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Long noncoding RNAs (lncRNAs) are a diverse and pervasive class of genes. Recent studies in the mammalian brain have uncovered several novel mechanisms. LncRNA loci are often located in proximity to developmental transcriptional factors. The lncRNA product may act like a transcription factor to control distantly located genes, or in other instances, the lncRNA loci contain DNA regulatory elements that act locally on neighboring genes. Circular RNAs are covalently closed single-stranded RNAs that can control neuronal function by acting as microRNA sponges and additional mechanisms. LncRNAs can also engage in target-directed microRNA degradation to shape the pool of microRNAs and translation. Thus, diverse mechanisms allow lncRNAs to act in the nucleus and cytoplasm to control neuronal fate and function.

The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages—that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix–loop–helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein–DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity. Lee, Mall et al. explore why the sequence-related transcription factors Myod1 and Ascl1 lead to different reprogramming outcomes when expressed in fibroblasts, despite binding similar targets.

Standard methods for the creation of neuronal cells via direct induction from primary tissue use perinatal fibroblasts, which hinders the important study of patient specific genetic lesions such as those underlying neuropsychiatric disorders. To address this we developed a novel method for the direct induction of neuronal cells (induced neuronal cells, iN cells) from adult human fibroblast cells. Reprogramming fibroblasts into iN cells via recombinant virus resulted in cells that stain for markers such as MAP2 and PSA-NCAM and exhibit electrophysiological properties such as action potentials and voltage dependent sodium- and potassium currents that reveal a neuronal phenotype. Transcriptome and chromatin analysis using RNA-Seq, microRNA-Seq and ATAC-Seq, respectively, further confirm neuronal character. 22q11.2 Deletion-Syndrome (22q11DS) is caused by a large 3 million base-pair heterozygous deletion on human chromosome 22 and is strongly associated with neurodevelopmental, neuropsychiatric phenotypes such as schizophrenia and autism. We leverage the direct-iN cell model for the study of genetic neurodevelopmental conditions by presenting gene-by-gene as well as networkwide effects of the 22q11DS deletion on gene expression in human neuronal cells, on several levels of functional genomics analysis. Some of the genes within the 22q11DS deletion boundary exhibit unexpected cell-type-specific changes in transcript levels, and genome-wide we can detect dysregulation of calcium channel subunit genes and other genes known to be involved in autism or schizophrenia, such as NRXN1, as well synaptic pathways. This genome-wide effect on gene expression can also be observed at the microRNA and chromatin levels, showing that the iN cells have indeed converted to a neuronal phenotype at several regulatory levels: chromatin, protein-coding RNAs and microRNAs, revealing relevant disease pathways and genes. We present this model of inducing neurons from fibroblasts as a useful general resource to study the genetic and molecular basis of normal and abnormal brain development and brain function.

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing1, but its influence on specific cells remains largely unknown. Fibroblasts are present in most tissues and contribute to wound healing2,3. They are also the most widely used cell type for reprogramming to induced pluripotent stem (iPS) cells, a process that has implications for regenerative medicine and rejuvenation strategies4. Here we show that fibroblast cultures from old mice secrete inflammatory cytokines and exhibit increased variability in the efficiency of iPS cell reprogramming between mice. Variability between individuals is emerging as a feature of old age5–8, but the underlying mechanisms remain unknown. To identify drivers of this variability, we performed multi-omics profiling of fibroblast cultures from young and old mice that have different reprogramming efficiencies. This approach revealed that fibroblast cultures from old mice contain ‘activated fibroblasts’ that secrete inflammatory cytokines, and that the proportion of activated fibroblasts in a culture correlates with the reprogramming efficiency of that culture. Experiments in which conditioned medium was swapped between cultures showed that extrinsic factors secreted by activated fibroblasts underlie part of the variability between mice in reprogramming efficiency, and we have identified inflammatory cytokines, including TNF, as key contributors. Notably, old mice also exhibited variability in wound healing rate in vivo. Single-cell RNA-sequencing analysis identified distinct subpopulations of fibroblasts with different cytokine expression and signalling in the wounds of old mice with slow versus fast healing rates. Hence, a shift in fibroblast composition, and the ratio of inflammatory cytokines that they secrete, may drive the variability between mice in reprogramming in vitro and influence wound healing rate in vivo. This variability may reflect distinct stochastic ageing trajectories between individuals, and could help in developing personalized strategies to improve iPS cell generation and wound healing in elderly individuals. Fibroblasts from old mice are heterogeneous, which affects the ability of these fibroblasts to reprogram into induced pluripotent stem cells in vitro and influences wound healing rate in vivo.

