Laminin 511 and WNT signalling sustain prolonged expansion of hiPSC-derived hippocampal progenitors.

Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200 days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses.

Translational control in cortical development.

Differentiation of specific neuronal types in the nervous system is worked out through a complex series of gene regulation events. Within the mammalian neocortex, the appropriate expression of key transcription factors allocates neurons to different cortical layers according to an inside-out model and endows them with specific properties. Precise timing is required to ensure the proper sequential appearance of key transcription factors that dictate the identity of neurons within the different cortical layers. Recent evidence suggests that aspects of this time-controlled regulation of gene products rely on post-transcriptional control, and point at micro-RNAs (miRs) and RNA-binding proteins as important players in cortical development. Being able to simultaneously target many different mRNAs, these players may be involved in controlling the global expression of gene products in progenitors and post-mitotic cells, in a gene expression framework where parallel to transcriptional gene regulation, a further level of control is provided to refine and coordinate the appearance of the final protein products. miRs and RNA-binding proteins (RBPs), by delaying protein appearance, may play heterochronic effects that have recently been shown to be relevant for the full differentiation of cortical neurons and for their projection abilities. Such heterochronies may be the base for evolutionary novelties that have enriched the spectrum of cortical cell types within the mammalian clade.

COTAN: scRNA-seq data analysis based on gene co-expression.

Estimating the co-expression of cell identity factors in single-cell is crucial. Due to the low efficiency of scRNA-seq methodologies, sensitive computational approaches are critical to accurately infer transcription profiles in a cell population. We introduce COTAN, a statistical and computational method, to analyze the co-expression of gene pairs at single cell level, providing the foundation for single-cell gene interactome analysis. The basic idea is studying the zero UMI counts' distribution instead of focusing on positive counts; this is done with a generalized contingency tables framework. COTAN can assess the correlated or anti-correlated expression of gene pairs, providing a new correlation index with an approximate p-value for the associated test of independence. COTAN can evaluate whether single genes are differentially expressed, scoring them with a newly defined global differentiation index. Similarly to correlation network analysis, it provides ways to plot and cluster genes according to their co-expression pattern with other genes, effectively helping the study of gene interactions, becoming a new tool to identify cell-identity markers. We assayed COTAN on two neural development datasets with very promising results. COTAN is an R package that complements the traditional single cell RNA-seq analysis and it is available at https://github.com/seriph78/COTAN.

A eutherian-specific microRNA controls the translation of Satb2 in a model of cortical differentiation.

Cerebral cortical development is controlled by key transcription factors that specify the neuronal identities in the different layers. The mechanisms controlling their expression in distinct cells are only partially known. We investigated the expression and stability of Tbr1, Bcl11b, Fezf2, Satb2, and Cux1 mRNAs in single developing mouse cortical cells. We observe that Satb2 mRNA appears much earlier than its protein and in a set of cells broader than expected, suggesting an initial inhibition of its translation, subsequently released during development. Mechanistically, Satb2 3'UTR modulates protein translation of GFP reporters during mouse corticogenesis. We select miR-541, a eutherian-specific miRNA, and miR-92a/b as the best candidates responsible for SATB2 inhibition, being strongly expressed in early and reduced in late progenitor cells. Their inactivation triggers robust and premature SATB2 translation in both mouse and human cortical cells. Our findings indicate RNA interference as a major mechanism in timing cortical cell identities.

Lysosome Dynamic Properties during Neuronal Stem Cell Differentiation Studied by Spatiotemporal Fluctuation Spectroscopy and Organelle Tracking.

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 µm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle.

miR-182-5p is an evolutionarily conserved Tbx5 effector that impacts cardiac development and electrical activity in zebrafish.

To dissect the TBX5 regulatory circuit, we focused on microRNAs (miRNAs) that collectively contribute to make TBX5 a pivotal cardiac regulator. We profiled miRNAs in hearts isolated from wild-type, CRE, Tbx5lox/+and Tbx5del/+ mice using a Next Generation Sequencing (NGS) approach. TBX5 deficiency in cardiomyocytes increased the expression of the miR-183 cluster family that is controlled by Kruppel-like factor 4, a transcription factor repressed by TBX5. MiR-182-5p, the most highly expressed miRNA of this family, was functionally analyzed in zebrafish. Transient overexpression of miR-182-5p affected heart morphology, calcium handling and the onset of arrhythmias as detected by ECG tracings. Accordingly, several calcium channel proteins identified as putative miR-182-5p targets were downregulated in miR-182-5p overexpressing hearts. In stable zebrafish transgenic lines, we demonstrated that selective miRNA-182-5p upregulation contributes to arrhythmias. Moreover, cardiac-specific down-regulation of miR-182-5p rescued cardiac defects in a zebrafish model of Holt-Oram syndrome. In conclusion, miR-182-5p exerts an evolutionarily conserved role as a TBX5 effector in the onset of cardiac propensity for arrhythmia, and constitutes a relevant target for mediating the relationship between TBX5, arrhythmia and heart development.

