngenes and is involved in the maintenance\nof cellular memory through epigenetic\nchromatin modifications. Recent studies\nhave implicated a role for PcG genes in\nthe self-renewal of stem cells, a process\nin which cellular memory is maintained\nthrough cell  division (19). ERAS (ES cell\nexpressed Ra) encodes a Ras- membrane\nprotein involved in proliferation and tu-\nmorigenicity of embryonic stem cells\n(20). TCL1 (T-cell leukemia/ lymphoma 1A)\nis an oncogene involved in regulation of\nproliferation of embryonic stem cells and\nis a downstream gene of OCT4 (POU\nclass 5 homeobox 1 [POU5F1, also known\nas OCT4]) (21). UTF1 (undifferentiated em-\nbryonic cell transcription factor 1) encodes\na tightly DNA-associated protein with\ntranscriptional repressor activity and is\nexpressed in embryonic pluripotent stem\ncells (22). All the other genes we ana-\nlyzed, including OCT4, SOX2 (SRY [sex\ndetermining region Y]-box 2), SOX15 (SRY\n[sex determining region Y]-box 15),\nNANOG (Nanog homeobox), SALL4 (sal-\nlike 4), DPP A2(developmental pluripotency\nassociated 2), GDF3 (growth differentiation\nfactor 3), ZFP42 (zinc finger protein 42 ho-\nmolog), and KLF4 (Kruppel-like factor 4),\ncode for transcription factors for genes\ninvolved in the preservation of stem cell\npluripotency (see also Supplementary\nFile 1 for additional references specific\nfor stemness-related genes).\nOur results highlight the expression of\nstem cell markers both in endometrial\nand endometriotic tissues, suggesting\nthat stem cells may play a role in disease\nprogression.\nMATERIALS AND METHODS\nPatients and Samples\nClinical samples of endometrial and\nendometriotic tissues were collected\nfrom 26 patients (endometrial tissues\nfrom n = 14 patients aged 29–58 years,\nmean 46.9 years; endometriosis samples\nfrom n = 12 patients, aged 24–46 years,\nmean 34.4 years) at the Department of\nGynaecology, Obstetrics and Reproduc-\ntive Medicine of the Second University\nof Naples. The patients were undergoing\nhysterectomy, laparoscopy, or laparo-\ntomy for benign pathologies. Informed\nwritten consent was obtained from each\npatient. Surgery was performed irrespec-\ntive of the day of the patient’s menstrual\ncycle. The patients had never received\nany hormonal treatment before surgery.\nAfter surgery\n, endometrial biopsies\nand excised ovarian endometriotic le-\nsions were formaldehyde fixed, and\nhematoxylin-stained cross sections were\nanalyzed by experienced histopatholo-\ngists for assessment of the grade of en-\ndometriosis (I–IV) and for determination\nof the stage of the menstrual cycle (pro-\nliferative or secretory), referring to estab-\nlished histological criteria (23). The clini-\ncal characteristics of the patients and\nsamples are shown in Table 1.\nThe samples from each patient were ei-\nther snap frozen and stored at –80°C or\nfixed in buffered formaldelyde 4% (Sigma-\nAldrich, St. Louis, MO, USA) and embed-\nded in paraffin using standard techniques\nfor immunohistochemical (IHC) analysis.\nRNA Extraction and Reverse\nTranscription–Polymerase Chain\nReaction\nTotal RNA was extracted from frozen\ntissue samples using TRIzol (Molecular\nResearch Center, Cincinnati, OH, USA)\nand from paraffin-embedded tissues\n(RNeasy minikit; Qiagen, Valencia, CA,\nUSA) according to manufacturer’s instruc-\ntions. RNA was treated with DNase I (Am-\nbion, Austin, TX, USA) to remove DNA\ncontamination. RNA concentration was\nmeasured using a NanoDrop ND-1000\nspectrophotometer (NanoDrop Technolo-\ngies). RNA integrity was verified by elec-\ntrophoresis on denaturing 1% agarose gel.\nAbsence of residual genomic DNA was\nverified by polymerase chain reation\n(PCR) on total RNA without reverse\ntranscription (RT). Genomic human\nDNA was used as a positive control of\nPCR reactions.\ncDNA was generated from 200 ng of\neach RNA sample. RT was done at 42°C\nfor 1 h in the presence of random exam-\ners and Moloney-murine leukemia virus\nreverse transcriptase (Finnzymes, Espoo,\nFinland). GeneBank sequences for human\nmRNAs SOX2, SOX15, ERAS, SALL4,\nOCT4, NANOG, UTF1, DPP A2, BMI1,\nGDF3, ZFP42, KLF4, TCL1 and Primer Ex-\n\n394 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009\nSTEM CELLS AND ENDOMETRIOSIS\npress software (Applied Biosystems, Fos-\nter City, CA, USA) were used to design\nprimer pairs for the genes and the house\nkeeping gene GAPDH. Primer sequences\nare listed in Table 2. They were chosen to\nyield 100–150 bp. Each PCR was repeated\nfor 35 cycles. PCR products were vali-\ndated by running the PCR products on\nagarose gel to confirm a single band.\nEach RT-PCR reaction was repeated at\nleast three times. A semiquantitative\nanalysis of mRNA levels was performed\nby the GEL DOC UV system (Bio-Rad,\nHercules, CA, USA) on agarose gels\ncontaining the GelStar nucleic acid gel\nstain (Lonza, Basel, Switzerland), a\nhighly sensitive fluorescent stain able to\ndetect as little as 20 pg of DNA, with a\nfour–sixteen-fold increase of sensitivity\ncompared with ethidium bromide.