Neurons are highly polarized cells with complex neurite morphology. Spatial organization and local translation of RNAs in dendrites and axons play an important role in many neuronal functions. Here we performed super-resolution spatial profiling of RNAs inside individual neurons at the genome scale using multiplexed error-robust fluorescencein situhybridization (MERFISH), and mapped the spatial organization of up to ∼4,200 RNA species (genes) across multiple length scales, ranging from sub-micrometer to millimeters. Our data generated a quantitative intra-neuronal atlas of RNAs with distinct transcriptome compositions in somata, dendrites, and axons, and revealed diverse sub-dendritic distribution patterns of RNAs. Moreover, our spatial analysis identified distinct groups of genes exhibiting specific spatial clustering of transcripts at the sub-micrometer scale that were dependent on protein synthesis and differentially dependent on synaptic activity. Overall, these data provide a rich resource for characterizing the subcellular organization of the transcriptome in neurons with high spatial resolution.

Generation of neurons of vast neurotransmitter, morphological and hodological subtype diversity involves early spatial and temporal patterning of the neuronal precursors by morphogenic gradients and combinatorial expression of homeotic transcription factors. Proneural basic-helix-loop-helix (bHLH) transcription factors play a key role in this process. However, it remains unknown how bHLH factors such as NGN2 and Ascl1, which are primarily proneural, contribute to subtype specification in a cell context-dependent manner. Here, we investigate the context-dependent molecular function of a pro-neural bHLH transcription factor NGN2; a factor which we previously showed to efficiently induce the neuronal fate in human embryonic stem (hES) cells. We tested its molecular function and lineage-inducing capacity in context of different chromatin environments and in combination with other transcription factors with spatially restricted expression patterns and evaluated NGN2 genomic localization, chromatin dynamics and transcriptional consequences using multi-omics methods. This work revealed that the combination of NGN2 and EMX1, a forebrain-restricted homeobox transcription factor, yields a highly homogeneous glutamatergic neuronal forebrain subpopulation without partial cholinergic and monoaminergic gene programs seen in NGN2 only cells. In contrast, modifying the chromatin environment that NGN2 is exposed to, strongly regulates the regional identity of resulting neurons that persists throughout reprogramming but does not influence neurotransmitter subtype specification. This shows how nature co-opts combinatorial strategies of using different chromatin landscapes and/or homeodomain transcription factors with spatially restricted expression to generate a vast variety of neuronal subtypes with a few transcription factors. Our work thus provides improved strategies to generate a plethora of defined and subtype specific neuronal cell populations from pluripotent stem cells for therapeutic or disease modeling purposes.

Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.

Approaches to differentiating pluripotent stem cells (PSCs) into neurons currently face two major challenges-(i) generated cells are immature, with limited functional properties; and (ii) cultures exhibit heterogeneous neuronal subtypes and maturation stages. Using lineage-determining transcription factors, we previously developed a single-step method to generate glutamatergic neurons from human PSCs. Here, we show that transient expression of the transcription factors Ascl1 and Dlx2 (AD) induces the generation of exclusively GABAergic neurons from human PSCs with a high degree of synaptic maturation. These AD-induced neuronal (iN) cells represent largely nonoverlapping populations of GABAergic neurons that express various subtype-specific markers. We further used AD-iN cells to establish that human collybistin, the loss of gene function of which causes severe encephalopathy, is required for inhibitory synaptic function. The generation of defined populations of functionally mature human GABAergic neurons represents an important step toward enabling the study of diseases affecting inhibitory synaptic transmission.

Summary Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.