Pluripotent Stem Cells for Brain Repair: Protocols and Preclinical Applications in Cortical and Hippocampal Pathologies.

Brain injuries causing chronic sensory or motor deficit, such as stroke, are among the leading causes of disability worldwide, according to the World Health Organization; furthermore, they carry heavy social and economic burdens due to decreased quality of life and need of assistance. Given the limited effectiveness of rehabilitation, novel therapeutic strategies are required to enhance functional recovery. Since cell-based approaches have emerged as an intriguing and promising strategy to promote brain repair, many efforts have been made to study the functional integration of neurons derived from pluripotent stem cells (PSCs), or fetal neurons, after grafting into the damaged host tissue. PSCs hold great promises for their clinical applications, such as cellular replacement of damaged neural tissues with autologous neurons. They also offer the possibility to create in vitro models to assess the efficacy of drugs and therapies. Notwithstanding these potential applications, PSC-derived transplanted neurons have to match the precise sub-type, positional and functional identity of the lesioned neural tissue. Thus, the requirement of highly specific and efficient differentiation protocols of PSCs in neurons with appropriate neural identity constitutes the main challenge limiting the clinical use of stem cells in the near future. In this Review, we discuss the recent advances in the derivation of telencephalic (cortical and hippocampal) neurons from PSCs, assessing specificity and efficiency of the differentiation protocols, with particular emphasis on the genetic and molecular characterization of PSC-derived neurons. Second, we address the remaining challenges for cellular replacement therapies in cortical brain injuries, focusing on electrophysiological properties, functional integration and therapeutic effects of the transplanted neurons.

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation.

The morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3'UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3'UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping.

Post-transcriptional Modulation of Sphingosine-1-Phosphate Receptor 1 by miR-19a Affects Cardiovascular Development in Zebrafish.

Sphingosine-1-phosphate is a bioactive lipid and a signaling molecule integrated into many physiological systems such as differentiation, proliferation and migration. In mammals S1P acts through binding to a family of five trans-membrane, G-protein coupled receptors (S1PRs) whose complex role has not been completely elucidated. In this study we use zebrafish, in which seven s1prs have been identified, to investigate the role of s1pr1. In mammals S1PR1 is the most highly expressed S1P receptor in the developing heart and regulates vascular development, but in zebrafish the data concerning its role are contradictory. Here we show that overexpression of zebrafish s1pr1 affects both vascular and cardiac development. Moreover we demonstrate that s1pr1 expression is strongly repressed by miR-19a during the early phases of zebrafish development. In line with this observation and with a recent study showing that miR-19a is downregulated in a zebrafish Holt-Oram model, we now demonstrate that s1pr1 is upregulated in heartstring hearts. Next we investigated whether defects induced by s1pr1 upregulation might contribute to the morphological alterations caused by Tbx5 depletion. We show that downregulation of s1pr1 is able to partially rescue cardiac and fin defects induced by Tbx5 depletion. Taken together, these data support a role for s1pr1 in zebrafish cardiovascular development, suggest the involvement of this receptor in the Tbx5 regulatory circuitry, and further support the crucial role of microRNAs in early phase of zebrafish development.

Neurons Generated by Mouse ESCs with Hippocampal or Cortical Identity Display Distinct Projection Patterns When Co-transplanted in the Adult Brain.

The capability of generating neural precursor cells with distinct types of regional identity in vitro has recently opened new opportunities for cell replacement in animal models of neurodegenerative diseases. By manipulating Wnt and BMP signaling, we steered the differentiation of mouse embryonic stem cells (ESCs) toward isocortical or hippocampal molecular identity. These two types of cells showed different degrees of axonal outgrowth and targeted different regions when co-transplanted in healthy or lesioned isocortex or in hippocampus. In hippocampus, only precursor cells with hippocampal molecular identity were able to extend projections, contacting CA3. Conversely, isocortical-like cells were capable of extending long-range axonal projections only when transplanted in motor cortex, sending fibers toward both intra- and extra-cortical targets. Ischemic damage induced by photothrombosis greatly enhanced the capability of isocortical-like cells to extend far-reaching projections. Our results indicate that neural precursors generated by ESCs carry intrinsic signals specifying axonal extension in different environments.

