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.

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.

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.

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.

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.

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.

MicroRNAs couple cell fate and developmental timing in retina.

Cell identity is acquired in different brain structures according to a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle-related microRNAs (miRNAs). Through expression and functional screenings, we selected 4 miRNAs--mir-129, mir-155, mir-214, and mir-222--that are highly expressed at early developmental stages in the embryonic retina and bind to the 3' UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of additional bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through the activity of a set of miRNAs.

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.

Noggin elicits retinal fate in Xenopus animal cap embryonic stem cells.

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse-transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c-fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells.

miR-290 acts as a physiological effector of senescence in mouse embryo fibroblasts.

The culture-induced senescence of mouse embryo fibroblasts (MEF) correlates with reduction of cell proliferation. In this work we found that the accumulation of cells with 4C DNA content and the transcriptional change of several microRNAs (miRNAs or miRs) are relevant events in culture senescence. By comparing the miRNA expression profiles of physiologically senescent MEF and that of senescent MEF induced by the downregulation of leukemia-related factor, we identified miR-290 as a common upregulated miRNA. When miR-290 was transfected in presenescent MEF, SA-beta-gal(+) cells and p16, two markers of culture senescence, increased compared with control, indicating that miR-290 is causally involved in senescence. Interestingly, nocodazole (NCZ), which induces G2/M block, increased the percentage of senescent cells as well as the expression of miR-290 and of the tumor suppressor p16, thus mimicking culture senescence. As miR-290 was overexpressed in NCZ-treated cells and it was able to induce senescence in proliferating MEF, we investigated whether miR-290 and NCZ could share common mechanisms of culture senescence. Whereas the induction of SA-beta-gal(+) by miR-290 was not strengthened by coupling its transfection with NCZ treatment, the transfection of the antagomir 290 (d-290) plus NCZ treatment, while blocking cells at G2/M, suppressed SA-beta-gal(+) and p16 induction. On the basis of these findings we conclude that miR-290 might act as a physiological effector of NCZ induced as well as culture senescence via p16 regulation expanding the role of this miRNA from embryonic stem to differentiated cells.

Timing the generation of distinct retinal cells by homeobox proteins.

The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR), we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b) and bipolar cells (Xvsx1 and Xotx2). The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

Dicer inactivation causes heterochronic retinogenesis in Xenopus laevis.

Maturation of miRNAs by dicer is required in vertebrates for normal neural development. Here we report that dicer inactivation in Xenopus affects cell cycle progression, survival and timing of the generation of retinal cells, resulting in small retinas with lamination defects. In particular, dicer inactivation delays the exit from the cell cycle and the translation of key genes of late neurogenesis, highlighting a crucial role of miRNAs in retinal 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.

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.

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.

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.

Cell cycle and cell fate interactions in neural development.

Mechanisms coupling cell cycle and cell fate operate at different steps during neural development. Intrinsic factors control the cell proliferation of distinct brain regions and changes of cell fate competence, whereas components of the cell cycle machinery could play a major role in setting the appropriate timing of the generation of different cell types.