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Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1–derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.

Summary Cellular identity is ultimately dictated by the interaction of transcription factors with regulatory elements (REs) to control gene expression. Advances in epigenome profiling techniques have significantly increased our understanding of cell-specific utilization of REs. However, it remains difficult to dissect the majority of factors that interact with these REs due to the lack of appropriate techniques. Therefore, we developed TINC: TALE-mediated isolation of nuclear chromatin. Using this new method, we interrogated the protein complex formed at the Nanog promoter in embryonic stem cells (ESCs) and identified many known and previously unknown interactors, including RCOR2. Further interrogation of the role of RCOR2 in ESCs revealed its involvement in the repression of lineage genes and the fine-tuning of pluripotency genes. Consequently, using the Nanog promoter as a paradigm, we demonstrated the power of TINC to provide insight into the molecular makeup of specific transcriptional complexes at individual REs as well as into cellular identity control in general., Graphical Abstract, Highlights • TINC allows the isolation of a specific locus for molecular analyses • TINC identified hundreds of proteins at the Nanog promoter • RCOR2 is a component of the pluripotency network in embryonic stem cells • RCOR2 is required for efficient differentiation, Here, Knaupp and colleagues describe TINC, an epigenetic method that allows interrogation of mammalian regulatory complexes at a single-locus resolution. TINC was applied to dissect the transcriptional complex at the Nanog promoter in embryonic stem cells, revealing hundreds of interactors, including RCOR2, hence redefining how this gene is regulated in pluripotency.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development 1–6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas 7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

Genome wide association studies provide statistical measures of gene–trait associations that reveal how genetic variation influences phenotypes. This study develops an unsupervised dimensionality reduction method called UnTANGLeD (Unsupervised Trait Analysis of Networks from Gene Level Data) which organizes 16,849 genes into discrete gene programs by measuring the statistical association between genetic variants and 1,393 diverse complex traits. UnTANGLeD reveals 173 gene clusters enriched for protein–protein interactions and highly distinct biological processes governing development, signalling, disease, and homeostasis. We identify diverse gene networks with robust interactions but not associated with known biological processes. Analysis of independent disease traits shows that UnTANGLeD gene clusters are conserved across all complex traits, providing a simple and powerful framework to predict novel gene candidates and programs influencing orthogonal disease phenotypes. Collectively, this study demonstrates that gene programs co-ordinately orchestrating cell functions can be identified without reliance on prior knowledge, providing a method for use in functional annotation, hypothesis generation, machine learning and prediction algorithms, and the interpretation of diverse genomic data.

Macrophages are key cellular contributors to COVID-19 pathogenesis. Whether SARS-CoV-2 can enter macrophages, replicate and release new viral progeny remains controversial. Similarly, whether macrophages need to sense replicating virus to drive cytokine release is also unclear. Macrophages are heterogeneous cells poised to respond to their local microenvironment, and accordingly, the SARS-CoV-2 entry receptor ACE2 is only present on a subset of macrophages at sites of human infection. Here, we use in vitro approaches to investigate how SARS-CoV-2 interacts with ACE2-negative and ACE2-positive human macrophages and determine how these macrophage populations sense and respond to SARS-CoV-2. We show that SARS-CoV-2 does not replicate within ACE2-negative human macrophages and does not induce pro-inflammatory cytokine expression. By contrast, ACE2 expression in human macrophages permits SARS-CoV-2 entry, replication, and virion release. ACE2-expressing macrophages sense replicating virus to trigger pro-inflammatory and anti-viral programs that limit virus release. These combined findings resolve several controversies regarding macrophage-SARS-CoV-2 interactions and identify a signaling circuit by which macrophages sense SARS-CoV-2 cell entry and respond by restricting viral replication.One sentence summaryLack of macrophage ACE2 expression precludes SARS-CoV-2 entry and sensing, while ACE2-expressing macrophages sense intramacrophage SARS-CoV-2 replication to induce rapid anti-viral responses that limit new virion release.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus–based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML., This study shows that the closely related transcription factors ZEB1 and ZEB2 cooperate to restrain myeloid and lymphoid differentiation programs in hematopoietic stem and progenitor cells, ensuring fidelity of differentiation in multiple lineages.