\nTo determine the lowest number of\nmolecules of a given mRNA in a pool that\ncan be detected by RT-PCR, it is war-\nranted to know the percentage of that\nmRNA in the pool. In many cases, it is not\npossible to determine this percentage.\nConsequently we established an alterna-\ntive method based on serial dilutions of\ntotal RNA, ranging from 1000 ng to 1 ng,\nused to carry out RT-PCR to detect high-\n(GAPDH),  medium- (HPRT) and low-\n expressed (E2F2) mRNAs after 35 cycles.\nHighly expressed mRNA was detected\nin all experimental conditions we used in\nthe presence of GelStar, whereas 10 ng of\ntotal RNA was the lowest quantity to de-\ntect medium- and low-expressed mRNAs.\nIn the RT-PCR analysis in this study\nwe used 200 ng of total RNA and 35 cy-\ncles for amplification, far above the limit\nof detection of low-expressed mRNAs.\nWhen minimal differences in gene ex-\npression were detected by PCR, experi-\nments were repeated using the real-time\nPCR assays, run on an Opticon 4 machine\n(Bio-Rad). Reactions were performed ac-\ncording to the manufacturer’s instructions\nusing the SYBR Green PCR master mix\n(Stratagene, La Jolla, CA, USA). Relative\nquantitative RT-PCR was used to deter-\nmine the fold difference for genes. Melt-\ning curves (65°C–94°C) were also gener-\nated to determine whether there were any\nspurious amplification products. The real-\ntime PCR efficiency was calculated for\neach primer pair using a dilution series\nand MJ Opticon II analysis software.\nImmunohistochemical Analysis\nTissue samples from patients were\nfixed in 4% buffered formaldehyde, dehy-\ndrated, and embedded in paraffin. Con-\nsecutive 5-μm cross sections were placed\non coated slides, deparaffinized through\na series of xylene and ethanol washes,\nand used for IHC analysis of SALL4 and\nOCT4 expression. We verified the IHC\nsignal for SALL4, using sections of mouse\nadult testis and heart as positive and neg-\native controls, respectively (Supplemental\nFigure 1). We verified the IHC signal for\nOCT4 using sections of mouse embryo\ntestis (E13.5) and mouse adult heart as\npositive and negative controls, respec-\ntively (Supplemental Figure 2).\nAntigen retrieval was obtained\nthrough incubation in citrate buffer at\npH 6.0 for 10 min followed by gradual\ncooling at room temperature for 20 min.\nAfter 1 h incubation in blocking solution\n(5% bovine serum albumin and 1% don-\nkey serum), slides were incubated\novernight at 4°C with SALL4 mouse\nmonoclonal antibody (1:100, Abnova,\nWalnut, CA, USA) or OCT4 rabbit poly-\nclonal antibody (1:250, Abcam, Cam-\nbridge, UK) diluted in blocking solution,\naccording to manufacturers’ instructions.\nIn negative controls the primary antibod-\nies were omitted.\nAfter being washed, slides were incu-\nbated with biotinylated antimouse or an-\ntirabbit secondary antibodies for 30 min\nat room temperature. The slides were\nthen washed again and incubated with\nstreptavidin-peroxidase (HRP) (Vector\nLaboratories, Burlingame, CA, USA) for\n30 min at room temperature. Finally, spe-\ncific hybridization of antibodies was\nTable 1. Patient clinical characteristics.*\nPhase of Grade of \nCase no. Age, years menstrual cycle endometriosis Pathology\nEndometrium 1 58 M Uterus fibromatosis\nEndometrium 2 29 PP Uterine myoma\nEndometrium 3 53 SP Endometrial polyp\nEndometrium 4 54 SP Uterus fibromatosis\nEndometrium 5 46 SP Uterus fibromatosis\nEndometrium 6 58 M Cystocele\nEndometrium 7 52 SP Uterus fibromatosis\nEndometrium 8 43 PP Uterine myoma\nEndometrium 9 41 PP Uterus fibromatosis\nEndometrium 10 37 SP Ovarian cyst\nEndometrium 11 51 M Endometrial polyp\nEndometrium 12 41 SP Uterine myoma\nEndometrium 13 52 SP Uterus fibromatosis\nEndometrium 14 41 PP Uterus fibromatosis\nEndometriosis 1 38 SP II\nEndometriosis 2 39 SP III\nEndometriosis 3 44 SP IV\nEndometriosis 4 29 SP II\nEndometriosis 5 26 SP III\nEndometriosis 6 28 PP III\nEndometriosis 7 31 PP III\nEndometriosis 8 46 SP IV\nEndometriosis 9 24 PP III\nEndometriosis 10 42 PP IV\nEndometriosis 11 33 PP III\nEndometriosis 12 33 PP I\n*PP, proliferative phase; SP, secretory phase; M, menopause.\n\nRESEARCH ARTICLE\nMOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 395\nhighlighted through incubation with di-\namino benzidine and HRP substrate\nbuffer (Vector). The diamino benzidine\nsubstrate solution gives a brown precipi-\ntate at the site of the target antigen recog-\nnized by the primary antibody. Nuclei\nwere counterstained blue with Mayer’s\nhematoxylin (Merck, Darmstadt, Ger-\nmany). Dried slides were immersed in\nxylene solution and coverslips applied\nusing ultramount.\nImage screening and photography of\nserial cross sections were performed\nusing a Leica IM 1000 System (Leica Mi-\ncrosystems, Wetzlar, Germany). Slides\nwere analyzed by two blinded indepen-\ndent observers.\nStatistical Analysis\nThe Multivariate Statistical Package\n(Kovach Computing Service, Isle of\n Anglesey, UK) was used for Ward’s mini-\nmum variance clustering method to eval-\nuate gene expression variability among\ndifferent samples.\nStatistical analyses (Fisher exact test;\nStudent t and Bonferroni tests) were\nevaluated using the GraphPad Software\n(Prism 4.0).