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Vascular retinopathy, characterized by abnormal blood vessel growth in the retina, frequently results in vision impairment or loss. Neovascular tufts, a distinctive pathologic feature of this condition, are highly leaky blood vessel structures, exacerbating secondary complications. Despite their clinical significance, the mechanisms underlying tuft development are not fully elucidated, posing challenges for effective management and treatment of vascular retinopathy. This study investigates the role of c-Src in neovascular tuft formation. Although c-Src has been acknowledged as a pivotal regulator in developmental angiogenesis within the retinal vasculature, its specific role in governing pathologic retinal angiogenesis remains to be fully understood. The oxygen-induced retinopathy model was used for neovascular tuft formation in both Cre-mediated vascular-specific c-Src knockout mice and wild-type littermates. High-resolution imaging and analysis of isolated retinas were conducted. c-Src depletion demonstrated a significant reduction in neovascular tufts within the oxygen-induced retinopathy model. This decrease in tuft formation was observed independently of any alterations in cell death, cell proliferation, or cell adhesion, and the absence of c-Src did not impact tuft pericyte coverage and junctional morphology. These findings underline the critical role of c-Src in the pathogenesis of neovascular tufts in vascular retinopathy. Understanding the molecular mechanisms involving c-Src may offer valuable insights for the development of targeted therapies aimed at mitigating vision-threatening complications associated with retinopathy., Competing Interests: Disclosure Statement None declared., (Copyright © 2024. Published by Elsevier Inc.)

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human cell reprogramming traditionally involves time-intensive, multistage, costly tissue culture polystyrene-based cell culture practices that ultimately produce low numbers of reprogrammed cells of variable quality. Previous studies have shown that very soft 2- and 3-dimensional hydrogel substrates/matrices (of stiffnesses ≤ 1 kPa) can drive ~2× improvements in human cell reprogramming outcomes. Unfortunately, these similarly complex multistage protocols lack intrinsic scalability, and, furthermore, the associated underlying molecular mechanisms remain to be fully elucidated, limiting the potential to further maximize reprogramming outcomes. In screening the largest range of polyacrylamide (pAAm) hydrogels of varying stiffness to date (1 kPa to 1.3 MPa), we have found that a medium stiffness gel (~100 kPa) increased the overall number of reprogrammed cells by up to 10-fold (10×), accelerated reprogramming kinetics, improved both early and late phases of reprogramming, and produced induced pluripotent stem cells (iPSCs) having more naïve characteristics and lower remnant transgene expression, compared to the gold standard tissue culture polystyrene practice. Functionalization of these pAAm hydrogels with poly-l-dopamine enabled, for the first-time, continuous, single-step reprogramming of fibroblasts to iPSCs on hydrogel substrates (noting that even the tissue culture polystyrene practice is a 2-stage process). Comparative RNA sequencing analyses coupled with experimental validation revealed that a novel reprogramming regulator, protein phosphatase and actin regulator 3, up-regulated under the gel condition at a very early time point, was responsible for the observed enhanced reprogramming outcomes. This study provides a novel culture protocol and substrate for continuous hydrogel-based cell reprogramming and previously unattained clarity of the underlying mechanisms via which substrate stiffness modulates reprogramming kinetics and iPSC quality outcomes., Competing Interests: Competing interests: The authors declare that they have no competing interests., (Copyright © 2024 Mohammad Mahfuz et al.)

Wnt signaling is a critical determinant of cell lineage development. This study used Wnt dose-dependent induction programs to gain insights into molecular regulation of stem cell differentiation. We performed single-cell RNA sequencing of hiPSCs responding to a dose escalation protocol with Wnt agonist CHIR-99021 during the exit from pluripotency to identify cell types and genetic activity driven by Wnt stimulation. Results of activated gene sets and cell types were used to build a multiple regression model that predicts the efficiency of cardiomyocyte differentiation. Cross-referencing Wnt-associated gene expression profiles to the Connectivity Map database, we identified the small-molecule drug, tranilast. We found that tranilast synergistically activates Wnt signaling to promote cardiac lineage differentiation, which we validate by in vitro analysis of hiPSC differentiation and in vivo analysis of developing quail embryos. Our study provides an integrated workflow that links experimental datasets, prediction models, and small-molecule databases to identify drug-like compounds that control cell differentiation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function 1-8 . These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory., (© 2023. The Author(s).)

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.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3 Δ/Δ ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans., (© 2023. Crown.)