The vertebral column of individual mammalian species often exhibits remarkable robustness in the number and identity of vertebral elements that form (known as axial formulae). The genetic mechanism(s) underlying this constraint however remain ill-defined. Here, we reveal the interplay of three regulatory pathways (Gdf11, miR-196 and Retinoic acid) is essential in constraining total vertebral number and regional axial identity in the mouse, from cervical through to tail vertebrae. All three pathways have differing control over Hox cluster expression, with heterochronic and quantitative changes found to parallel changes in axial identity. However, our work reveals an additional role for Hox genes in supporting axial elongation within the tail region, providing important support for an emerging view that mammalian Hox function is not limited to imparting positional identity as the mammalian body plan is laid down. More broadly, this work provides a molecular framework to interrogate mechanisms of evolutionary change and congenital anomalies of the vertebral column., Vertebral column length and shape exhibits remarkable robustness within a species but diversity across species. Here the authors reveal the molecular logic constraining vertebral number in mouse and a novel role for posterior Hox genes in this context.

The impact of aging on intestinal stem cells (ISCs) has not been fully elucidated. In this study, we identified widespread epigenetic and transcriptional alterations in old ISCs. Using a reprogramming algorithm, we identified a set of key transcription factors (Egr1, Irf1, FosB) that drives molecular and functional differences between old and young states. Overall, by dissecting the molecular signature of aged ISCs, our study identified transcription factors that enhance the regenerative capacity of ISCs.

DNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

Background: Stem cells-derived neuronal cultures hold great promise for in vitro disease modelling and drug screening. However, currently stem cells-derived neuronal cultures do not recapitulate the functional properties of primary neurons, such as network properties. Cultured primary murine neurons develop networks which are synchronised over large fractions of the culture, whereas neurons derived from mouse embryonic stem cells (ESCs) display only partly synchronised network activity and human pluripotent stem cells-derived neurons have mostly asynchronous network properties. Therefore, strategies to improve correspondence of derived neuronal cultures with primary neurons need to be developed to validate the use of stem cell-derived neuronal cultures as in vitro models.New method: By combining serum-free derivation of ESCs from mouse blastocysts with neuronal differentiation of ESCs in morphogen-free adherent culture we generated neuronal networks with properties recapitulating those of mature primary cortical cultures.Results: After 35 days of differentiation ESC-derived neurons developed network activity very similar to that of mature primary cortical neurons. Importantly, ESC plating density was critical for network development. Comparison with existing method(s): Compared to the previously published methods this protocol generated more synchronous neuronal networks, with high similarity to the networks formed in mature primary cortical culture.Conclusion: We have demonstrated that ESC-derived neuronal networks recapitulating key properties of mature primary cortical networks can be generated by optimising both stem cell derivation and differentiation. This validates the approach of using ESC-derived neuronal cultures for disease modelling and in vitro drug screening. (C) 2017 Elsevier B.V. All rights reserved.

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

Intestinal stem cells are responsible for the extremely rapid renewal of the entire epithelial lining. The maintenance of the critical absorptive function of the gut relies on the appropriate control of the stem cell pool by the local micro-environment or niche cells. Aging causes dramatic changes in the local regulation of stem cells and alters their function, resulting in absorptive defects and development of diseases. Mechanistically, aging induces both profound cellular and molecular intrinsic alterations of the stem cell pool and mediates aberrant behavior of their associated niche cells. Importantly, some of these identified changes can be therapeutically targeted, which offers the possibility to restore gut function in aging.

With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types.

Reprogrammable mouse models engineered to conditionally express Oct-4, Klf-4, Sox-2 and c-Myc (OKSM) have been instrumental in dissecting molecular events underpinning the generation of induced pluripotent stem cells. However, until now these models have been reported in the context of the m2 reverse tetracycline-controlled transactivator, which results in low reprogramming efficiency and consequently limits the number of reprogramming intermediates that can be isolated for downstream profiling. Here, we describe an improved OKSM mouse model in the context of the reverse tetracycline-controlled transactivator 3 with enhanced reprogramming efficiency (>9-fold) and increased numbers of reprogramming intermediate cells albeit with similar kinetics, which we believe will facilitate mechanistic studies of the reprogramming process.