\nAll supplementary materials are available\nonline at www.molmed.org.\nRESULTS\nRT-PCR Analysis of Stemness-Related\nGenes\nWe analyzed by RT-PCR the expression\nof a set of 13 stemness-related genes\n(Table 1) in endometrial (n = 14) and en-\ndometriotic (n = 12) biopsy samples.\nOverall results are shown in Table 3. The\nhistogram in Figure 1A shows the per-\ncentage of expression of each gene in the\nendometrium and endometriotic sample\ngroups and the histogram in Figure 1B re-\nports the number of expressed stemness-\nrelated genes in endometrial and endo -\nmetriotic samples.\nResults indicated that SOX2 mRNA\nwas not expressed in any of the samples\nwe analyzed (Table 3, Figure 1A). Con-\nversely, OCT4, KFL4, and BMI1 mRNAs\nwere expressed in all the endometrium\nand endometriotic samples we examined\n(Table 3, Figure 1A).\nOther genes, such as DPP A2and\nSOX15, were found to be expressed in\nthe same percentage of patients in endo-\nmetrial and endometriotic sample groups\n(Figure 1A).\nERAS, NANOG, and GDF3 showed a\nslightly higher (but not statistically sig-\nnificant) frequency of expression in en-\ndometrial than in endometriotic samples\n(Figure 1A).\nTable 2. Summary of RT-PCR primer sequences, position, annealing temperature, and chromosome mapping position of the stemness-\nrelated target genes.\nPrimer Annealing PCR Chromosome mapping\nGene position Primer sequence temperature, °C product, bp of the gene\nGAPDH 472 5′-GCATCCTGCACCACCACCTG -3′ 55 347 12p13\n799 5′-GCCTGGTTCACGACGTTCTT -3′\nSOX2 1563 5′-CCATCCACACTCACGCAAAA-3′ 59 139 3q27\n1701 5′-TATACAAGGTCCATTCCCCCG -3′\nOCT4 1121 5′-TCCCATGCATTCAAACTGAGG -3′ 60 103 6p21,31\n1223 5′-CCAAAAACCCTGGCACAAACT-3′\nNANOG 1169 5′-TGGACACTGGCTGAATCCTTC -3′ 59 142 12p13,31\n1310 5′-CGTTGATTAGGCTCCAACCAT -3′\nKLF4 1508 5′-CTGCGGCAAAACCTACACAA-3′ 60 182 9q31\n1689 5′-GGTCGCA TTTTTGGCACTG-3′\nERAS 969 5′-AATGTAGACCTTTCCCCAGGC -3′ 58 135 Xp11,23\n1103 5′-AAAGCCCCTCACCAAGTGAA-3′\nGDF3 778 5′-AAAAGGAAGAGCAGCCATCCCT-3′ 60 110 12p13.1\n887 5′-GCAATGATCCACTTGTGCCAA -3′\nSOX15 315 5′-GAACAGGTTGGAAGCAAAGGC –3′ 59 127 17p13\n441 5′-GCGTCGATCCTGAAAATGGA -3′\nDPPA2 798 5′-AGCCATGTTGGCATCATGG -3′ 58 108 3q13,13\n905 5′-GAGGCTTGCAGCAAAAAGGC -3′\nSALL4 2394 5′-GCCCAG ATATCCTGGAAACCA–3′ 60 115 20q13,13/13,2\n250 5′-TTCTCGGAGCTCTCTGCTTTG -3′\nTCL1 667 5′-CTCGGC TTTTTCTCAGCTGGAT-3′ 59 127 14q32,1\n793 5′-GGTGAATCGGCTGTGTTCTCA -3′\nZFP42 953 5′-ATGACAGTCTGAGCGCAATCG -3′ 60 133 4q35,2\n1085 5′-AACGCTTTCCCACATTCCG -3′\nUTF1 876 5′-CGACATCGCGAACATCCTG -3′ 64 117 10q26\n992 5′-AGAATGAAGCCCACGGCCA -3′\nBMI1 437 5′-AATGTCTTTT CCGCCCGCT-3′ 59 139 10p11,23\n575 5′-ACCCTCCACAAAGCACACACAT-3′\n\n396 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009\nSTEM CELLS AND ENDOMETRIOSIS\nThe remaining genes we analyzed\n(SALL4, UTF1, and TCL1) showed a dif-\nferent percentage of expression in en-\ndometrium and endometriotic sample\ngroups, with a trend for higher percent-\nages of expression in endometriotic sam-\nples than in endometrium samples. In\nmore detail, UTF1 (also known as undif-\nferentiated embryonic cell transcription\nfactor 1) showed a significantly higher\nfrequency of expression in endometriotic\nsamples than in endometrium (83% ver-\nsus 43%, P < 0.05). Also TCL1 showed a\nremarkable difference in the percentage\nof expression between endometrial and\nendometriotic samples (50% versus 83%),\nalthough the difference was not statisti-\ncally significant. Of note, ZFP42 was ex-\npressed in only 25% of endometriotic\n tissues (classified as III and IV grade)\nand in none of the endometrial biopsy\nsamples.\nThe 12 endometriotic samples coex-\npressed a minimum of 6 to a maximum of\n10 stemness-related genes (Figure 1B).\nConversely, the 14 endometrial samples\ncoexpressed a minimum of 4 to a maximum\nof 11 stemness-related genes (Figure 1B).\nNo significant differences were observed\nin the number of expressed genes between\nthe two groups of samples.\nFor this study we report only qualita-\ntive RT-PCR data about the expression of\na panel of 13 stemness-related genes, be-\ncause the endometrial and endometriotic\nbiopsies were harvested during the last\ndecade and in some cases the quality of\nRNA extracted from frozen or paraffin-\nembedded tissues did not allow us to ob-\ntain fully reliable quantitative RT-PCR\ndata. Nevertheless, in some patients we\nfound a correlation between the expres-\nsion level of stemness-related genes and\nthe grade of endometriosis, as well as a\ntrend (not statistically significant) for a\nhigher expression level of some genes\n(for example, SALL4) in endometriotic\ntissues rather than in endometrium sam-\nples (data not shown).