human adult germline stem cells in question

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Human adult germline stem cells in question Arising from: S. Conrad et al. Nature 456, 344349 (2008) Conrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs) 1 . Here we investigate the pluripotency of haGSCs by using global gene- expression analysis based on their gene array data 1 and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues’ haGSCs is therefore called into question. Fibroblasts can be easily established from human testicular cul- tures 2 . Considering the similarities between haGSCs and non-testis fibroblasts, we isolated fibroblasts from human testicular biopsies and derived clusters of cells (human testicular fibroblast cells, hTFCs) from them (Fig. 1a, b). haGSCs were found to be morpho- logically similar, if not identical, to these hTFCs but not to hESCs (Fig. 1c). Gene expression relative to human ESCs 0.0001 0.001 0.01 0.1 1 10 OCT4 SOX2 NANOG C-MYC KLF4 hESCs hTFC-1 hTFC-2 OCT4 α-TUBULIN NANOG hESCs hTFC-1 hTFC-2 hTFCs hESCs c b a f e d i h g hTFCs STAT3 CDH2/N-cadherin CDH1/E-cadherin DPPA3 TERT AFP DAZL DDX4/VASA hESC H1L H7 H9 H13B H14A haGSC hTFC FF FP FC Human ESC ines Human fibroblast cells Human ES cell-enriched genes Fibroblast cell-enriched genes TDGF1 LIN28 NANOG SOX2 L1TD1 DPPA4 CXADR ZFP42 PTPRZ1 HOOK1 DNMT3B LRRN1 SALL2 TERF1 PHC1 SALL4 PRDM14 TERT ZIC5 SALL3 POU5F1 CLDN6 ZSCAN10 EMID2 LEFTY2 DPPA2 LEFTY1 NODAL GDF3 SOX21 DCN CNTN3 LOC399959 PRRX1 PSG5 DKK1 NR2F2 PDGFRA DPYD IL1R1 LRRC15 SNAI2 PTX3 NRN1 EMP1 LOX NNMT GBP3 ARID5B COL3A1 LUM MMP1 GREM1 k j H1L, H7, H9, H13B and H14A H1 1–3 hSC 1–3 haGSC 1–6 hTFC 1–3 FF 1–2 FP 1–2 FC 1–3 B cell 1-3 hESC H1L H7 H9 H13B H14A haGSC hTFC FF FP FC Human ESC lines Human fibroblast cells hESCs hTFCs hTFCs haGSCs hESCs haGSCs 16 12 10 14 8 6 4 4 6 8 10 12 14 16 2 2 4 6 8 10 12 14 16 2 4 6 8 10 12 14 16 2 16 12 10 14 8 6 4 2 16 12 10 14 8 6 4 2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 –0.2 –0.18 –0.16 –0.15 0.4 0.2 0.2 0.1 0 –0.1 –0.2 0 –0.2 –0.19 –0.17 PC1 PC3 PC2 Figure 1 | Characterization of human testis- derived fibroblast cells. ac, Phase-contrast images of human testicular fibroblast cells (hTFCs) at high (a) and low (b) magnification and of human embryonic stem cells (hESCs) (line H1) (c); scale bars, 200 mm. df, Scatter plots of pairwise global gene-expression comparisons: hESCs versus human adult germline stem cells (haGSCs) (d); hESCs versus hTFCs (e); and haGSCs versus hTFCs (f). g, Real-time RT–PCR analysis of hESCs and hTFCs; error bars represent standard deviation. h, Western blot analysis of OCT4, NANOG and a-TUBULIN. i, Heat map of the microarray gene-expression data of the genes discussed by Conrad et al. 1 . j, Principal component (PC) analysis of the global gene-expression profiles of hESCs, haGSCs, hTFCs and fibroblasts. k, Heat map of microarray gene expression of a list of human ESC- and fibroblast-enriched genes 3 . Gene-expression colour key is shown at the top in log 2 scale. Microarray data were downloaded from the GEO database; accession numbers GSE11350 (haGSCs, hESCs (ES line H1), human spermatogonial cells (hSCs)) 1 , GSE12583 (foreskin fibroblasts (FF)) 8 , GSE15322 (normal colon fibroblasts (FC)), GSE15148 (ES lines H1L, H7, H9, H13B and H14A, and parental foreskin cells (FP)) 3 and GSE10831 (B cells) 9 . NATURE | Vol 465 | 24 June 2010 BRIEF COMMUNICATIONS ARISING E1 Macmillan Publishers Limited. All rights reserved ©2010

