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Differentiation-Defective Phenotypes Revealed by Large-Scale Analyses of Human Pluripotent Stem Cells

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Differentiation-Defective Phenotypes Revealed by Large-Scale Analyses of Human Pluripotent Stem Cells Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells Michiyo Koyanagi-Aoia,1, Mari Ohnukia,1, Kazutoshi Takahashia, Keisuke Okitaa, Hisashi Nomab, Yuka Sawamuraa, Ito Teramotoa, Megumi Naritaa, Yoshiko Satoa, Tomoko Ichisakaa, Naoki Amanoa, Akira Watanabea, Asuka Morizanea, Yasuhiro Yamadaa,c, Tosiya Satod, Jun Takahashia,e, and Shinya Yamanakaa,f,2 aCenter for iPS Cell Research and Application, eDepartment of Biological Repair, Institute for Frontier Medical Sciences, and cInstitute for Integrated Cell- Material Sciences, Kyoto University, Kyoto 606-8507, Japan; bDepartment of Data Science, The Institute of Statistical Mathematics, Tokyo 190-8562, Japan; dDepartment of Biostatistics, Kyoto University School of Public Health, Kyoto 606-8501, Japan; and fGladstone Institute of Cardiovascular Disease, San Francisco, CA 94158 Contributed by Shinya Yamanaka, October 30, 2013 (sent for review September 13, 2013) We examined the gene expression and DNA methylation of 49 at least three passages. In addition, we analyzed the original somatic human induced pluripotent stem cells (hiPSCs) and 10 human cells, two human embryonic carcinoma cell (hECC) lines (NTera2 embryonic stem cells and found overlapped variations in gene cloneD1 and 2102Ep 4D3), and three cancer cell lines (HepG2, expression and DNA methylation in the two types of human MCF7, and Jurkat). pluripotent stem cell lines. Comparisons of the in vitro neural The mRNA microarray analyses (Fig. 1A) identified 61 probes fi differentiation of 40 hiPSCs and 10 human embryonic stem cells with signi cant differences in expression between hESCs and < showed that seven hiPSC clones retained a significant number of hiPSCs [t test, false discovery rate (FDR) 0.05]. Each of the 61 undifferentiated cells even after neural differentiation culture and probes showed variable expression among both the hESCs and formed teratoma when transplanted into mouse brains. These hiPSCs, and the distributions of the expression levels in the two differentiation-defective hiPSC clones were marked by higher groups overlapped (Fig. 1D). Of note, hESCs established at expression levels of several genes, including those expressed from Kyoto University (Kyoto hESCs) were more similar to hiPSCs than to the remaining hESCs (other hESCs) in their expression long terminal repeats of specific human endogenous retroviruses. of 15 probes that were differentially expressed between hESCs These data demonstrated a subset of hiPSC lines that have aberrant and hiPSCs [FDR <0.05 and fold change (FC) >3] (Fig. S1A). In gene expression and defective potential in neural differentiation, fi contrast, hierarchical clustering using all probes showed no clear- which need to be identi ed and eliminated before applications in cut separation among Kyoto hESCs, other ESCs, and iPSCs, regenerative medicine. indicating that the similarities between Kyoto ESCs and iPSCs are confined to a small set of genes (Fig. S1B). In addition, the uman pluripotent stem cells possess a robust potential for miRNA array analyses (Fig. 1B) did not find any significant Hproliferation and provide useful sources of cells for re- differences between hESCs and hiPSCs (t test, FDR <0.05). The generative medicine and drug discovery. Two types of human expressions of hsa-miR-886-3p and hsa-miR-142-3p tended pluripotent stem cells have been generated: human embryonic to be higher in hiPSCs, but the expression levels of these stem cells (hESCs) derived from blastocysts (1) and induced pluripotent stem cells (hiPSCs), which are generated from so- Significance matic cells by factor-mediated reprogramming (2, 3). fi In the past few years, ndings have been controversial in In the past few years, findings have been controversial in regard to whether hESCs and hiPSCs are distinct cell types. regard to whether human induced pluripotent stem cells Some reports have argued that they could not be clearly distin- – (hiPSCs) are distinct from human embryonic stem cells (hESCs) guished (4 6), whereas others have reported that they have dif- in their molecular signatures and differentiation properties. In ferences in their gene expression (7–10), DNA methylation (10– this study, hiPSCs and hESCs have overlapping variations in 13), and capacity for differentiation (14). In the latter papers, molecular signatures such as RNA expression and DNA meth- relatively small numbers of cell lines were generally compared. fi In addition, most comparisons used pluripotent cell lines from ylation. However, some hiPSC clones retained a signi cant various laboratories, so the observed differences may be