\nThe RT-PCR data concerning the pres-\nence or absence of gene expression in the\n26 samples under analysis were used to\ncarry out a minimum variance test to\nevaluate gene expression variability\namong different patients. Our goal was\nto obtain a minimum variance clustering\nbased on a matrix constructed with the\npresence/absence of gene expression\npoints, such that patients having similar\npatterns of expressed/not expressed\ngenes fall in the same cluster and have\nmore genetic homogeneity compared\nwith those showing different expression\npatterns, which are then classified in\ndistinct clusters. We did not find any\ncorrelation between the phase of the\nTable 3. Qualitative RT-PCR analysis of stemness-related genes in 14 endometrial tissues and in 12 endometriotic samples.*\nCase no. Gene\n(cycle phase or endometriosis grade) SOX2 DPPA2 GDF3 TCL1 ZFP42 UTF1 ERAS SALL4 NANOG SOX15 OCT4 KFL4 BMI1\nEndometrium 1 (M) – – – – – – – – + + + + +\nEndometrium 2 (PP) – – – – – – – + + + + + +\nEndometrium 3 (SP) – – – + – – – + + + + + +\nEndometrium 4 (SP) – – – – – + + + + + + + +\nEndometrium 5 (SP) – – + + – + + + + + + + +\nEndometrium 6 (M) – – – – – – – + + + + + +\nEndometrium 7 (SP) – – + – – + + + + + + + +\nEndometrium 8 (PP) – – – + – – – + + + + + +\nEndometrium 9 (PP) – – + – – + + + + + + + +\nEndometrium 10 (SP) – – + + – – + – + + + + +\nEndometrium 11 (M) – – + + – – + + + + + + +\nEndometrium 12 (SP) – – – – – – + – – – + + +\nEndometrium 13 (SP) – + + + – + + + + + + + +\nEndometrium 14 (PP) – + + + – + + + + + + + +\nEndometriosis 1 (II ) – – + + – + + + + + + + +\nEndometriosis 2 (III) – – + + – + + + + + + + +\nEndometriosis 3 (IV) – – – + + + + + + + + + +\nEndometriosis 4 (II) – – – – – + + – + + + + +\nEndometriosis 5 (III) – – – + + – – + + + + + +\nEndometriosis 6 (III) – – – + + + + + + + + + +\nEndometriosis 7 (III) – – – + – + – + – + + + +\nEndometriosis 8 (IV) – – + + – + – + – + + + +\nEndometriosis 9 (III) – – – + – – – + – + + + +\nEndometriosis 10 (IV) – – – + – + – + + + + + +\nEndometriosis 11 (III) – + – – – + + + + – + + +\nEndometriosis 12 (I) – + – + – + – + + + + + +\n*Summary of the results on the presence (+) or absence (–) of gene expression for each patient. PP, proliferative phase; SP, secretory\nphase; M, menopause.\n\nRESEARCH ARTICLE\nMOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 397\nmenstrual cycle and the number of ex-\npressed stemness-related genes (Table 3).\nSimilarly, no significant evidence was\ndetected for a correlation between the\ngrade of endometriosis and the number\nof expressed stemness-related genes\n(Table 3).\nImmunohistochemical Detection of\nSALL4 and OCT4 Proteins in\nEndometrial and Endometriotic\nSamples\nEndometrial (n = 14) and endometri-\notic (n = 12) samples embedded in paraf-\nfin were submitted to IHC-mediated\nanalysis of the expression of SALL4 and\nOCT4. We selected these two proteins for\nIHC analysis because these proteins play\nan important role in stemness preserva-\ntion (24), because they may clarify possi-\nble misleading results deriving from RT-\nPCR analysis of OCT4 expression, and\nfinally, because quantitative RT-PCR data\nindicated a trend for a higher expression\nlevel for their mRNAs in endometriosis\nsamples rather than in endometrium,\neven though the difference was not sta-\ntistically significant.\nWe analyzed at least five consecutive\ncross sections for each tissue sample.\nOnly cross sections of endometrial and\nendometriotic tissues with markedly\nbrown-stained cells, showing a clear\nstructure, were scored positive for\nSALL4 and OCT4 protein expression.\nPositive cells for SALL4 and OCT4\nwere detectable in different consecutive\ncross sections of the tissue samples we\nanalyzed (Figures 2 and 3). The staining\nfor both SALL4 and OCT4 showed nu-\nclear localization.\nCells positive for SALL4 were found in\nall the endometriotic tissues we analyzed\n(Figure 2). None of the endometrial sam-\nples revealed cells positive for SALL4. To\nfurther confirm these data, IHC detection\nof SALL4 was also conducted on paired\nectopic and eutopic endometrium from\nthe same patient (sample endometriosis 8,\nTables 1 and 3), revealing SALL4-positive\ncells only in endometriotic tissue.\nCells positive for OCT4 were found in\nthe stroma of all the endometriotic tis-\nsues we analyzed. Stromal cells positive\nfor OCT4 were also detected in the endo-\nmetrial samples (Figure 3).\nWe observed only single stromal cells\npositive for OCT4 immunostaining both\nin endometrium and in endometriotic\nsamples. Conversely, SALL4-positive\ncells in endometriotic tissues were also\nlocated in a periglandular position and\nin the stromal vasculature.\nControl IHC reaction for SALL4 was\npositive on mouse adult testis and nega-\ntive on mouse adult heart (Supplemental\nFigure 1). Control IHC reaction for OCT4\nwas positive on mouse embryo testis\nFigure 1. (A) The histogram shows the frequency of expression of stemness-related genes\nin endometrial tissues (white columns) and in endometriotic samples (gray columns). (B)\nThe histogram shows the number of expressed stemness-related genes in endometrial tis-\nsues (white columns) and in endometriotic samples (gray columns).\n\n398 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009\nSTEM CELLS AND ENDOMETRIOSIS\n(E13.5) and negative on mouse adult\nheart (Supplemental Figure 2).