Epidermal growth factor (EGF) maintains intestinal stem cell (ISC) proliferation and is a key component of organoid growth media yet is dispensable for intestinal homeostasis, suggesting roles for multiple EGF family ligands in ISC function. Here, we identified neuregulin 1 (NRG1) as a key EGF family ligand that drives tissue repair following injury. NRG1, but not EGF, is upregulated upon damage and is expressed in mesenchymal stromal cells, macrophages, and Paneth cells. NRG1 deletion reduces proliferation in intestinal crypts and compromises regeneration capacity. NRG1 robustly stimulates proliferation in crypts and induces budding in organoids, in part through elevated and sustained activation of mitogen-activated protein kinase (MAPK) and AKT. Consistently, NRG1 treatment induces a proliferative gene signature and promotes organoid formation from progenitor cells and enhances regeneration following injury. These data suggest mesenchymal-derived NRG1 is a potent mediator of tissue regeneration and may inform the development of therapies for enhancing intestinal repair after injury.

Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irradiated mouse embryonic fibroblasts (iMEFs). This culture system reliably sustains mESC pluripotency and self-renewal capacity, allowing their use in a wide range of experimental settings.

Lentiviral gene transfer technologies exploit the natural efficiency of viral transduction to integrate exogenous genes into mammalian cells. This provides a simple research tool for inducing transgene expression or endogenous gene knockdown in both dividing and nondividing cells. This chapter describes an improved protocol for polyethylenimine (PEI)-mediated multi-plasmid transfection and polyethylene glycol (PEG) precipitation to generate and concentrate lentiviral vectors.

Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of four transcription factors: Oct4, Klf4, Sox2, and c-Myc (OKSM). These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) the characterization of the pluripotent potential of iPS cell lines using alkaline phosphatase staining, flow cytometry, and the teratoma formation assays.

Mouse embryonic stem cells (mESCs) are pluripotent cells derived from preimplantation embryos that have the capacity to self-renew indefinitely in vitro. mESCs are an indispensable tool for studying cellular differentiation in vitro, generating disease in a dish models, and have been used extensively for the generation of transgenic animals. Therefore, maintaining their pluripotent state, even after extended culture, is crucial for their utility. Herein, we describe in detail a protocol for the culture of mESCs in the presence of fetal calf serum (FCS), leukemia inhibitory factor (LIF), and a layer of irradiated mouse embryonic fibroblasts (iMEFs). This culture system reliably sustains mESC pluripotency and self-renewal capacity, allowing their use in a wide range of experimental settings.

Lentiviral gene transfer technologies exploit the natural efficiency of viral transduction to integrate exogenous genes into mammalian cells. This provides a simple research tool for inducing transgene expression or endogenous gene knockdown in both dividing and nondividing cells. This chapter describes an improved protocol for polyethylenimine (PEI)-mediated multi-plasmid transfection and polyethylene glycol (PEG) precipitation to generate and concentrate lentiviral vectors.

Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of four transcription factors: Oct4, Klf4, Sox2, and c-Myc (OKSM). These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) the characterization of the pluripotent potential of iPS cell lines using alkaline phosphatase staining, flow cytometry, and the teratoma formation assays.

Peripheral blood mononuclear cells were donated by a male teenager with clinically diagnosed Attention Deficit Hyperactivity Disorder under the Diagnostic and Statistical Manual of Mental Disorders IV criteria and his unaffected male sibling. Induced pluripotent stem cells were developed using integration-free Sendai Reprogramming factors containing OCT, SOX2, KLF4, and c-MYC. All four iPSC lines displayed pluripotent cell morphology, pluripotency-associated factors at protein level, alkaline phosphatase enzymatic activity, male karyotype of 46, XY, and in vitro differentiation capacity into all the three germ layers and negative for Mycoplasma.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

The establishment and maintenance of pluripotency depend on precise coordination of gene expression. We establish serine-arginine-rich splicing factor 3 (SRSF3) as an essential regulator of RNAs encoding key components of the mouse pluripotency circuitry, SRSF3 ablation resulting in the loss of pluripotency and its overexpression enhancing reprogramming. Strikingly, SRSF3 binds to the core pluripotency transcription factor Nanog mRNA to facilitate its nucleo-cytoplasmic export independent of splicing. In the absence of SRSF3 binding, Nanog mRNA is sequestered in the nucleus and protein levels are severely downregulated. Moreover, SRSF3 controls the alternative splicing of the export factor Nxf1 and RNA regulators with established roles in pluripotency, and the steady-state levels of mRNAs encoding chromatin modifiers. Our investigation links molecular events to cellular functions by demonstrating how SRSF3 regulates the pluripotency genes and uncovers SRSF3-RNA interactions as a critical means to coordinate gene expression during reprogramming, stem cell self-renewal and early development.