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Page 1: Human adult germline stem cells in question

Human adult germline stem cells in questionArising from: S. Conrad et al. Nature 456, 344–349 (2008)

Conrad et al. have generated human adult germline stem cells(haGSCs) from human testicular tissue, which they claim have similarpluripotent properties to human embryonic stem cells (hESCs)1. Herewe investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data1 and comparingthe expression of pluripotency marker genes in haGSCs and hESCs,and in haGSCs and human fibroblast samples derived from differentlaboratories, including our own. We find that haGSCs and fibroblastshave a similar gene-expression profile, but that haGSCs and hESCs do

not. The pluripotency of Conrad and colleagues’ haGSCs is thereforecalled into question.

Fibroblasts can be easily established from human testicular cul-tures2. Considering the similarities between haGSCs and non-testisfibroblasts, we isolated fibroblasts from human testicular biopsiesand derived clusters of cells (human testicular fibroblast cells,hTFCs) from them (Fig. 1a, b). haGSCs were found to be morpho-logically similar, if not identical, to these hTFCs but not to hESCs(Fig. 1c).

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Figure 1 | Characterization of human testis-derived fibroblast cells. a–c, Phase-contrastimages of human testicular fibroblast cells(hTFCs) at high (a) and low (b) magnificationand of human embryonic stem cells (hESCs) (lineH1) (c); scale bars, 200mm. d–f, Scatter plots ofpairwise global gene-expression comparisons:hESCs versus human adult germline stem cells(haGSCs) (d); hESCs versus hTFCs (e); andhaGSCs versus hTFCs (f). g, Real-time RT–PCRanalysis of hESCs and hTFCs; error barsrepresent standard deviation. h, Western blotanalysis of OCT4, NANOG and a-TUBULIN.i, Heat map of the microarray gene-expressiondata of the genes discussed by Conrad et al.1.j, Principal component (PC) analysis of the globalgene-expression profiles of hESCs, haGSCs,hTFCs and fibroblasts. k, Heat map of microarraygene expression of a list of human ESC- andfibroblast-enriched genes3. Gene-expressioncolour key is shown at the top in log2 scale.Microarray data were downloaded from the GEOdatabase; accession numbers GSE11350(haGSCs, hESCs (ES line H1), humanspermatogonial cells (hSCs))1, GSE12583(foreskin fibroblasts (FF))8, GSE15322 (normalcolon fibroblasts (FC)), GSE15148 (ES lines H1L,H7, H9, H13B and H14A, and parental foreskincells (FP))3 and GSE10831 (B cells)9.

NATURE | Vol 465 | 24 June 2010 BRIEF COMMUNICATIONS ARISING

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Page 2: Human adult germline stem cells in question

We confirmed this similarity by comparing the global gene-expression profile of hTFCs and haGSCs using the gene array dataof Conrad et al.1. Scatter plots reveal that haGSCs were remarkablysimilar to hTFCs and to previously described fibroblasts, but not tohESCs (Fig. 1d–f). Conrad et al. did not present a scatter plot analysis,which would have helped visualization of any differences betweenhaGSCs and hESCs.

The pluripotency marker genes POU5F1/OCT4, SOX2, NANOGand LIN28, which are expressed in hESCs, were not expressed inhaGSCs or in hTFCs, whereas the fibroblast marker genes SNAI2and ACTA2 (refs 3, 4) were markedly overexpressed. Real-time ana-lysis using polymerase chain reaction with reverse transcription (RT–PCR) confirmed the absence of OCT4, SOX2 and NANOG expressionin hTFCs (Fig. 1g).

Messenger RNA expression of OCT4 and NANOG in hTFCs cor-related with protein abundance, as determined by western blotting(Fig. 1h). By contrast, the western blot results of Conrad et al.1 indi-cate that the amounts of OCT4 and NANOG protein in their haGSCswere virtually identical to those in hESCs. However, OCT4 andNANOG mRNA levels, extracted from their microarray data1, wereas low in haGSCs as in hTFCs and other human fibroblasts.

This inconsistency between mRNA and protein levels in haGSCs—whether analysed by western blot or immunohistochemistry—wasalso evident for other genes, such as CDH1/E-cadherin, DAZL andDDX4/VASA (Fig. 1i). Conrad et al. show that STAT3 and CDH2/N-cadherin are highly expressed in haGSCs, consistent with our findings(Fig. 1i). However, expression of STAT3 and CDH2/N-cadherin inhuman ESCs was nearly identical to that in human fibroblasts(Fig. 1i). Therefore, the expression of these genes cannot be used todistinguish pluripotent cells from fibroblasts.