\nDISCUSSION\nIn this study, we have characterized at\nthe mRNA level the expression of a panel\nof 13 embryonic stemness-related genes\nin two sets of human endometrium and\nendometriotic samples, together with the\nIHC verification for a subgroup of two\nfactors, to evaluate which of them were\npresent in endometrial and endometri-\notic tissues.\nVarious studies have highlighted the\npresence of stem cells in endometrium. In\nparticular, Du et al. (14) demonstrated\nthat lethally irradiated female mice re-\nceiving bone marrow transplantation\nfrom male donors show male-derived\ncells incorporated into the endometrium.\nThe presence of stem cells has also been\ndemonstrated in women who received\nbone marrow transplants from mis-\nmatched donors (25). The bone marrow\ncompartment can be subdivided into two\ninterdependent spaces: the hematopoietic\ncell compartment and the stroma. The\nstroma is composed of mesenchymal\nstem cells, fibroblasts, adipocytes, nerves,\nand the bone marrow’s vascular system.\nMesenchymal stem cells are quite rare,\ncomprising between 0.01% and 0.001% of\nnucleated cells in adult human bone mar-\nrow, depending on the age of individuals\n(26). Nonhemapoietic stem cells from\nbone marrow can potentially contribute\nto the preservation of multiple tissues.\nSome studies indicate that stem cells in\nendometrium are of bone marrow origin\nand share many characteristics with mes-\nenchymal stem cells, because they are\nable to differentiate into condrocytes, os-\nteocytes, and adipocytes and express pe-\nculiar antigens (27).\nOther recent studies have revealed the\npresence of stem cells in the menstrual\nblood, characterized by a high prolifera-\ntive rate in vitro, high differentiation abil-\nity, expression of a number of stemness-\nrelated nonhematopoietic markers\n(including OCT4), and production of ma-\ntrix metalloproteases, cytokine growth\nfactors, and angiogenic factors (28,29).\nNevertheless, the presence of hematopoi-\netic stem cells has also been demon-\nstrated immunologically in endometrium\n(30). The endometrial stem cells, both of\nhematopoietic or nonhematopoietic na-\nture, probably contribute to the de novo\nformation of stroma, glands, and vascu-\nlature in the reproductive cycle.\nIn this study, we highlighted the possi-\nble presence of stem cells in all the en-\ndometrium and endometriotic samples\nthrough the expression of 13 stemness-\nrelated genes.\nOur RT-PCR data highlight a signifi-\ncantly higher number of endometriotic\nsamples expressing UTF1 mRNA com-\npared to endometrial biopsy samples \n(P < 0.05). UTF1 is highly and almost ex-\nclusively expressed during embryogene-\nsis (31). In more detail, UTF1 is specifi-\ncally expressed in the inner cell mass\nand primitive ectoderm and is downreg-\nulated at early primitive streak stages\n(32). Of interest, it has been reported\nthat UTF1 expression is maintained in\nthe primordial germ cells in developing\nembryos and in the gonads in adult\n animals (33).\nZFP42 (also known as REX-1) is ex-\npressed only in about 25% of endometri-\notic samples, classified as III and IV\ngrade (Table 3). A recent study by Kris-\ntensen et al. (34) showed that ZFP42 and\nUTF1 are expressed throughout human\ntestes development and in testicular\ngerm cell tumors and in testicular carci-\nnoma, showing similarities with pluripo-\ntent embryonic stem cells.\nFigure 2. Representative IHC staining of SALL4 in human endometrium and in endometri-\notic tissue. Hematoxylin counterstaining. Endometriotic tissue (A, B) is compared with en-\ndometrial tissue (E, F). IHC staining of serial sections of the tissue used in A without primary\nantibody was done as negative control of the reaction (C, D). Black arrow in B indicates a\nrepresentative SALL4 IHC-positive cell. Subparts (B, D, F) represent 100× magnification of\nthe area enclosed in the black perimeter in A, C, E (40× magnification).\n\nRESEARCH ARTICLE\nMOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 399\nPromoter analysis indicated that the\nmurine UTF1 gene is transcriptionally\nregulated by OCT4 and SOX2 (35). Fi-\nnally, a recent study indicated that UTF1\nis a stably chromatin-associated tran-\nscriptional repressor protein involved in\nthe initiation of embryonic stem cell dif-\nferentiation, but not in embryonic stem\ncell self-renewal (36).\nRT-PCR data also indicate a trend for a\nhigher frequency of expression of TCL1in\nendometriotic samples. TCL1 (also known\nas T cell leukemia 1) is a protooncogene\nhighly activated in various human neo-\nplastic diseases, whereas its physiological\nexpression is tightly limited to early de-\nvelopmental cells as well as various de-\nvelopmental stages of immune cells (37).\nOne of the analyzed genes (SOX2) was\ndetected in neither endometrial nor en-\ndometriotic tissue, whereas DPP A2was\nexpressed only in samples from two pa-\ntients for each group. This result is not\nsurprising, because embryonic stem cells\nhave broader stemness properties (self-\nrenewal, pluripotency) compared with\nadult stem cells.