Samantha J. Ross

Multiple mechanisms disrupt the let-7 microRNA family in neuroblastoma

Poor prognosis in neuroblastoma is associated with genetic amplification of MYCN. MYCN is itself a target of let-7, a tumour suppressor family of microRNAs implicated in numerous cancers. LIN28B, an inhibitor of let-7 biogenesis, is overexpressed in neuroblastoma and has been reported to regulate MYCN. Here we show, however, that LIN28B is dispensable in MYCN-amplified neuroblastoma cell lines, despite de-repression of let-7. We further demonstrate that MYCN messenger RNA levels in amplified disease are exceptionally high and sufficient to sponge let-7, which reconciles the dispensability of LIN28B. We found that genetic loss of let-7 is common in neuroblastoma, inversely associated with MYCN amplification, and independently associated with poor outcomes, providing a rationale for chromosomal loss patterns in neuroblastoma. We propose that let-7 disruption by LIN28B, MYCN sponging, or genetic loss is a unifying mechanism of neuroblastoma development with broad implications for cancer pathogenesis.

Effect of Developmental Stage of HSC and Recipient on Transplant Outcomes

The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs.

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the reports that stress (such as exposure to acid) can coax somatic cells into a novel state of pluripotency have been retracted, the validity of stimulus-triggered acquisition of pluripotency (STAP) remains unclear (http://dx.doi.org/10.1038/protex. 2014.008 and Supplementary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

Flow-induced protein kinase A–CREB pathway acts via BMP signaling to promote HSC emergence

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta– gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response elementbinding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB ge nomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VEcadherin+ cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway down stream of shear stress that regulates HSC emergence in the AGM via the endothelial-tohematopoietic transition.

Developmental regulation of myeloerythroid progenitor function by the Lin28b–let-7–Hmga2 axis

Daley and collaborators show that endogenous Lin28b drives erythroid-dominant fetal hematopoiesis and that decreases in Lin28b activate adult granulocyte-predominant hematopoiesis.

Interferon-α signaling promotes embryonic HSC maturation.

In the developing mouse embryo, the first hematopoietic stem cells (HSCs) arise in the aorta-gonad-mesonephros (AGM) and mature as they transit through the fetal liver (FL). Compared with FL and adult HSCs, AGM HSCs have reduced repopulation potential in irradiated adult transplant recipients but mechanisms underlying this deficiency in AGM HSCs are poorly understood. By co-expression gene network analysis, we deduced that AGM HSCs show lower levels of interferon-α (IFN-α)/Jak-Stat1-associated gene expression than FL HSCs. Treatment of AGM HSCs with IFN-α enhanced long-term hematopoietic engraftment and donor chimerism. Conversely, IFN-α receptor-deficient AGMs (Ifnαr1(-/-)), had significantly reduced donor chimerism. We identify adenine-thymine-rich interactive domain-3a (Arid3a), a factor essential for FL and B lymphopoiesis, as a key transcriptional co-regulator of IFN-α/Stat1 signaling. Arid3a occupies the genomic loci of Stat1 as well as several IFN-α effector genes, acting to regulate their expression. Accordingly, Arid3a(-/-) AGM HSCs had significantly reduced transplant potential, which was rescued by IFN-α treatment. Our results implicate the inflammatory IFN-α/Jak-Stat pathway in the developmental maturation of embryonic HSCs, whose manipulation may lead to increased potency of reprogrammed HSCs for transplantation.

Engineered Murine HSCs Reconstitute Multi-lineage Hematopoiesis and Adaptive Immunity

Hematopoietic stem cell (HSC) transplantation is curative for malignant and genetic blood disorders, but is limited by donor availability and immune-mismatch. Deriving HSCs from patient-matched embryonic/induced-pluripotent stem cells (ESCs/iPSCs) could address these limitations. Prior efforts in murine models exploited ectopic HoxB4 expression to drive self-renewal and enable multi-lineage reconstitution, yet fell short in delivering robust lymphoid engraftment. Here, by titrating exposure of HoxB4-ESC-HSC to Notch ligands, we report derivation of engineered HSCs that self-renew, repopulate multi-lineage hematopoiesis in primary and secondary engrafted mice, and endow adaptive immunity in immune-deficient recipients. Single-cell analysis shows that following engraftment in the bone marrow niche, these engineered HSCs further specify to a hybrid cell type, in which distinct gene regulatory networks of hematopoietic stem/progenitors and differentiated hematopoietic lineages are co-expressed. Our work demonstrates engineering of fully functional HSCs via modulation of genetic programs that govern self-renewal and lineage priming.

Corrigendum: Failure to replicate the STAP cell phenomenon.

This corrects the article DOI: 10.1038/nature15513