Benjamin E. Powell

Systematic identification of culture conditions for induction and maintenance of naive human pluripotency.

Summary Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, “naive” state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.

Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in various embryonic and adult tissues. Mice mutant for either Tet1 or Tet2 are viable, raising the question of whether these enzymes have overlapping roles in development. Here we have generated Tet1 and Tet2 double-knockout (DKO) embryonic stem cells (ESCs) and mice. DKO ESCs remained pluripotent but were depleted of 5hmC and caused developmental defects in chimeric embryos. While a fraction of double-mutant embryos exhibited midgestation abnormalities with perinatal lethality, viable and overtly normal Tet1/Tet2-deficient mice were also obtained. DKO mice had reduced 5hmC and increased 5mC levels and abnormal methylation at various imprinted loci. Nevertheless, animals of both sexes were fertile, with females having smaller ovaries and reduced fertility. Our data show that loss of both enzymes is compatible with development but promotes hypermethylation and compromises imprinting. The data also suggest a significant contribution of Tet3 to hydroxylation of 5mC during development.

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells.

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.

Genome editing reveals a role for OCT4 in human embryogenesis.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR–Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR–Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Failure to replicate the STAP cell phenomenon.

Although the reports that stress (such as exposure to acid) can coax somatic cells into a novel state of pluripotency have been retracted, the validity of stimulus-triggered acquisition of pluripotency (STAP) remains unclear (http://dx.doi.org/10.1038/protex. 2014.008 and Supplementary Information). Here we describe the efforts of seven laboratories to replicate STAP, including experiments performed within the laboratory where STAP first originated, as well as re-analysis of the sequencing data from the STAP reports. Neonatal cells treated with two STAP protocols exhibited artefactual autofluoresence rather than bona fide reactivation of an Oct4 (also known as Pou5f1) and green fluorescent protein (GFP) transgene reporter, did not reactivate pluripotency markers towards embryonic stem (ES)-cell-like levels, and failed to generate teratomas or chimaerize blastocysts. Re-analysis of the original RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data identified discrepancies in the sex and genetic composition of parental donor cells and converted stem cells, and revealed a STAP-derived cell line to be a mixture containing trophoblast stem cells, attesting to the importance of validating the properties and provenance of pluripotent stem cells using a wide range of criteria. To assess the reprogramming capacity of STAP protocols, we used a transgenic Oct4-GFP reporter, which shows GFP reactivation during Oct4/Sox2/Klf4 reprogramming, in established induced pluripotent stem (iPS) cells and in the gonads of mid-gestation ‘all iPS cell’ embryos generated by tetraploid complementation (Extended Data Figs 1 and 2a). Working within the Vacanti laboratory where the concept of STAP cells originated, and assisted by a co-author of the STAP papers, a Daley laboratory member (A.D.L.A.) attempted to replicate two reported STAP protocols: (1) mechanical trituration and acid treatment of mouse lung cells (Brigham and Women’s Hospital (BWH) protocol; see Supplementary Information), and (2) acid treatment of mouse splenocytes (RIKEN protocol; Methods and Extended Data Fig. 2b). Seventy-two hours after stress treatment of lung cells, floating spheres appeared amidst cellular debris. Fluorescence microscopy revealed that both Oct4-GFP and wild-type spheres emitted lowlevel broad spectrum fluorescence detectable within both green and red filters, indicating autofluorescence (Fig. 1a). Untreated Oct4-GFP ES cells did not emit the same low-level broad spectrum fluorescence as STAP-treated cells. STAP-treated splenocytes formed spheres with lower efficiency, but also appeared autofluorescent. Flow cytometry indicated STAP-treated Oct4-GFP cells did not exhibit Oct4-GFP reactivation at levels comparable to control Oct4GFP mouse ES cells, and were indistinguishable from stressed wildtype controls (Fig. 1b). Absence of ES-cell-like levels of Oct4, Sox2 and Nanog transcripts and nonspecific immunofluorescence corroborated flow cytometry data (Extended Data Fig. 2c, d). Rare pluripotent cells should generate teratomas in immunocompromised mice, but STAP cells could not, unlike control ES cells (Extended Data Fig. 2e, f). Replication of the poly-L-glycolic acid (PLGA)-based teratoma production method described in the original STAP reports with GFP cells to distinguish host and donor contribution produced distinct masses of connective tissue, muscle and scar, with minimal GFP content, indicating primarily host origin (Fig. 1c, d and Extended Data Fig. 2g). Rare GFP-positive clusters did not form differentiated tissues characteristic of ES-cell-derived teratomas (Fig. 1d). Autofluorescent spheres failed to enter development after morula aggregation or blastocyst injection (Fig. 1e and Extended Data Fig. 2h–j). Therefore, pluripotency was undetectable in STAP experiments. Six other laboratories (Deng, Hanna, Hochedlinger, Jaenisch, Pei and Wernig) also attempted to generate STAP cells (Table 1) and made the following observations. First, autofluorescent sphere-like aggregates after STAP treatment were universally seen. Second, transgenic reporters used by Obokata and colleagues (GOF18-Oct4-GFP, containing the 18-kilobase genomic Oct4 fragment (GOF18)) and by the Daley, Pei and Hanna laboratories (GOF18-Oct4DPE-GFP, lacking the Oct4 proximal enhancer (PE) element) both exhibit activity in pre-implantation embryos, early post-implantation epiblast cells (embryonic day (E) 5.5), germ cells, and mouse ES/iPS cells; however, differential activity in late post-implantation epiblast (E6.5) and early passage mouse epiblast-derived stem cells has been ascribed to the Oct4 proximal enhancer. Using the same reporter as Obokata and colleagues, the Deng laboratory observed that the GFP signal in chemical iPS cells was easily distinguishable from the autofluorescence of STAP-treated cells (Extended Data Fig. 2k). The Jaenisch, Wernig and Hochedlinger laboratories failed to observe GFP reactivation with Oct4 or Nanog knock-in reporters, excluding a scenario of uncoupling between GFP and endogenous pluripotency expression. Despite a range of tested reporters, no group documented authentic Oct4/Nanog reporter activation that resembled bona fide ES cells. Third, the Deng laboratory failed to observe Oct4, Sox2 and Nanog induction 3 and 7 days after STAP treatment, reducing the likelihood that pluripotency was transiently activated and silenced by day 7 (Extended Data Fig. 2l). Finally, the Hanna, Wernig and Hochedlinger laboratories failed to generate stem-cell lines by culturing STAP-treated cells in leukaemia inhibitory factor (LIF) and adrenocorticotropic hormone (ACTH)-supplemented medium. In summary, 133 replicate attempts failed to document generation of ES-cell-like cells, corroborating and extending a recent report. We re-examined the high-throughput sequencing data from the STAP reports to investigate the genetic provenance of parental CD45 cells and converted STAP cells, STAP stem cells and Fgf4-induced stem cells (FI-SCs) (Fig. 1f). Comparative genomic hybridization array data mentioned in the original paper were not publicly released. Copy number variation (CNV) analysis conducted using ChIP-seq input samples revealed a discrepancy in sex across samples as well as chromosomal aberrations (Fig. 1g). In the original STAP reports, the authors stated that they mixed CD45 cells from male and female mice owing to the small number of CD45 cells retrieved from individual neonatal spleens. However, our analysis indicates that CD45 cells were female, whereas the derived cells (STAP cells, STAP stem cells and FI-SCs) were all male, a clear inconsistency. We note that control ES cells were also male (Fig. 1g). FI-SCs possessed trisomy 8, which renders mouse ES cells germline-incompetent (Fig. 1g). Inferred single nucleotide variants (SNVs) from RNA-seq data allowed classification of samples as genetically similar or dissimilar (Fig. 1h). Control ES cells, parental donor female CD45 cells, STAP cells, and STAP stem cells all possessed similar SNV profiles, consistent with their derivation from a first generation hybrid of C57BL6/129 strains, the reported genotype (Fig. 1h and Extended Data Fig. 3). By contrast, FI-SCs had an SNV profile that matched a single nucleotide polymorphism (SNP) profile of C57BL6 strain origin, indicating