Transdifferentiation, the process of converting from one cell type to another without going through a pluripotent state, has great promise for regenerative medicine. The identification of key transcription factors for reprogramming is currently limited by the cost of exhaustive experimental testing of plausible sets of factors, an approach that is inefficient and unscalable. Here we present a predictive system (Mogrify) that combines gene expression data with regulatory network information to predict the reprogramming factors necessary to induce cell conversion. We have applied Mogrify to 173 human cell types and 134 tissues, defining an atlas of cellular reprogramming. Mogrify correctly predicts the transcription factors used in known transdifferentiations. Furthermore, we validated two new transdifferentiations predicted by Mogrify. We provide a practical and efficient mechanism for systematically implementing novel cell conversions, facilitating the generalization of reprogramming of human cells. Predictions are made available to help rapidly further the field of cell conversion.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1− Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues., Sustained spermatogenesis depends on stem cell activity which is contained within a population of undifferentiated spermatogonia. Here, the authors identify a new population of undifferentiated spermatogonia in adult testis that expresses the transcription factor PDX1 and has stem cell capacity.

Our current understanding of induced pluripotent stem (iPS) cell generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether or not the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By functionally and molecularly characterizing the reprogramming pathways of fibroblasts, neutrophils and keratinocytes, we unveil that while some aspects of the reprogramming process are conserved, the key events, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion and reactivation of the pluripotency network are to a large degree cell type specific. Thus we reveal universal and cell type specific features of the reprogramming process.

Summary: Our current understanding of induced pluripotent stem cell (iPSC) generation has almost entirely been shaped by studies performed on reprogramming fibroblasts. However, whether the resulting model universally applies to the reprogramming process of other cell types is still largely unknown. By characterizing and profiling the reprogramming pathways of fibroblasts, neutrophils, and keratinocytes, we unveil that key events of the process, including loss of original cell identity, mesenchymal to epithelial transition, the extent of developmental reversion, and reactivation of the pluripotency network, are to a large degree cell-type specific. Thus, we reveal limitations for the use of fibroblasts as a universal model for the study of the reprogramming process and provide crucial insights about iPSC generation from alternative cell sources. : Nefzger et al. find that the molecular reprogramming trajectories of fibroblasts, neutrophils, and keratinocytes have a cell-type-specific component that only fully converges in induced pluripotent stem cells. The authors also identify universal changes shared by all three cell types, including two transcriptional waves and a conserved transcriptional program involving Egr1 downregulation. Keywords: reprogramming, induced pluripotent stem cells, fibroblasts, neutrophils, keratinocytes, transcriptional dynamics, Egr1

Induced pluripotent stem cells (iPSCs) are characterised by their ability to differentiate into any cell type of the body. Accordingly, iPSCs possess immense potential for disease modelling, pharmaceutical screening and autologous cell therapies. The most common source of iPSCs derivation is skin fibroblasts. However, from a clinical point of view, skin fibroblasts may not be ideal, as invasive procedures such as skin biopsies are required for their extraction. Moreover, fibroblasts are highly heterogeneous with a poorly defined developmental pathway, which makes studying reprogramming mechanistics difficult. Granulocytes, on the other hand, are easily obtainable, their developmental pathway has been extensively studied and fluorescence activated cell sorting allows for the isolation of these cells at high purity; thus iPSCs derivation from granulocytes could provide an alternative to fibroblast-derived iPSCs. Previous studies succeeded in producing iPSC colonies from mouse granulocytes but with the use of a mitotically inactivated feeder layer, restricting their use for studying reprogramming mechanistics. As granulocytes display poor survival under culture conditions, we investigated the influence of haematopoietic cytokines to stabilise this cell type in vitro and allow for reprogramming in the absence of a feeder layer. Our results show that treatment with MEF-conditioned media and/or initial exposure to GM-CSF allows for reprogramming of granulocytes under feeder-free conditions. This work can serve as a basis for future work aimed at dissecting the reprogramming mechanism as well as obtaining large numbers of iPSCs from a clinically relevant cell source.