Principal component analysis of global gene-expression datarevealed that haGSCs clustered closely with hTFCs and with previ-ously described human fibroblasts, but not with hESCs (Fig. 1j).Heat-map analysis with pluripotent and fibroblastic genes3 showedthat haGSCs were fibroblastic, but not pluripotent, in their gene-expression profile (Fig. 1k).

Our results indicate that haGSCs are by no means similar to hESCsbut that they do strongly resemble our hTFCs, which are not pluripo-tent. These findings are at odds with those of Conrad et al.1, who reportthat their haGSCs are able to form teratomas, a defining feature ofhuman pluripotent cells.

METHODS SUMMARYhTFCs were obtained from routine biopsies taken during testicular surgery (with

patients’ informed written consent). Following mechanical tissue disruption and

enzymatic digestion, cells were cultured in the medium described by Conrad et

al.1. These experiments were approved by the local ethics council. Microarrayanalysis in hTFCs was performed using Human Genome U133 Plus 2.0 chip

(Affymetrix) and processed as in ref. 5. The array data were deposited in the Gene

Expression Omnibus (GEO) database (accession number GSE17772). All micro-

arrays used in this study were globally normalized with the RMA algorithm

implemented in R-Bioconductor6.

Authors’ note: We contacted Conrad et al. to obtain their haGSC lines1; however,

the established haGSCs are not accessible at present to other laboratories because

consent between Conrad et al. and donor patients did not include permission to

distribute cells derived from the biopsies7. At the start of 2009, T. Skutella agreed

to send us the haGSCs once he receives permission for their distribution, but we

have not so far received them. We have been informed that the original consent

form stipulates that all derived cell lines must be destroyed after a period of three

years following biopsy.

Kinarm Ko1, Marcos J. Arauzo-Bravo1, Natalia Tapia1, Julee Kim1,

Qiong Lin2,3, Christof Bernemann1, Dong Wook Han1, Luca Gentile1,Peter Reinhardt1, Boris Greber1, Rebekka K. Schneider2,3,

Sabine Kliesch4, Martin Zenke2,3 & Hans R. Scholer1,5

1Department of Cell and Developmental Biology, Max Planck Institute forMolecular Biomedicine, Munster 48149, Germany.2Institute for Biomedical Engineering, Department of Cell Biology, RWTHAachen University Medical School, Aachen 52074, Germany.3Helmholtz Institute for Biomedical Engineering, RWTH AachenUniversity, Aachen 52074, Germany.4Department of Clinical Andrology, Centre for Reproductive Medicineand Andrology, University of Munster, Munster 48149, Germany.5Faculty of Medicine, University of Munster, Munster 48149, Germany.e-mail: [email protected]

Received 11 September 2009; accepted 23 March 2010.

1. Conrad, S. et al. Generation of pluripotent stem cells from adult human testis. Nature456, 344–349 (2008).

2. Chen, A. T., Fu, Y. S. & Reidy, J. A. Human testicular cultures. II. Sertoli cells. In Vitro 11,313–321 (1975).

3. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgenesequences. Science 324, 797–801 (2009).

4. Nakagawa, H. et al. Role of cancer-associated stromal fibroblasts in metastatic coloncancer to the liver and their expression profiles. Oncogene 23, 7366–7377 (2004).

5. Ko, K. et al. Induction of pluripotency in adult unipotent germline stem cells. Cell StemCell 5, 87–96 (2009).

6. Irizarry, R. A. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic AcidsRes. 31, e15 (2003).

7. Conrad, S. et al. Generation of pluripotent stem cells from adult human testis. Nature460, 1044 (2009).

8. Aasen, T. et al. Efficient and rapid generation of induced pluripotent stem cells fromhuman keratinocytes. Nature Biotechnol. 26, 1276–1284 (2008).

9. Vockerodt, M. et al. The Epstein–Barr virus oncoprotein, latent membrane protein-1,reprograms germinal centre B cells towards a Hodgkin’s Reed–Sternberg-likephenotype. J. Pathol. 216, 83–92 (2008).

Competing financial interests: declared none.

doi:10.1038/nature09089

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Page 3: Human adult germline stem cells in question

Conrad et al. replyReplying to: K. Ko et al. Nature 465, doi:10.1038/nature09089 (2010)

Ko et al.1 challenge our description of human adult germline stemcells (haGSCs)2, indicating that we have instead cultured testis fibro-blasts. However, they do not follow the experimental procedures wedescribe2 and so fail to reproduce our findings.