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Chromatin falls into one of two major subtypes: closed heterochromatin and euchromatin which is accessible, transcriptionally active, and occupied by transcription factors (TFs). The most widely used approach to interrogate differences in the chromatin state landscape is the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). While library generation is relatively inexpensive, sequencing depth requirements can make this assay cost-prohibitive for some laboratories. Findings: Here, we benchmark data from Beijing Genomics Institute's (BGI) DNBSEQ-G400 low-cost sequencer against data from a standard Illumina instrument (HiSeqX10). For comparisons, the same bulk ATAC-seq libraries generated from pluripotent stem cells (PSCs) and fibroblasts were sequenced on both platforms. Both instruments generate sequencing reads with comparable mapping rates and genomic context. However, DNBSEQ-G400 data contained a significantly higher number of small, sub-nucleosomal reads (>30% increase) and a reduced number of bi-nucleosomal reads (>75% decrease), which resulted in narrower peak bases and improved peak calling, enabling the identification of 4% more differentially accessible regions between PSCs and fibroblasts. The ability to identify master TFs that underpin the PSC state relative to fibroblasts (via HOMER, HINT-ATAC, TOBIAS), namely, foot-printing capacity, were highly similar between data generated on both platforms. Integrative analysis with transcriptional data equally enabled direct recovery of three published 3-factor combinations that have been shown to induce pluripotency. Conclusion: Other than a small increase in peak calling sensitivity for DNBSEQ-G400 data (BGI), both platforms enable comparable levels of open chromatin identification for ATAC-seq library sequencing, yielding similar analytical outcomes, albeit at low-data generation costs in the case of the BGI instrument., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Naval-Sanchez, Deshpande, Tran, Zhang, Alhomrani, Alsanie, Nguyen and Nefzger.)

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., (© 2022. The Author(s).)

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly., (© 2022. The Author(s).)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Regulatory T cell (Treg) reconstitution is essential for reestablishing tolerance and maintaining homeostasis following stem-cell transplantation. We previously reported that bone marrow (BM) is highly enriched in autophagy-dependent Treg and autophagy disruption leads to a significant Treg loss, particularly BM-Treg. To correct the known Treg deficiency observed in chronic graft-versus-host disease (cGVHD) patients, low dose IL-2 infusion has been administered, substantially increasing peripheral Treg (pTreg) numbers. However, as clinical responses were only seen in ∼50% of patients, we postulated that pTreg augmentation was more robust than for BM-Treg. We show that BM-Treg and pTreg have distinct characteristics, indicated by differential transcriptome expression for chemokine receptors, transcription factors, cell cycle control of replication and genes linked to Treg function. Further, BM-Treg were more quiescent, expressed lower FoxP3, were highly enriched for co-inhibitory markers and more profoundly depleted than splenic Treg in cGVHD mice. In vivo our data are consistent with the BM and not splenic microenvironment is, at least in part, driving this BM-Treg signature, as adoptively transferred splenic Treg that entered the BM niche acquired a BM-Treg phenotype. Analyses identified upregulated expression of IL-9R, IL-33R, and IL-7R in BM-Treg. Administration of the T cell produced cytokine IL-2 was required by splenic Treg expansion but had no impact on BM-Treg, whereas the converse was true for IL-9 administration. Plasmacytoid dendritic cells (pDCs) within the BM also may contribute to BM-Treg maintenance. Using pDC-specific BDCA2-DTR mice in which diptheria toxin administration results in global pDC depletion, we demonstrate that pDC depletion hampers BM, but not splenic, Treg homeostasis. Together, these data provide evidence that BM-Treg and splenic Treg are phenotypically and functionally distinct and influenced by niche-specific mediators that selectively support their respective Treg populations. The unique properties of BM-Treg should be considered for new therapies to reconstitute Treg and reestablish tolerance following SCT., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nicholls, Cao, Le Texier, Xiong, Hunter, Llanes, Aguliar, Schroder, Phipps, Lynch, Cao, Heazlewood, Williams, Clouston, Nefzger, Polo, Nilsson, Blazar and MacDonald.)

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.

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., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 (ADHD) 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 OCT4, SOX2, KLF4, and c-MYC. All four iPSC lines displayed pluripotent cell morphology, pluripotency-associated factors at the DNA and protein level, alkaline phosphatase enzymatic activity and a male karyotype of 46, XY. All lines had capacity for in vitro differentiation into all the three germ layers. All were negative for Mycoplasma., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (A undiff ); however, only a fraction of this population act as stem cells in undisturbed testis and A undiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult A undiff fractions and dissect A undiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing A undiff 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- A undiff states upon transplant and culture. We conclude that A undiff 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.

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. Stem Cells 2018;36:822-833., (© AlphaMed Press 2018.)

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., Competing Interests: MR, SA, CD, IR, TB, AK, CN, JP, MA No competing interests declared, (© 2018, Ratnadiwakara et al.)