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.

Efficient homing of hematopoietic stem cells (HSC) from blood to the bone marrow (BM) following transplant is critical for achieving optimal engraftment. Indeed, HSC mobilized using the growth factor G-CSF are known to have reduced engraftment potential compared to native BM HSC, potentially due to impaired homing ability. Thus, the characterization of novel homing mechanisms will allow identification of improved mobilization strategies that are not solely reliant on HSC number but also HSC quality. In this study, we demonstrate junctional adhesion molecule A (JAM-A) has multi-faceted roles in HSC regulation during steady state and following mobilization. Specifically, JAM-A is critical for HSC homing and engraftment post-transplant and contributes to the maintenance of HSC quiescence in the endosteal niche. Furthermore, G-CSF mobilization significantly reduces JAM-A expression on HSC as a result of JAM-A cleavage by the enzyme TACE, which is upregulated following G-CSF. In addition, the significantly impaired homing potential of HSC mobilized with G-CSF is attributed to lower levels of JAM-A, CXCR4 as well as α4β1 and α9β1 integrins. In contrast, we show that HSC mobilized using the combination of the α4β1/α9β1 integrin antagonist BOP and the CXCR4 antagonist AMD3100 had significantly greater homing and long-term multi-lineage engraftment potential; in part due to higher levels of JAM-A, CXCR4 and α4β1/α9β1 integrins on mobilized cells. In addition, compared to G-CSF, which requires multiple doses over several days, mobilization with BOP plus AMD3100 occurred 1h following a single injection. Thus, our results identify JAM-A as a critical regulator of stem and progenitor trafficking to and maintenance in the BM and suggests the use of G-CSF independent mobilization regimes that do not abrogate JAM-A expression/function should result in improved stem cell transplants.

Summary Self-renewal and pluripotency of the embryonic stem cell (ESC) state are established and maintained by multiple regulatory networks that comprise transcription factors and epigenetic regulators. While much has been learned regarding transcription factors, the function of epigenetic regulators in these networks is less well defined. We conducted a CRISPR-Cas9-mediated loss-of-function genetic screen that identified two epigenetic regulators, TAF5L and TAF6L, components or co-activators of the GNAT-HAT complexes for the mouse ESC (mESC) state. Detailed molecular studies demonstrate that TAF5L/TAF6L transcriptionally activate c-Myc and Oct4 and their corresponding MYC and CORE regulatory networks. Besides, TAF5L/TAF6L predominantly regulate their target genes through H3K9ac deposition and c-MYC recruitment that eventually activate the MYC regulatory network for self-renewal of mESCs. Thus, our findings uncover a role of TAF5L/TAF6L in directing the MYC regulatory network that orchestrates gene expression programs to control self-renewal for the maintenance of mESC state.

Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.

The canonical Wnt/β-catenin pathway is crucial for early embryonic patterning, tissue homeostasis, and regeneration. While canonical Wnt/β-catenin stimulation has been used extensively to modulate pluripotency and differentiation of pluripotent stem cells (PSCs), the mechanism of these two seemingly opposing roles has not been fully characterized and is currently largely attributed to activation of nuclear Wnt target genes. Here, we show that low levels of Wnt stimulation via ectopic expression of Wnt1 or administration of glycogen synthase kinase-3 inhibitor CHIR99021 significantly increases PSC differentiation into neurons, cardiomyocytes and early endodermal intermediates. Our data indicate that enhanced differentiation outcomes are not mediated through activation of traditional Wnt target genes but by β-catenin's secondary role as a binding partner of membrane bound cadherins ultimately leading to the activation of developmental genes. In summary, fine-tuning of Wnt signaling to subthreshold levels for detectable nuclear β-catenin function appears to act as a switch to enhance differentiation of PSCs into multiple lineages. Our observations highlight a mechanism by which Wnt/β-catenin signaling can achieve dosage dependent dual roles in regulating self-renewal and differentiation.