\nThe analysis of minimum variance did\nnot reveal any homogeneous clusters of\nsamples on the basis of gene expression\ndata, possibly because of the relatively\nlow number of samples we analyzed or\nbecause of the heterogeneity of samples\nin relation to the number and type of\ncells they contain.\nRecently, it has been discovered that\nrare cells in the endometrial stroma of\nabout 44% of women are positive for\nOCT4 (also known as OCT3/4,OCT3\nand POU5f1) (38), a protein member of\nthe POU transcription factor family.\nOCT4 is expressed in pluripotent cells,\nand its downregulation is associated\nwith loss of pluripotency. The results of\nthe mentioned study are in agreement\nwith our RT-PCR and IHC data, because\nwe highlighted the expression of OCT4\nmRNA and protein in all the eutopic en-\ndometrium samples we analyzed.\nThe latest results about OCT4 isoforms\nreveal the presence of three alternative\nsplice variants (OCT4-A, OCT4-B, and\nOCT4-B1) (39).\nThe PCR primers we used for OCT4\nmRNA analysis (Table 2) are both en-\nclosed within the exon 5 sequence and\ncannot be used to distinguish among the\nvariants OCT4-A, OCT4-B, and OCT-4B1\nand the RNA transcribed by the two\npseudogenes identified by the GeneBank\nnumbers NG_005793 and NG_006104.\nFor this reason, together with our obser-\nvation that the OCT4 RT-PCR signal was\nhigher in samples from patients with en-\ndometriosis samples than in samples\nfrom the endometrium group, we de-\ncided to further analyze the OCT4 ex-\npression in the two sets of human endo-\nmetrial and endometriotic samples at the\nprotein level. The antibody for OCT4 we\nused was obtained using a synthetic\nFigure 3. Representative IHC staining of OCT4 in human endometrium and in endometri-\notic tissue. Hematoxilin counterstaining. Endometriotic tissue (A, B) is compared with endo-\nmetrial tissue (E, F). IHC staining of serial sections of the tissue used in A without antibody\nwas done as a negative control of the reaction (C, D). Immunohistochemical staining of\nserial sections of the tissue used in E without antibody was done as a negative control of\nthe reaction (H, G). Black arrows in B and F indicate representative OCT4 IHC-positive\ncells. Subparts (B, D, F , H) represent 100×magnification of the area enclosed in the black\nperimeter in A, C, E, G (40×magnification).\n\n400 | FORTE ET AL. | MOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009\nSTEM CELLS AND ENDOMETRIOSIS\npeptide derived from within residues\n300 to the C-terminus of human OCT4\nas immunogene. The OCT4 splice vari-\nants OCT4-A and OCT4-B share an iden-\ntical C-terminal domain, whereas the re-\ncently discovered OCT4-B1 lacks the\nC-terminal domain because of a stop\ncodon in the cryptic exon 2b, and conse-\nquently the IHC data we obtained were\npotentially related to the OCT4-A and -B\nisoforms. Nonetheless, because we ob-\ntained a clear nuclear localization of the\nIHC signal for OCT4 (Figure 3), we can\nargue that it corresponds to the OCT4-A\nvariant, as it has been reported that the\nOCT-4B variant is localized in the cyto-\nplasm (40,41).\nThe variant OCT4-B1 has been discov-\nered very recently (39), and consequently\nall the currently available literature data\nconcerning the expression of OCT4 pro-\ntein involve the isoforms A and B, be-\ncause the antibody specific for the puta-\ntive truncated protein translated by the\nOCT4-B1 splice variant is not available.\nThe translation of the OCT4-B1 mRNA\nvariant identified by Atlasi et al. has not\nyet been demonstrated, and its putative\nrole in stemness and in carcinogenesis\nhas been only suggested, but not demon-\nstrated experimentally (for example,\nthrough RNA interference assays). More-\nover, nothing is known about the cellular\nlocalization (at the nuclear or cytoplas-\nmatic level) of the protein possibly ex-\npressed by the novel OCT4 mRNA splice\nvariant.\nIt should be underlined that OCT4 has\nbeen considered for a long time a reli-\nable marker for stemness, but a recent\nstudy demonstrated the expression of\nOCT4 also in normal differentiated adult\ncells from human peripheral blood, thus\nsuggesting that the presence of OCT4\nalone can no longer be considered suffi-\ncient to define a cell as pluripotent (42).\nNevertheless, in our experiments we\nsupported the presence of OCT4 as a\nmarker of stemness with the expression\ndata of adjunctive 12 stemness-related\ngenes.\nParallel experiments revealed the pres-\nence of SALL4 mRNA both in eutopic\nand ectopic endometrium samples, but\nrevealed the presence of SALL4 protein\nonly in endometriotic samples.\nIt should be underlined that we were\nalso able to analyze the SALL4 expres-\nsion in paired ectopic and eutopic endo-\nmetrial tissue from the same patient\n(sample endometriosis 8 in Table 1),\nidentifying SALL4-positive cells only in\nectopic endometrium. The direct com-\nparison between autologous ectopic and\neutopic endometrium can exclude vari-\nables related to individual genetic vari-\nability and to various effects of hormonal\nstimulation during the menstrual cycle,\nand thus such comparison can further\nclarify the contribution of stem cells to\nthe pathogenesis of endometriosis.