Assessment of antibody library diversity through next generation sequencing and technical error compensation.

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its complexity (also known as diversity), i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library complexity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Complexity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring complexity from such a small sampling is, however, very rudimental and gives limited information about the real diversity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library complexity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the complexity, taking in consideration the sequencing error.

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

MicroRNA 19a replacement partially rescues fin and cardiac defects in zebrafish model of Holt Oram syndrome.

Holt-Oram Syndrome (HOS) is an autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene, a transcription factor capable of regulating hundreds of cardiac-specific genes through complex transcriptional networks. Here we show that, in zebrafish, modulation of a single miRNA is sufficient to rescue the morphogenetic defects generated by HOS. The analysis of miRNA-seq profiling revealed a decreased expression of miR-19a in Tbx5-depleted zebrafish embryos compared to the wild type. We revealed that the transcription of the miR-17-92 cluster, which harbors miR-19a, is induced by Tbx5 and that a defined dosage of miR-19a is essential for the correct development of the heart. Importantly, we highlighted that miR-19a replacement is able to rescue cardiac and pectoral fin defects and to increase the viability of HOS zebrafish embryos. We further observed that miR-19a replacement shifts the global gene expression profile of HOS-like zebrafish embryos towards the wild type condition, confirming the ability of miR-19a to rescue the Tbx5 phenotype. In conclusion our data demonstrate the importance of Tbx5/miR-19a regulatory circuit in heart development and provide a proof of principle that morphogenetic defects associated with HOS can be rescued by transient miRNA modulation.

Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization.

Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs) toward eye field fates. Inhibition of Wnt/ß-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

Noggin-Mediated Retinal Induction Reveals a Novel Interplay Between Bone Morphogenetic Protein Inhibition, Transforming Growth Factor ß, and Sonic Hedgehog Signaling.

It has long been known that the depletion of bone morphogenetic protein (BMP) is one of the key factors necessary for the development of anterior neuroectodermal structures. However, the precise molecular mechanisms that underlie forebrain regionalization are still not completely understood. Here, we show that Noggin1 is involved in the regionalization of anterior neural structures in a dose-dependent manner. Low doses of Noggin1 expand prosencephalic territories, while higher doses specify diencephalic and retinal regions at the expense of telencephalic areas. A similar dose-dependent mechanism determines the ability of Noggin1 to convert pluripotent cells in prosencephalic or diencephalic/retinal precursors, as shown by transplant experiments and molecular analyses. At a molecular level, the strong inhibition of BMP signaling exerted by high doses of Noggin1 reinforces the Nodal/transforming growth factor (TGF)ß signaling pathway, leading to activation of Gli1 and Gli2 and subsequent activation of Sonic Hedgehog (SHH) signaling. We propose a new role for Noggin1 in determining specific anterior neural structures by the modulation of TGFß and SHH signaling.

The double inhibition of endogenously produced BMP and Wnt factors synergistically triggers dorsal telencephalic differentiation of mouse ES cells.

Embryonic stem (ES) cells are becoming a popular model of in vitro neurogenesis, as they display intrinsic capability to generate neural progenitors that undergo the known steps of in vivo neural development. These include the acquisition of distinct regional fates, which depend on growth factors and signals that are present in the culture medium. The control of the intracellular signaling that is active at different steps of ES cell neuralization, even when cells are cultured in chemically defined medium, is complicated by the endogenous production of growth factors. However, this endogenous production has been poorly investigated so far. To address this point, we performed a high-throughput analysis of the expression of morphogens during mouse ES cell neuralization in minimal medium. We found that during their neuralization, ES cells increased the expression of members of Wnt, Fibroblast Growth Factor (FGF), and BMP families. Conversely, the expression of Activin/Nodal and Shh ligands was low in early steps of neuralization. In this experimental condition, neural progenitors and neurons generated by ES cells expressed a gene expression profile that was consistent with a midbrain identity. We found that endogenous BMP and Wnt signaling, but not FGF signaling, synergistically affected ES cell neural patterning, by turning off a profile of dorsal/telencephalic gene expression. Double BMP and Wnt inhibition allowed neuralized ES cells to sequentially activate key genes of cortical differentiation. Our findings are consistent with a novel synergistic effect of Wnt and BMP endogenous signaling of ES cells in inhibiting a cortical differentiation program.

From pluripotency to forebrain patterning: an in vitro journey astride embryonic stem cells.