The on-target pioneer factors Ascl1 and Myod1 are sequence-related but induce two developmentally unrelated lineages-that is, neuronal and muscle identities, respectively. It is unclear how these two basic helix-loop-helix (bHLH) factors mediate such fundamentally different outcomes. The chromatin binding of Ascl1 and Myod1 was surprisingly similar in fibroblasts, yet their transcriptional outputs were drastically different. We found that quantitative binding differences explained differential chromatin remodelling and gene activation. Although strong Ascl1 binding was exclusively associated with bHLH motifs, strong Myod1-binding sites were co-enriched with non-bHLH motifs, possibly explaining why Ascl1 is less context dependent. Finally, we observed that promiscuous binding of Myod1 to neuronal targets results in neuronal reprogramming when the muscle program is inhibited by Myt1l. Our findings suggest that chromatin access of on-target pioneer factors is primarily driven by the protein-DNA interaction, unlike ordinary context-dependent transcription factors, and that promiscuous transcription factor binding requires specific silencing mechanisms to ensure lineage fidelity.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Age-associated chronic inflammation (inflammaging) has emerged as a central hallmark of aging1-3, but its impact on specific cells is still largely unknown. Fibroblasts are present in all tissues and contribute to wound healing4-6. They are also the cell type that is mostly used for induced pluripotent stem cell (iPSC) reprogramming7 – a process that has implications for regenerative medicine and rejuvenation strategies8-17. Here we show that primary fibroblasts from old mice secrete inflammatory cytokines and that there is an increased variability in reprogramming efficiency between fibroblast cultures from old individuals. Individual-to-individual variability is emerging as a key feature of old age18-21, which could reflect distinct aging trajectories, but the underlying causes remain unknown. To identify drivers of this variability, we perform a multi-omic assessment of young and old fibroblast cultures with different reprogramming efficiency. This approach, coupled with single cell transcriptomics, reveals that old fibroblast cultures are heterogeneous and show a greater proportion of ‘activated fibroblasts’ that secrete inflammatory cytokines, which correlates with reprogramming efficiency. We experimentally validate that activated fibroblasts express inflammatory cytokines in vivo and that their presence is linked to enhanced reprogramming efficiency in culture. Conditioned-media swapping experiments show that extrinsic factors secreted by activated fibroblasts are more critical than intrinsic factors for the individual-to-individual variability in reprogramming efficiency, and we identify TNFα as a key inflammatory cytokine underlying this variability. Interestingly, old mice also exhibit variability in wound healing efficiency in vivo and old wounds show an increased subpopulation of activated fibroblasts with a unique TNFα signature. Our study shows that a switch in fibroblast composition, and the ratio of inflammatory cytokines they secrete, drives variability in reprogramming in vitro and may influence wound healing in vivo. These findings could help identify personalized strategies to improve iPSC generation and wound healing in older individuals.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Long noncoding RNAs (lncRNAs) have been shown to act as important cell biological regulators including cell fate decisions but are often ignored in human genetics. Combining differential lncRNA expression during neuronal lineage induction with copy number variation morbidity maps of a cohort of children with autism spectrum disorder/intellectual disability versus healthy controls revealed focal genomic mutations affecting several lncRNA candidate loci. Here we find that a t(5:12) chromosomal translocation in a family manifesting neurodevelopmental symptoms disrupts specifically lnc-NR2F1. We further show that lnc-NR2F1 is an evolutionarily conserved lncRNA functionally enhances induced neuronal cell maturation and directly occupies and regulates transcription of neuronal genes including autism-associated genes. Thus, integrating human genetics and functional testing in neuronal lineage induction is a promising approach for discovering candidate lncRNAs involved in neurodevelopmental diseases.

Global transcription factor activity is measured by screening cell extracts with an oligonucleotide library.

SummaryHow transcription factors (TFs) reprogram one cell lineage to another remains unclear. Here, we define chromatin accessibility changes induced by the proneural TF Ascl1 throughout conversion of fibroblasts into induced neuronal (iN) cells. Thousands of genomic loci are affected as early as 12 hr after Ascl1 induction. Surprisingly, over 80% of the accessibility changes occur between days 2 and 5 of the 3-week reprogramming process. This chromatin switch coincides with robust activation of endogenous neuronal TFs and nucleosome phasing of neuronal promoters and enhancers. Subsequent morphological and functional maturation of iN cells is accomplished with relatively little chromatin reconfiguration. By integrating chromatin accessibility and transcriptome changes, we built a network model of dynamic TF regulation during iN cell reprogramming and identified Zfp238, Sox8, and Dlx3 as key TFs downstream of Ascl1. These results reveal a singular, coordinated epigenomic switch during direct reprogramming, in contrast to stepwise cell fate transitions in development.

Summary Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage., Graphical Abstract, Highlights • ASCL1 alone generates functional neurons from fibroblast and embryonic stem cells • ASCL1-induced 1F-iN cells display slow maturation kinetics • ASCL1 overexpression induces endogenous expression of Myt1l and Brn2 • ASCL1-induced 1F-iN cells are predominantly excitatory, Direct reprogramming of nonneuronal cells into functional neurons is often considered to require overexpression of multiple transcription factors, but their functional hierarchy remained unknown. Here, Wernig, Südhof, and colleagues show that overexpression of an “on target” pioneer factor, ASCL1, is sufficient to generate induced neuronal cells from fibroblast and embryonic stem cells of both mouse and human origin.

Cellular differentiation processes during normal embryonic development are guided by extracellular soluble factors such as morphogen gradients and cell contact signals, eventually resulting in induction of specific combinations of lineage-determining transcription factors. The young field of epigenetic reprogramming takes advantage of this knowledge and uses cell fate determination factors to convert one lineage into another such as the conversion of fibroblasts into pluripotent stem cells or neurons. These induced cell fate conversions open up new avenues for studying disease processes, generating cell material for therapeutic intervention such as drug screening and potentially also for cell-based therapies. However, there are still limitations that have to be overcome to fulfill these promises, centering on reprogramming efficiencies, cell identity, and maturation. In this review, we discuss the discovery of induced neuronal reprogramming, ways to improve the conversion process, and finally how to define properly the identity of those converted neuronal cells.

We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O₂). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O₂ tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O₂ conditions compared to 50% under high O₂ growth conditions, confirming the beneficial aspect of reprogramming under low O₂. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3β and Smad-mediated pathways. Our results showed that lowering the O₂ tension at the initiation of reprogramming is a simple and efficient strategy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

Age-associated chronic inflammation (inflammageing) is a central hallmark of ageing

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

SummaryMutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.