LETTER TO THE EDITOR Human Induced Pluripotent Stem Cells Develop Teratoma More Efﬁciently and Faster Than Human Embryonic Stem Cells Regardless the Site of Injection IVAN GUTIERREZ-ARANDA, VERONICA RAMOS-MEJIA, CLARA BUENO, MARTIN MUNOZ-LOPEZ, PEDRO. J. REAL, ANGELA MA´ CIA, LAURA SANCHEZ, GERTRUDIS LIGERO, JOSE L. GARCIA-PAREZ, PABLO MENENDEZ Andalusian Stem Cell Bank, Centro de Investigacio´ n Biome´dica, CSJA-UGR. Granada, Spain Human embryonic stem cell (hESC) and reprogrammed/induced pluripotent stem cell (iPSC) research is becoming the ‘‘ﬂavor of the month’’ for downstream applications such as drug screening, disease modeling, and future regenerative medicine and cell thera- pies [1–4]. Pluripotency (the ability to give rise to any cell type of the three germ layers: mesoderm, ectoderm, and endoderm) is the deﬁning feature of hESCs and iPSCs [5]. In vivo teratoma formation in immune-compromised mice is the ‘‘gold-standard’’ assay to deﬁne bona ﬁde pluripotent stem cells capable of gener- ating tumoral disorganized structures containing tissues repre- senting the three germ layers [5, 6]. Despite the importance of teratoma assay as an extended screen for the pluripotency of hESCs and iPSCs and as in vivo assay to explore molecular and cellular mechanisms underlying the biology of human teratomas and their transition to teratocarcinomas, there are no standard procedures for performing this assay [5–7]. Different studies on hESCs have correlated the site of implantation with the efﬁ- ciency of teratoma formation and histology tissue composition [6, 8]. However, limited data are available regarding the tera- toma development latency. More importantly, no study so far has compared side-by-side the efﬁciency, latency, and histologi- cal tumor composition of hESCs- and iPSCs-derived teratomas. In addition, a new generation of immunodeﬁcient mice has been developed: the NOD/SCID IL2Rc / mouse. This strain carries a IL2Rc-chain deﬁciency that blocks signaling through multiple cytokine receptors leading to many innate immune defects [9, 10]. The non obese diabetic/severe combined immune-deﬁcient (NOD/SCID) IL2Rc / strain facilitates engraftment and tumor formation and does not develop thymic lymphoma, ensuring a longer lifespan of inoculated mice. Here, we followed the improved teratoma protocol previ- ously developed by Prokhorova et al. [6, 11–13] to transplant side-by-side as few as 1 106 of either fully characterized un- differentiated hESCs or iPSCs in 6- to 8-week-old non obese di- abetic/severe combined immune-deﬁcient (NOD/SCID) IL2Rc / mice [11, 13–15]. The following hESC lines were used: H9, H1, AND1, AND2, AND3, HS181, and ECAT. The following iPSC lines were used: MSHU-001, iAND4, CB- CD34þ iPSC1, and CB-CD34þ iPSC2. These lines have been fully characterized and deposited according to Spanish Legisla- tion at The Spanish Stem Cell Bank (http://www.isciii.es/ htdocs/terapia/terapia_lineas.jsp) [16]. Brieﬂy, cells were resus- pended in phosphate buffered saline (PBS) supplemented with 30% matrigel (Becton Dickinson, San Jose, CA, http:// www.bd.com) [6] and transplanted subcutaneously (200 ll vol- ume) or by intratesticular injection (60 ll volume). Figure 1A depicts the experimental strategy used. We then analyzed efﬁ- ciency, latency, and histological tumor composition. In hESCs, the rate of teratoma formation was 81% subcutaneously versus 94% intratesticularly (n ¼ 30 mice; Fig. 1B). However, the in- tratesticular injection, despite showing higher efﬁciency of tera- toma formation, displayed a slightly longer latency (66 vs. 59 days; p-value > 0.05). There were no site-speciﬁc differences in the teratoma composition at the histological level (Fig. 1C). Interestingly, when iPSCs were transplanted the rate of tera- toma formation was 100% (n ¼ 16 mice), regardless the type of injection. More importantly, iPSCs seem more aggressive in vivo as the latency was shortened 52% (from 59 days to 31 days) upon subcutaneous injection and 26% (from 66 days to 49 days) upon intratesticular injection. As with hESCs, no differences in teratoma composition were observed either. To the best of our knowledge, this is the ﬁrst study compar- ing side-by-side the efﬁciency, latency, and teratoma composi- tion between hESCs and iPSCs. We found clear differences in the efﬁciency and latency but not in the teratoma histological composition. Further experiments are still demanded to gain insights into the higher aggressiveness in vivo of iPSCs as com- pared with hESCs. Ploidy, analyzed by conventional G-banding karyotype, could not explained these differences because all but two pluripotent stem cell lines were euploid: the aneuploid lines were one hESC (AND1) and one iPSC (iAND4). It is worth emphasizing, however, that karyotype analysis is not a high-re- solution technique detecting ﬁne genomic aberrations, with a euploid karyotype not being therefore indicative of an overall cellular genomic stability. Whether or not speciﬁc tiny genomic insults (detectable by high-resolution methods such as compar- ative genomic hybridazation (CGH)-arrays and single-nucleo- tide polymorphism analysis) or epigenetic differences may explain the higher aggressiveness in vivo of iPSCs still needs to be elucidated. We envision that these data may be useful not only for stem cells scientists addressing pluripotency issues and studying mechanisms underlying speciﬁc germ-layer/tissue Author Contributions: I.G.-A: conception and design, performed research, data collection, data analysis and interpretation; L.S., G.L., C.B., P.J.R., A.M., M.M.-L., and V.R.: performed research, data collection and data analysis; J.L.G.-P.: provision of study material, manuscript writing and ﬁnancial support; P.M.: data analysis and interpretation, conception and design, ﬁnancial support and manuscript writing. All authors read and approved the ﬁnal version of the manuscript. Correspondence: Pablo Menendez, Ph.D., M.B.A., Andalusian Stem Cell Bank, Instituto de Investigacio´n Biome´dica. Parque Tecnolo´gico de la Salud, Avda. del Conocimiento s/n. 18,100 Granada, Spain. Telephone: 34-958-894-672; Fax: 34-958-894-652; e-mail: pablo.menendez@juntadeandalucia.es Received May 17, 2010; accepted for publication June 22, 2010; ﬁrst published online in STEM CELLS EXPRESS 16 July, 2010. VC AlphaMed Press 1066-5099/2009/$30.00/0 doi: 10.1002/stem.471 STEM CELLS 2010;28:1568–1570 www.StemCells.com