Embryonic stem cells (ESCs) have been used extensively as in vitro models of neural development and disease, with special efforts towards their conversion into forebrain progenitors and neurons. The forebrain is the most complex brain region, giving rise to several fundamental structures, such as the cerebral cortex, the hypothalamus, and the retina. Due to the multiplicity of signaling pathways playing different roles at distinct times of embryonic development, the specification and patterning of forebrain has been difficult to study in vivo. Research performed on ESCs in vitro has provided a large body of evidence to complement work in model organisms, but these studies have often been focused more on cell type production than on cell fate regulation. In this review, we systematically reassess the current literature in the field of forebrain development in mouse and human ESCs with a focus on the molecular mechanisms of early cell fate decisions, taking into consideration the specific culture conditions, exogenous and endogenous molecular cues as described in the original studies. The resulting model of early forebrain induction and patterning provides a useful framework for further studies aimed at reconstructing forebrain development in vitro for basic research or therapy.

MicroRNAs and cell fate in cortical and retinal development.

MicroRNAs (miRNAs) are involved in crucial steps of neurogenesis, neural differentiation, and neuronal plasticity. Here we review experimental evidence suggesting that miRNAs may regulate the histogenesis of the cerebral cortex and neural retina. Both cortical and retinal early progenitor cells are multipotent, that is, they can generate different types of cortical or retinal cells, respectively, in one lineage. In both cortical and retinal development, the precise timing of activation of cell fate transcription factors results in a stereotyped schedule of generation of the different types of neurons. Emerging evidence indicates that miRNAs may play an important role in regulating such temporal programing of neuronal differentiation. Neuronal subtypes of the cortex and retina exhibit distinct miRNA signatures, implying that miRNA codes may be used to specify different types of neurons. Interfering with global miRNA activity changes the ratio of the different types of neurons produced. In fact, there are examples of cell fate genes that are regulated at the translational level, both in retinogenesis and in corticogenesis. A model depicting how miRNAs might orchestrate both the type and the birth of different neurons is presented and discussed. Glossary. • Lineage: the temporally ordered cell progeny of an individual progenitor cell. • Specification: the (reversible) process by which a cell becomes capable of, and biased toward, a particular fate. • Commitment: the process by which cell fate is fully determined and can no longer be affected by external cues. • Potency: the entire complement of cells that a progenitor can ultimately produce. • Multipotency: the ability to give rise to more than one cell type. • Progenitor: a dividing cell that, in contrast to a stem cell, cannot proliferate indefinitely. • Antago-miR: modified antisense oligonucleotide that blocks the activity of a miRNA. • Heterochronic neuron: type of neurons that is generated at inappropriate times of development. • Neuron birth date: the time of the last mitosis of a neuronal cell.

The positional identity of mouse ES cell-generated neurons is affected by BMP signaling.

We investigated the effects of bone morphogenetic proteins (BMPs) in determining the positional identity of neurons generated in vitro from mouse embryonic stem cells (ESCs), an aspect that has been neglected thus far. Classical embryological studies in lower vertebrates indicate that BMPs inhibit the default fate of pluripotent embryonic cells, which is both neural and anterior. Moreover, mammalian ESCs generate neurons more efficiently when cultured in a minimal medium containing BMP inhibitors. In this paper, we show that mouse ESCs produce, secrete, and respond to BMPs during in vitro neural differentiation. After neuralization in a minimal medium, differentiated ESCs show a gene expression profile consistent with a midbrain identity, as evaluated by the analysis of a number of markers of anterior-posterior and dorsoventral identity. We found that BMPs endogenously produced during neural differentiation mainly act by inhibiting the expression of a telencephalic gene profile, which was revealed by the treatment with Noggin or with other BMP inhibitors. To better characterize the effect of BMPs on positional fate, we compared the global gene expression profiles of differentiated ESCs with those of embryonic forebrain, midbrain, and hindbrain. Both Noggin and retinoic acid (RA) support neuronal differentiation of ESCs, but they show different effects on their positional identity: whereas RA supports the typical gene expression profile of hindbrain neurons, Noggin induces a profile characteristic of dorsal telencephalic neurons. Our findings show that endogenously produced BMPs affect the positional identity of the neurons that ESCs spontaneously generate when differentiating in vitro in a minimal medium. The data also support the existence of an intrinsic program of neuronal differentiation with dorsal telencephalic identity. Our method of ESC neuralization allows for fast differentiation of neural cells via the same signals found during in vivo embryonic development and for the acquisition of cortical identity by the inhibition of BMP alone.