\nNevertheless, it should be considered\nthat this differential expression of SALL4\nprotein between endometrial and en-\ndometriotic tissues could be related not\nnecessarily to a translational mechanism\nof regulation of SALL4 expression, but\ncould be related to the very low expres-\nsion of SALL4 protein in endometrium.\nThe presence of OCT4- and SALL4-\npositive cells mainly in the stroma of en-\ndometrial and endometriotic samples is\nin agreement with results of other stud-\nies based on stem cell detection through\nthe analysis of stemness markers (38,16).\nNevertheless, we found some SALL4-\npositive cells also in the vasculature and\nin periglandular positions.\nSALL4 and OCT4 work as essential\nstemness factors. Our choice to analyze at\nthe protein level both SALL4 and OCT4\nrelies also on experimental evidence that\nSALL4 forms a crucial interconnected au-\ntoregulatory network with OCT4 in em-\nbryonic stem cells (43). It has also been\ndemonstrated in mouse embryonic stem\ncells that SALL4 is a transcriptional regu-\nlator of OCT4 and has a critical role in the\nmaintenance of stem cell pluripotency by\nmodulating OCT4 expression (44).\nCONCLUSIONS\nOur data indicating an increased pres-\nence of stem cell markers in endometri-\notic samples are in agreement with the\nrecent studies revealing an increased ex-\npression of the adult stem cell marker\nMusashi-1 in endometriosis and endo-\nmetrial carcinoma (16). Our preliminary\nresults indicate that the percentages of\nsingle cells positive for SALL4 and\nOCT4 we detected in the stroma of en-\ndometriotic tissues are comparable to\nthose found by Gotte M et al. for\nMusashi-1–positive cells (data not\nshown). The contribution of stem cells to\nendometriosis has been hypothesized in\nmany reports of studies and reviews\n(45,14).\nIf further verified, the presence of stem\ncells in ectopic and eutopic endometrium\ncan provide new insights into the mecha-\nnisms at the basis of gynecological dis-\neases related to cell proliferation, includ-\ning endometrial carcinoma.\nTo our knowledge, this is the first\nstudy highlighting the expression of a\npanel of stemness-related genes in\nhuman endometrial and endometriotic\nsamples, with a particular relevance for\nUTF1 and TCL1. Moreover, we report for\nthe first time the expression of SALL4\nand OCT4 proteins in endometriotic\nsamples. Overall data obtained in this\nstudy suggest a possible role for stem\ncells in the pathogenesis of endometrio-\nsis, even if further data are warranted to\nsupport this hypothesis.\nDISCLOSURE\nThe authors declare that they have no\ncompeting interests as defined by Molec-\nular Medicine, or other interests that\nmight be perceived to influence the re-\nsults and discussion reported in this\npaper.\nREFERENCES\n1. Maruyama T, Yoshimura Y. (2008) Molecular and\ncellular mechanisms for differentiation and re-\ngeneration of the uterine endometrium. Endocr. J.\n55:795–810.\n2. Bulun SE. (2009) Endometriosis. N. Engl. J. Med.\n360:268–79.\n3. Nezhat F, Datta MS, Hanson V , Pejovic T, Nezhat\nC. (2008) The relationship of endometriosis and\novarian malignancy: a review. Fertil. Steril.\n90:1559–70.\n4. Ohlsson Teague EM, et al. (2009) MicroRNA-\n egulated pathways associated with endometrio-\nsis. Mol. Endocrinol. 23:265–75.\n\nRESEARCH ARTICLE\nMOL MED 15(11-12)392-401, NOVEMBER-DECEMBER 2009 | FORTE ET AL. | 401\n5. Xue Q, et al. (2007) Transcriptional activation of\nsteroidogenic factor-1 by hypomethylation of the\n5′CpG island in endometriosis. J. Clin. Endocrinol.\nMetab. 92:3261–7.\n6. Xue Q, et al. (2007) Promoter methylation regu-\nlates estrogen receptor 2 in human endometrium\nand endometriosis. Biol. Reprod. 77:681–7.\n7. Bischoff FZ, Heard M, Simpson JL. (2002) So-\nmatic DNA alterations in endometriosis: high\nfrequency of chromosome 17 and p53 loss in late-\nstage endometriosis. J. Reprod. Immunol. 55:49–64.\n8. Zhou HE, Nothnick WB. (2005) The relevancy of\nthe matrix metalloproteinase system to the\npathophysiology of endometriosis. Front. Biosci.\n10:569–75.\n9. Simpson JL, Elias S, Malinak LR, Buttram VC Jr.\n(1980) Heritable aspects of endometriosis, I: ge-\nnetic studies. Am. J. Obstet. Gynecol. 137:327–31.\n10. Roobrouck VD, Ulloa-Montoya F, Verfaillie CM.\n(2008) Self-renewal and differentiation capacity\nof young and aged stem cells. Exp. Cell. Res.\n314:1937–44.\n11. Chan RW, Schwab KE, Gargett CE. (2004) Clono-\ngenicity of human endometrial epithelial and\nstromal cells. Biol. Reprod. 70:1738–50.\n12. Cervello I, Martinez-Conejero JA, Horcajadas JA,\nPellicer A, Simon C. (2007) Identification, charac-\nterization and co-localization of label-retaining\ncell population in mouse endometrium with typ-\nical undifferentiated markers. Hum. Reprod.\n22:45–51.\n13. Chan RW, Gargett CE. (2006) Identification of\nlabel-retaining cells in mouse endometrium. Stem\nCells. 24:1529–38.\n14. Du H, Taylor HS. (2007) Contribution of bone\nmarrow-derived stem cells to endometrium and\nendometriosis. Stem Cells. 25:2082–6.