Alejandro De Los Angeles

Optimization of scarless human stem cell genome editing

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

Hallmarks of pluripotency

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.

Accessing naïve human pluripotency

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: naïve and primed. Naïve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the naïve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether naïve human pluripotent stem cells with bona fide features of both a pluripotent state and naïve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of naïve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture naïve pluripotency in human cells. Unraveling how to stably induce naïve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.

NF-κB activation impairs somatic cell reprogramming in ageing.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor–Guillermo progeria syndrome and Hutchinson–Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Distinct and Combinatorial Functions of Jmjd2b/Kdm4b and Jmjd2c/Kdm4c in Mouse Embryonic Stem Cell Identity

Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.

Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition

During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin+ cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.

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

Genome-Wide Profiling of Pluripotent Cells Reveals a Unique Molecular Signature of Human Embryonic Germ Cells

Human embryonic germ cells (EGCs) provide a powerful model for identifying molecules involved in the pluripotent state when compared to their progenitors, primordial germ cells (PGCs), and other pluripotent stem cells. Microarray and Principal Component Analysis (PCA) reveals for the first time that human EGCs possess a transcription profile distinct from PGCs and other pluripotent stem cells. Validation with qRT-PCR confirms that human EGCs and PGCs express many pluripotency-associated genes but with quantifiable differences compared to pluripotent embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), and embryonal carcinoma cells (ECCs). Analyses also identified a number of target genes that may be potentially associated with their unique pluripotent states. These include IPO7, MED7, RBM26, HSPD1, and KRAS which were upregulated in EGCs along with other pluripotent stem cells when compared to PGCs. Other potential target genes were also found which may contribute toward a primed ESC-like state. These genes were exclusively up-regulated in ESCs, IPSCs and ECCs including PARP1, CCNE1, CDK6, AURKA, MAD2L1, CCNG1, and CCNB1 which are involved in cell cycle regulation, cellular metabolism and DNA repair and replication. Gene classification analysis also confirmed that the distinguishing feature of EGCs compared to ESCs, ECCs, and IPSCs lies primarily in their genetic contribution to cellular metabolism, cell cycle, and cell adhesion. In contrast, several genes were found upregulated in PGCs which may help distinguish their unipotent state including HBA1, DMRT1, SPANXA1, and EHD2. Together, these findings provide the first glimpse into a unique genomic signature of human germ cells and pluripotent stem cells and provide genes potentially involved in defining different states of germ-line pluripotency.

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA.

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells.

Direct Reprogramming of Human Primordial Germ Cells into Induced Pluripotent Stem Cells: Efficient Generation of Genetically Engineered Germ Cells.

Primordial germ cells (PGCs) share many properties with embryonic stem cells (ESCs) and innately express several key pluripotency-controlling factors, including OCT4, NANOG, and LIN28. Therefore, PGCs may provide a simple and efficient model for studying somatic cell reprogramming to induced pluripotent stem cells (iPSCs), especially in determining the regulatory mechanisms that fundamentally define pluripotency. Here, we report a novel model of PGC reprogramming to generate iPSCs via transfection with SOX2 and OCT4 using integrative lentiviral. We also show the feasibility of using nonintegrative approaches for generating iPSC from PGCs using only these two factors. We show that human PGCs express endogenous levels of KLF4 and C-MYC protein at levels similar to embryonic germ cells (EGCs) but lower levels of SOX2 and OCT4. Transfection with both SOX2 and OCT4 together was required to induce PGCs to a pluripotent state at an efficiency of 1.71%, and the further addition of C-MYC increased the efficiency to 2.33%. Immunohistochemical analyses of the SO-derived PGC-iPSCs revealed that these cells were more similar to ESCs than EGCs regarding both colony morphology and molecular characterization. Although leukemia inhibitory factor (LIF) was not required for the generation of PGC-iPSCs like EGCs, the presence of LIF combined with ectopic exposure to C-MYC yielded higher efficiencies. Additionally, the SO-derived PGC-iPSCs exhibited differentiation into representative cell types from all three germ layers in vitro and successfully formed teratomas in vivo. Several lines were generated that were karyotypically stable for up to 24 subcultures. Their derivation efficiency and survival in culture significantly supersedes that of EGCs, demonstrating their utility as a powerful model for studying factors regulating pluripotency in future studies.