We repeated the procedures used by Ko et al. that producedfibroblasts1 by using parts of our own protocol2 with human testistissue, but omitting the array of selection procedures describedthere — such as MACS (magnetic-activated cell separation) andmatrix selection of germ stem cells. We confirmed that it is indeedimpossible to establish a germ-cell culture in this way, as Ko et al.know from their mouse studies. In the resulting cultures, the platesare overgrown with somatic cells that aggregate to clumps of cells:most of these cells are probably fibroblasts. The populations ofhuman testicular fibroblast cells (hTFCs) described by Ko et al.1

are a completely different cell population and are not comparableto haGSCs.

Fibroblasts are not germ stem cells and are unable to differentiateinto cells of all germ layers, as the cells we describe did2. However, themicroarrays on which Ko et al. place emphasis were only one ofseveral molecular and functional tests we used2 to detect similaritiesto human embryonic stem cells (hESCs).

According to the Microarray Facility Tubingen, one of the criticalpoints of the challenge by Ko et al.1 is that they use our data set forcomparison with their own expression profiles from different celltypes. The use of microarrays that are generated several days or monthsapart introduces systematic batch effects or non-biological differences,which make it meaningless to compare samples from different batchesdirectly. Batch effects are inevitable when new samples or replicates areadded incrementally to an existing array data set, or when a meta-analysis of multiple studies pools microarray data across differentlaboratories3. Statements about comparisons between the differentdata set are therefore not constructive.

We are now working on the selection and amplification of ourhaGSCs so that we have sufficient cell material to share with collea-gues. Detailed protocols are available from us and the authors of refs2, 4–6 for those who wish to investigate haGSCs further.Sabine Conrad1, Markus Renninger3, Jorg Hennenlotter3, Tina Wiesner1,Lothar Just1, Michael Bonin4, Wilhelm Aicher5,6, Hans-Jorg Buhring7,

Ulrich Mattheus2, Andreas Mack2, Hans-Joachim Wagner2,

Stephen Minger8, Matthias Matzkies9, Michael Reppel9,

Jurgen Hescheler9, Karl-Dietrich Sievert3, Arnulf Stenzl3 &

Thomas Skutella1,6

1Institute of Anatomy, Department of Experimental Embryology,

Osterbergstraße 3, 72074 Tubingen, Germany.2Institute of Anatomy, Department of Cellular Neurobiology,

Osterbergstraße 3, 72074 Tubingen, Germany.3Department of Urology, University Clinic Tubingen,

Hoppe-Seyler-Straße 3, Tubingen 72076, Germany.4Institute of Anthropology and Human Genetics, Microarray Facility,

University Clinic, Calwerstraße 7, 72076 Tubingen, Germany.5ZMF Research Laboratories, University Clinic Tubingen,

Waldhornlestraße 22, 72072 Tubingen, Germany.6Center for Regenerative Biology and Medicine (ZRM),

Paul-Ehrlich-Straße 15, 72076 Tubingen, Germany.7Department of Internal Medicine II, University Clinic Tubingen,

Otfried-Muller-Straße 10, 72076 Tubingen, Germany.8Stem Cell Biology Laboratory, Wolfson Centre for Age-Related

Diseases, King’s College London, King’s College, London SE1 1UL, UK.9Institute of Neurophysiology, University of Cologne,

Robert-Koch-Straße 39, 50931 Cologne, Germany.

e-mail: [email protected]

1. Ko, K. et al. Human adult germline stem cells in question. Nature 465, doi:10.1038/nature09089 (2010).

2. Conrad, S. et al. Generation of pluripotent stem cells from adult human testis. Nature456, 344–349 (2008).

3. Rhodes, D. R. et al. Large-scale meta-analysis of cancer microarray data identifiescommon transcriptional profiles of neoplastic transformation and progression. Proc.Natl Acad. Sci. USA 101, 9309–9314 (2004).

4. Golestaneh, N. et al. Pluripotent stem cells derived from adult human testes. StemCells Dev. 18, 1115–1126 (2009).

5. Kossack, N. et al. Isolation and characterization of pluripotent human spermatogonialstem cell-derived cells. Stem Cells 27, 138–149 (2009).

6. Mizrak, S. C. et al. Embryonic stem cell-like cells derived from adult human testis.Hum. Reprod. 25, 158–167 (2010).

Competing financial interests: declared none.

doi:10.1038/nature09090

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