Erratum: Systematic identification of culture conditions for induction and maintenance of naive human pluripotency (Cell Stem Cell (2014) 15 (471-487))

Thorold W. Theunissen, Benjamin E. Powell, Haoyi Wang, Maya Mitalipova, Dina A. Faddah, Jessica Reddy, Zi Peng Fan, Dorothea Maetzel, Kibibi Ganz, Linyu Shi, Tenzin Lungjangwa, Sumeth Imsoonthornruksa, Yonatan Stelzer, Sudharshan Rangarajan, Ana D’Alessio, Jianming Zhang, Qing Gao, Meelad M. Dawlaty, Richard A. Young, Nathanael S. Gray, and Rudolf Jaenisch* *Correspondence: jaenisch@wi.mit.edu http://dx.doi.org/10.1016/j.stem.2014.08.002

A dynamic multi-compartmental model of DNA methylation with demonstrable predictive value in hematological malignancies

Recent advances have highlighted the central role of DNA methylation in leukemogenesis and have led to clinical trials of epigenetic therapy, notably hypomethylating agents, in myelodysplasia and acute myeloid leukemia. However, despite these advances, our understanding of the dynamic regulation of the methylome remains poor. We have attempted to address this shortcoming by producing a dynamic, six-compartmental model of DNA methylation levels based on the activity of the Dnmt methyltransferase proteins. In addition, the model incorporates the recently discovered Tet family proteins which enzymatically convert methylcytosine to hydroxymethylcytosine. A set of first order, partial differential equations comprise the model and were solved via numerical integration. The model is able to predict the relative abundances of unmethylated, hemimethylated, fully methylated, and hydroxymethylated CpG dyads in the DNA of cells with fully functional Dnmt and Tet proteins. In addition, the model accurately predicts the experimentally measured changes in these abundances with disruption of Dnmt function. Furthermore, the model reveals the mechanism whereby CpG islands are maintained in a hypomethylated state via local modulation of Dnmt and Tet activities without any requirement for active demethylation. We conclude that this model provides an accurate depiction of the major epigenetic processes involving modification of DNA.

Fertile offspring from sterile sex chromosome trisomic mice

Trisomic animals lose third chromosome Generally, when a third sex chromosome is added to the normal two in mammals (XX for female and XY for male), developmental defects result. Mice that are trisomic for the sex chromosomes are infertile. Hirota et al. demonstrate that reprogramming cells from sterile mice with chromosome trisomies XXY or XYY generates XY stem cells. Sperm generated from these XY stem cells could give rise to healthy, fertile offspring. Reprogramming also promoted loss of the extra chromosome in cells from patients with Klinefelter (XXY) or Down (trisomy 21) syndrome. Science, this issue p. 932 Extra chromosomes can be eliminated during stem cell reprogramming to correct sterile sex chromosome trisomies in mice. Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts from sterile trisomic XXY and XYY mice lose the extra sex chromosome through a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes.

Erratum: Genome editing reveals a role for OCT4 in human embryogenesis

This corrects the article DOI: 10.1038/nature24033

Corrigendum: Failure to replicate the STAP cell phenomenon.

This corrects the article DOI: 10.1038/nature15513