attrib- number of undifferentiated cells even after neural differenti- utable to laboratory-specific variations owing to technical dif- ation culture and formed teratoma when transplanted into ferences (15). mouse brains. These differentiation-defective hiPSC clones were To overcome these problems, we compared the mRNA and marked by higher expression levels of several genes, including microRNA (miRNA) expression and DNA methylation between those expressed from long terminal repeats of specifichuman CELL BIOLOGY 10 hESCs and 49 hiPSCs that had been cultured under the same endogenous retroviruses. They need to be identified and elimi- conditions. Furthermore, we compared the in vitro directed nated prior to applications in regenerative medicine. neural differentiation of these pluripotent stem cells. Author contributions: M.K.-A., M.O., K.T., and S.Y. designed research; M.K.-A., M.O., K.T., Results K.O., Y. Sawamura, I.T., M.N., Y. Sato, and T.I. performed research; A.M. and J.T. sup- Overlapped Variations of mRNA Expression and DNA Methylation in ported the neural differentiation experiments; Y.Y. supported the pathological analysis; H.N. and T.S. supported the statistical analysis; M.K.-A., M.O., H.N., N.A., and A.W. ana- hiPSCs and hESCs. We analyzed a total of 49 hiPSCs derived from lyzed data; and M.K.-A., M.O., K.T., and S.Y. wrote the paper. fi four types of somatic cells, including human dermal broblasts Conflict of interest statement: S.Y. is a member without salary of the scientific advisory (HDFs), dental-pulp stem cells (DP), cord blood cells (CB), and boards of iPierian, iPS Academia Japan, Megakaryon Corporation, and HEALIOS K. K. peripheral blood mononuclear cells (PBMN), generated using Japan. three gene delivery methods, including those using retroviruses, Data deposition: Gene expression, miRNA expression, DNA methylation, and exon array nonintegration episomal plasmids, and Sendai viruses (Table 1 data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi. and Dataset S1). Most clones were generated in our own labo- nlm.nih.gov/geo (accession no. GSE49053). ratory, except for three clones that were established in another 1M.K.-A. and M.O. contributed equally to this work. laboratory (16). Before the analyses of gene/miRNA expression 2To whom correspondence should be addressed. E-mail: [email protected]. (Fig. 1 A and B) and DNA methylation (Fig. 1C) we cultured these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. hiPSCs, as well as 10 hESCs, under the same culture conditions for 1073/pnas.1319061110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319061110 PNAS | December 17, 2013 | vol. 110 | no. 51 | 20569–20574 Downloaded by guest on September 29, 2021 Table 1. Summary of the iPSC clones used in this study examined, we did not identify significantly differentially meth- Method used to generate clones ylated CpG regions (CG-DMR) between the hESCs and hiPSCs (Mann–Whitney U test, FDR <0.05) (Fig. 1C). Origin Retrovirus Episomal plasmid Sendaivirus Total We then validated the CG-DMRs reported in previous stud- ies. Three studies identified a total of 205 regions as CG-DMRs, HDFs 22 (5, 0) 3 (0, 0) 0 (0, 0) 25 (5, 0) including 130, 71, and 4 regions identified by comparing five DP 1 (0, 0) 2 (1, 1) 0 (0, 0) 3 (1, 1) hiPSCs and two hESCs (13), three versus three (12), and nine CB 3 (1, 2) 4 (0, 0) 5 (0, 0) 12 (1, 2) versus three (10) cell lines, respectively. Of the 205 regions, 46 PBMN 0 (0, 0) 5 (0, 1) 4 (0, 0) 9 (0, 1) regions containing 66 CpG dinucleotides were covered by the Total 26 (6, 2) 14 (1, 2) 9 (0, 0) 49 (7, 4) Infinium platform used in our study (Table S1A). Based on the Total clone numbers with type-1 defective clone numbers and type-2 methylation levels in our hiPSCs and hESCs, these CpGs were defective clone numbers in parentheses are shown. clustered into three groups (Fig. 1E). Two-thirds of these CpGs belonged to group A: They tended to be highly methylated in hESCs, ECCs, and cancer cell lines and to be hypomethylated in miRNAs showed overlapped variations among hiPSCs and hiPSCs, as well as somatic cells. However, they were also hypo- hESCs (Fig. S1A). methylated in Kyoto ESCs (17). The methylation status of the We next compared the global DNA methylation status be- upstream region of the paraoxonase 3 (PON3), a representative tween hiPSCs and hESCs by the Illumina Infinium Human example of CpGs in group A, was confirmed by pyrosequencing Methylation27 BeadChip assay. Among 27,445 CpG dinucleotides (Fig. 1F). Thus, the CpG methylation status in group A may A DE0.5-0.50 F DPYS mRNA expression mRNA expression PON3 PON3 (Group A) -10-8-6 -4 -206 2 4 PON3 8 DCDC2 100 SDR42E1 DCDC2 6 LOC387647 MOS (high) TRIM4 CALCB 80 4 RGPD1 CTHRC1 TUSC1 2 TRIM4 ANKRD7 GRM1 60 0 TMEM132C IRAK4 KLF8 ACOT4 -2 FRMD3 CCDC68 40 DNAJC15 KAAG1 -4 NPY Methylation (%) average
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