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 35days 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Somatic cell reprogramming into induced pluripotent stem cells (iPSCs) induces changes in genome architecture reflective of the embryonic stem cell (ESC) state. However, only a small minority of cells typically transition to pluripotency, which has limited our understanding of the process. Here, we characterize the DNA regulatory landscape during reprogramming by time-course profiling of isolated sub-populations of intermediates poised to become iPSCs. Widespread reconfiguration of chromatin states and transcription factor (TF) occupancy occurs early during reprogramming, and cells that fail to reprogram partially retain their original chromatin states. A second wave of reconfiguration occurs just prior to pluripotency acquisition, where a majority of early changes revert to the somatic cell state and many of the changes that define the pluripotent state become established. Our comprehensive characterization of reprogramming-associated molecular changes broadens our understanding of this process and sheds light on how TFs access and change the chromatin during cell-fate transitions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The Authors. Published by Elsevier Inc. 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.

This corrects the article DOI: 10.1038/ni.3713.

Gut dysbiosis might underlie the pathogenesis of type 1 diabetes. In mice of the non-obese diabetic (NOD) strain, we found that key features of disease correlated inversely with blood and fecal concentrations of the microbial metabolites acetate and butyrate. We therefore fed NOD mice specialized diets designed to release large amounts of acetate or butyrate after bacterial fermentation in the colon. Each diet provided a high degree of protection from diabetes, even when administered after breakdown of immunotolerance. Feeding mice a combined acetate- and butyrate-yielding diet provided complete protection, which suggested that acetate and butyrate might operate through distinct mechanisms. Acetate markedly decreased the frequency of autoreactive T cells in lymphoid tissues, through effects on B cells and their ability to expand populations of autoreactive T cells. A diet containing butyrate boosted the number and function of regulatory T cells, whereas acetate- and butyrate-yielding diets enhanced gut integrity and decreased serum concentration of diabetogenic cytokines such as IL-21. Medicinal foods or metabolites might represent an effective and natural approach for countering the numerous immunological defects that contribute to T cell-dependent autoimmune diseases.

The role of RNA binding proteins (RBPs) during nuclear reprogramming is poorly characterized. In this issue of Cell Stem Cell,Li et al. (2017) show that DEAD-box RBP DDX5 acts as a reprogramming roadblock and give important mechanistic insights into the establishment of pluripotency by characterizing the intricate downstream events., (Copyright © 2017. Published by Elsevier Inc.)

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. Stem Cells 2017;35:626-640., (© 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

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., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The isolation of pure populations of mouse intestinal stem cells (ISCs) is essential to facilitate functional studies of tissue homeostasis, tissue regeneration, and intestinal diseases. However, the purification of ISCs has relied predominantly on the use of transgenic reporter alleles in mice. Here, we introduce a combinational cell surface marker-mediated strategy that allows the isolation of an ISC population transcriptionally and functionally equivalent to the gold standard Lgr5-GFP ISCs. Used on reporter-free mice, this strategy allows the isolation of functional, transcriptionally distinct ISCs uncompromised by Lgr5 haploinsufficiency., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Snail family members regulate epithelial-to-mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation-induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage., (© 2015 The Authors.)

Mature cells of the body can be reprogrammed towards a pluripotent state by forced expression of the transcription factors Oct-4, Klf-4, Sox2, and C-Myc (OKSM) at very low efficiency. To study the reprogramming process in detail the rare intermediates of the reaction need to be separated from the bulk population. Using a genetically engineered reprogrammable mouse strain we describe how to isolate intermediates from reprogramming cultures of mouse embryonic fibroblasts via antibody labeling of cell surface markers and fluorescence-activated cell sorting (FACS).

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

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., (Copyright © 2014. Published by Elsevier B.V.)

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., (Copyright © 2013 AlphaMed Press.)

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.

Factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is inefficient, complicating mechanistic studies. Here, we examined defined intermediate cell populations poised to becoming iPSCs by genome-wide analyses. We show that induced pluripotency elicits two transcriptional waves, which are driven by c-Myc/Klf4 (first wave) and Oct4/Sox2/Klf4 (second wave). Cells that become refractory to reprogramming activate the first but fail to initiate the second transcriptional wave and can be rescued by elevated expression of all four factors. The establishment of bivalent domains occurs gradually after the first wave, whereas changes in DNA methylation take place after the second wave when cells acquire stable pluripotency. This integrative analysis allowed us to identify genes that act as roadblocks during reprogramming and surface markers that further enrich for cells prone to forming iPSCs. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming., (Copyright © 2012 Elsevier Inc. All rights reserved.)