\n15. Prowse AH, et al. (2006) Molecular genetic evi-\ndence that endometriosis is a precursor of ovar-\nian cancer. Int. J. Cancer. 119:556–62.\n16. Gotte M, et al. (2008) Increased expression of the\nadult stem cell marker Musashi-1 in endometrio-\nsis and endometrial carcinoma. J. Pathol.\n215:317–29.\n17. Cai J, Weiss ML, Rao MS. (2004) In search of\n“stemness.” Exp. Hematol. 32:585–98.\n18. T\nakahashi K, Yamanaka S. (2006) Induction of\npluripotent stem cells from mouse embryonic\nand adult fibroblast cultures by defined factors.\nCell. 126:663–76.\n19. Iwama A, Oguro H, Negishi M, Kato Y,\nNakauchia H. (2005) Epigenetic regulation of\nhematopoietic stem cell self-renewal by poly-\ncomb group genes. Int. J. Hematol. 81:294–300.\n20. Kameda T, Thomson JA. (2005) Human ERas\ngene has an upstream premature polyadenyla-\ntion signal that results in a truncated, noncoding\ntranscript. Stem Cells. 23:1535–40.\n21. Matoba R, et al. (2006) Dissecting Oct3/4-regulated\ngene networks in embryonic stem cells by expres-\nsion profiling. PLoS One. 1:e26.\n22. Kooistra SM, Thummer RP , Eggen BJ. (2009)\nCharacterization of human UTF1, a chromatin-\nassociated protein with repressor activity ex-\npressed in pluripotent cells. Stem Cell Res . 2009,\nFeb 12 [Epub ahead of print].\n23. Noyes EW HA, Rock J. (1950) Dating the endo-\nmetrial biopsy. Fertil. Steril. 1:3–25.\n24. Yang J, et al. (2008) Genome-wide analysis re-\nveals Sall4 to be a major regulator of pluripo-\ntency in murine-embryonic stem cells. Proc. Natl.\nAcad. Sci. U. S. A. 105:19756–61.\n25. Taylor HS. (2004) Endometrial cells derived from\ndonor stem cells in bone marrow transplant re-\ncipients. JAMA. 292:81–5.\n26. Apel A, et al. (2009) Suitability of human mes-\nenchymal stem cells for gene therapy depends on\nthe expansion medium. Exp. Cell. Res.\n315:498–507.\n27. Gargett CE, Schwab KE, Zillwood RM, Nguyen\nHP , Wu D. (2009) Isolation and culture of epithe-\nlial progenitors and mesenchymal stem cells\nfrom human endometrium. Biol. Reprod.\n80:1136–45.\n28. Meng X, et al. (2007) Endometrial regenerative\ncells: a novel stem cell population. J. Transl. Med.\n5:57.\n29. Musina RA, Belyavski AV , Tarusova OV , Solovy-\nova EV , Sukhikh GT. (2008) Endometrial mes-\nenchymal stem cells isolated from the menstrual\nblood. Bull. Exp. Biol. Med. 145:539–43.\n30. Lynch L, Golden-Mason L, Eogan M, O’Herlihy C,\nO’Farrelly C. (2007) Cells with haematopoietic stem\ncell phenotype in adult human endometrium: rele-\nvance to infertility? Hum. Reprod. 22:919–26.\n31.\nNishimoto M, et al. (2001) Structural analyses of\nthe UTF1 gene encoding a transcriptional coacti-\nvator expressed in pluripotent embryonic stem\ncells. Biochem. Biophys. Res. Commun. 285:945–53.\n32. Okuda A, et al. (1998) UTF1, a novel transcrip-\ntional coactivator expressed in pluripotent em-\nbryonic stem cells and extra-embryonic cells.\nEMBO J. 17:2019–32.\n33. Chuva de Sousa Lopes SM, et al. (2005) Altered\nprimordial germ cell migration in the absence of\ntransforming growth factor beta signaling via\nALK5. Dev. Biol. 284:194–203.\n34. Kristensen DM, et al. (2008) Presumed pluripo-\ntency markers UTF-1 and REX-1 are expressed in\nhuman adult testes and germ cell neoplasms.\nHum. Reprod. 23:775–82.\n35. Nishimoto M, Fukushima A, Okuda A, Mura-\nmatsu M. (1999) The gene for the embryonic\nstem cell coactivator UTF1 carries a regulatory\nelement which selectively interacts with a com-\nplex composed of Oct-3/4 and Sox-2. Mol. Cell.\nBiol. 19:5453–65.\n36. van den Boom V , et al. (2007) UTF1 is a chromatin-\nassociated protein involved in ES cell differentia-\ntion. J. Cell. Biol. 178:913–24.\n37. Noguchi M, Ropars V , Roumestand C, Suizu F.\n(2007) Proto-oncogene TCL1: more than just a\ncoactivator for Akt. FASEB J. 21:2273–84.\n38. Matthai C, et al. (2006) Oct-4 expression in\nhuman endometrium. Mol. Hum. Reprod. 12:7–10.\n39. Atlasi Y, Mowla SJ, Ziaee SA, Gokhale PJ, An-\ndrews PW. (2008) OCT4 spliced variants are dif-\nferentially expressed in human pluripotent and\nnonpluripotent cells. Stem Cells. 26:3068–74.\n40. Lee J, Kim HK, Rho JY, Han YM, Kim J. (2006)\nThe human OCT-4 isoforms differ in their ability\nto confer self-renewal. J. Biol. Chem. 281:33554–65.\n41. Cauffman G, Liebaers I, Van Steirteghem A, Van\nde Velde H. (2006) POU5F1 isoforms show differ-\nent expression patterns in human embryonic\nstem cells and preimplantation embryos. Stem\nCells. 24:2685–91.\n42. Zangrossi S, et al. (2007) Oct-4 expression in\nadult human differentiated cells challenges its\nrole as a pure stem cell marker. Stem Cells.\n25:1675–80.\n43. Lim CY, et al. (2008) Sall4 regulates distinct tran-\nscription circuitries in different blastocyst-derived\nstem cell lineages. Cell Stem Cell. 3:543–54.\n44. Zhang J, et al. (2006) Sall4 modulates embryonic\nstem cell pluripotency and early embryonic de-\nvelopment by the transcriptional regulation of\nPou5f1. Nat. Cell\nBiol. 8:1114–23.\n45. Starzinski-Powitz A, Zeitvogel A, Schreiner A,\nBaumann R. (2003) Endometriosis—a stem cell\ndisease [in German]? Zentralbl. Gynakol. 125:235–8.","source_license":"CC0","license_restricted":false}