LIM homeobox transcription factor 1 alpha (Lmx1a) is required for the development of midbrain dopaminergic neurons, roof plate formation, and cortical hem development. We generated a reporter embryonic stem cell (ESC) line for Lmx1a and used it to track differentiation and extract neural progenitors from differentiating mouse ESCs. Lmx1a(+) cells gave rise to functional cortical upper layer GABAergic neurons or dopaminergic neurons depending on the culture conditions used for differentiation. Under chemically defined neurobasal conditions, ESC differentiation resulted in widespread and transient expression of Lmx1a, without the addition of exogenous factors such as sonic hedgehog (Shh), Wnts, and/or bone morphogenic proteins (BMPs). Under neutral conditions, Lmx1a(+) cells express genes known to be downstream of Lmx1a and cortical hem markers Wnt3a and p73. The majority of these cells did not express the ventral midbrain dopaminergic marker Foxa2 or dorsal roof plate marker BMP-2. Lmx1a(+) -Foxa2(-) cells were primed to become SatB2(+) GABAergic neurons and appeared to be resistant to dopaminergic patterning cues. PA6 coculture produced a substantial population of Lmx1a(+) progenitors that also expressed Foxa2 and on further differentiation gave rise to dopaminergic neurons at high frequency. We conclude that Lmx1a is a useful marker for the extraction of progenitors of GABAergic or dopaminergic neurons. We caution against the assumption that it indicates dopaminergic commitment during in vitro differentiation of ESCs. Indeed, in monolayer culture under neurobasal conditions, with or without the addition of Shh and fibroblast growth factor 8 (FGF8), Lmx1a(+) cells were predominantly progenitors of forebrain GABAergic neurons. We obtained dopaminergic cells in large numbers only by coculture with PA6 cells., (Copyright © 2012 AlphaMed Press.)

Neurons differentiated in vitro from embryonic stem cells (ESCs) have the potential to serve both as models of disease states and in drug discovery programs. In this study, we use sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF-8) to enrich for forebrain and midbrain phenotypes from mouse ESCs. We then investigate, using Ca(2+) imaging and [(3)H]-GABA release studies, whether the GABAergic neurons produced exhibit distinct functional phenotypes. At day 24 of differentiation, reverse transcriptase-PCR showed the presence of both forebrain (Bf-1, Hesx1, Pgc-1α, Six3) and midbrain (GATA2, GATA3) selective mRNA markers in developing forebrain-enriched cultures. All markers were present in midbrain cultures except for Bf-1 and Pgc-1α. Irrespective of culture conditions all GABA immunoreactive neurons were also immunoreactive to neuropeptide Y (NPY) antibodies. Forebrain and midbrain GABAergic neurons responded to ATP (1 mM), L-glutamate (30 μM), noradrenaline (30 μM), acetylcholine (30 μM) and dopamine (30 μM), with similar elevations of intracellular Ca(2+)([Ca(2+)](i)). The presence of GABA(A) and GABA(B) antagonists, bicuculline (30 μM) and CGP55845 (1 μM), increased the elevation of [Ca(2+)](i) in response to dopamine (30 μM) in midbrain, but not forebrain GABAergic neurons. All agonists, except dopamine, elicited similar [(3)H]-GABA release from forebrain and midbrain cultures. Dopamine (30 μM) did not stimulate significant [(3)H]-GABA release in midbrain cultures, although it was effective in forebrain cultures. This study shows that differentiating neurons toward a midbrain fate restricts the expression of forebrain markers. Forebrain differentiation results in the expression of forebrain and midbrain markers. All GABA(+) neurons contain NPY, and show similar agonist-induced elevations of [Ca(2+)](i) and [(3)H]-GABA release. This study indicates that the pharmacological phenotype of these particular neurons may be independent of the addition of the patterning factors that direct neurons toward forebrain and midbrain fates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Investigation of serotonergic neuronal activity and its relationship to disease has been limited by a lack of physiologically relevant in vitro cell models. Serotonergic neurons derived from embryonic stem cells (ESCs) could provide a platform for such studies and provide models for use in drug discovery. Here, we report enhancement of serotonergic differentiation using a genetic approach. Expression of Gata2 increased the yield of serotonergic neurons. Enhancement was only achieved when Gata2 was expressed under the control of the tissue-specific promoter of the transcription factor Nkx6.1. High levels of Gata2 expression in ESCs compromised pluripotency and induced non-neuronal differentiation. Combined directed expression of Gata2, proneural gene Mash1, and forkhead transcription factor Foxa2 further enhanced serotonergic neural differentiation, resulting in a 10-fold increase in serotonin content. These neurons were also capable of depolarization (KCl, 30 mM)-induced elevations of intracellular Ca(2+) . The presence of sonic hedgehog during differentiation produced a further modest increase in numbers (1.5-fold). Transgene expression did not influence the number of tyrosine hydroxylase positive neurons in the cultures after 20 days, implying that Gata2, Mash1, and Foxa2 modulate in vitro differentiation at a time beyond the decision-point for dopaminergic or nondopaminergic commitment. This study demonstrates that the directed expression of specific transcription factors enhances serotonergic neuron differentiation in vitro and highlights the importance of transgene expression at the right stage of ESC differentiation to effect the generation of a desired neural subtype., (Copyright © 2011 AlphaMed Press.)