Neurogenin Research Articles

Reduced Neurog3 Gene Dosage Shifts Enteroendocrine Progenitor Towards Goblet Cell Lineage in the Mouse Intestine.

Background & AimsTransient expression of Neurog3 commits intestinal secretory progenitors to become enteroendocrine-biased progenitors and hence drive enteroendocrine differentiation. Loss of Neurog3 in mouse resulted in the depletion of intestinal enteroendocrine cells (EECs) and an increase in goblet cells. Earlier studies in developing mouse pancreas identified a role of Neurog3 gene dosage in endocrine and exocrine cell fate allocation. We aimed to determine whether Neurog3 gene dosage controls fate choice of enteroendocrine progenitors.MethodsWe acquired mutant Neurog3 reporter mice carrying 2, 1, or null Neurog3 alleles to study Neurog3 gene dosage effect by lineage tracing. Cell types arising from Neurog3+ progenitors were determined by immunohistochemistry using antibodies against intestinal lineage-specific markers. RNA sequencing of sorted Neurog3+/+, Neurog3+/-, or bulk intestinal cells were performed and differentially expressed genes were analyzed.ResultsWe identified 2731 genes enriched in sorted Neurog3+/+-derived cells in the Neurog3+/+EYFP mouse intestine when compared with bulk duodenum epithelial cells. In the intestine of Neurog3+/-EGFP heterozygous mouse, we observed a 63% decrease in EEC numbers. Many Neurog3-derived cells stained for goblet marker Mucin 2. RNA sequencing of sorted Neurog3+/- cells uncovered enriched expression of genes characteristic for both goblet and enteroendocrine cells, indicating the mixed lineages arose from Neurog3+ progenitors. Consistent with this hypothesis, deletion of both Neurog3 alleles resulted in the total absence of EECs. All Neurog3+-derived cells stained for Mucin 2.ConclusionsWe identified that the fate of Neurog3+ enteroendocrine progenitors is dependent on Neurog3 gene dosage. High Neurog3 gene dosage enforces the commitment of secretory progenitors to an EE lineage, while constraining their goblet cell lineage potential. Transcriptome profiling data was deposited to Gene Ontology omnibus, accession number: GSE149203.

Enteric anendocrinosis attributable to a novel Neurogenin-3 variant

Congenital diarrhea and enteropathies (CODEs) are a group of monogenic disorders that often present with severe diarrhea in the first weeks of life. Enteric anendocrinosis (EA), an extremely rare cause of CODE, is characterized by a marked reduction of intestinal enteroendocrine cells (EC). EA is associated with recessively inherited variants in Neurogenin-3 (NEUROG3) gene. Here we investigate a case of a male infant who presented with mysterious severe malabsorptive diarrhea since birth. Thorough clinical assessments and laboratory tests were successful to exclude the majority of differential diagnosis categories. However, the patient's diagnosis was not established until the genetic test using whole-exome sequencing (WES) was performed. We identified a novel homozygous missense disease-causing variant (DCV) in NEUROG3 (c.413C>G, p.Thr138Arg). Moreover, molecular dynamic simulation analysis showed that (p.Thr138Arg) led to a global change of the NEUROG3 orientation affecting its DNA binding capacity. To the best of our knowledge, this is the first time to apply WES to reach a differential diagnosis of patients with CODEs. Our study not only expands our knowledge about NEUROG3 variants and their clinical consequences but also proves that WES is a very effective tool for the diagnosis of CODEs. This might be of value in early diagnosis of diseases and prenatal CODEs detection.

Transplantation of human dental pulp stem cells in streptozotocin-induced diabetic rats.

Type 1 diabetes mellitus (T1DM) is a chronic metabolic disease caused by the destruction of pancreatic β-cells. Human dental pulp stem cells represent a promising source for cell-based therapies, owing to their easy, minimally invasive surgical access, and high proliferative capacity. It was reported that human dental pulp stem cells can differentiate into a pancreatic cell lineage in vitro; however, few studies have investigated their effects on diabetes. Our study aimed to investigate the therapeutic potential of intravenous and intrapancreatic transplantation of human dental pulp stem cells in a rat model of streptozotocin-induced type 1 diabetes. Forty Sprague Dawley male rats were randomly categorized into four groups: control, diabetic (STZ), intravenous treatment group (IV), and intrapancreatic treatment group (IP). Human dental pulp stem cells (1 × 106cells) or vehicle were injected into the pancreas or tail vein 7days after streptozotocin injection. Fasting blood glucose levels were monitored weekly. Glucose tolerance test, rat and human serum insulin and C-peptide, pancreas histology, and caspase-3, vascular endothelial growth factor, and Ki67 expression in pancreatic tissues were assessed 28days post-transplantation. We found that both IV and IP transplantation of human dental pulp stem cells reduced blood glucose and increased levels of rat and human serum insulin and C-peptide. The cells engrafted and survived in the streptozotocin-injured pancreas. Islet-like clusters and scattered human dental pulp stem cells expressing insulin were observed in the pancreas of diabetic rats with some difference in the distribution pattern between the two injection routes. RT-PCR analyses revealed the expression of the human-specific pancreatic β-cell genes neurogenin 3 (NGN3), paired box 4 (PAX4), glucose transporter2 (GLUT2), and insulin in the pancreatic tissues of both the IP and IV groups. In addition, the transplanted cells downregulated the expression of caspase-3 and upregulated the expression of vascular endothelial growth factor and Ki67, suggesting that the injected cells exerted pro-angiogenetic and antiapoptotic effects, and promoted endogenous β-cell replication. Our study is the first to show that human dental pulp stem cells can migrate and survive within streptozotocin-injured pancreas, and induce antidiabetic effects through the differentiation and replacement of lost β-cells and paracrine-mediated pancreatic regeneration. Thus, human dental pulp stem cells may have therapeutic potential to treat patients with long term T1DM.

Transcriptional activity of FIGLA, NEUROG2, and EGR1 transcription factors associated with polymorphisms in the proximal regulatory region of GPR54 gene in cattle

Activity of transcription factors affect synthesis of G-protein coupled receptor 54 (GPR54), an important factor in regulation of initiation of puberty. Expression of the GPR54 gene in cattle is associated with polymorphisms in the proximal regulatory region (PRR) of the GPR54 gene. Transcription resulting in production of GPR54 mRNA transcript occurs as a result of transcription factor (TF) interactions in the PRR. Polymorphisms in the PRR may be associated with extent of activity of these TFs. Folliculogenesis-specific BHLH TF (FIGLA), neurogenin 2 (NEUROG2), and early growth response 1 (EGR1) are important in modulation of ovarian follicle development and neurons synthesizing GnRH, thus, regulating biosynthesis of luteinizing hormone. The aim of this study, therefore, was to assess the transcription-activating potential of binding sites for FIGLA, NEUROG2, and EGR1 TFs in the GPR54 promoter of cattle. Two luciferase-based promoters, ATC and CCT, which contain three single nucleotide polymorphisms (SNPs), A/C-794, T/C-663, and C/T-601, in the GPR54 PRR, were analyzed to evaluate gene expression and activation of different promoters by FIGLA, NEUROG2, and EGR1. The FIGLA induced GPR54 transcription through the CCT, whereas NEUROG2 and EGR1 induced GPR54 transcription through the ATC promoter-binding site. The CCT-activating effects of FIGLA were greater (2.56-fold) than the ATC-activating effects (P < 0.05). The ATC-activating effects of NEUROG2 and EGR1 were markedly greater (12.91- and 8.41-fold; P < 0.01) than CCT-activating effects. The polymorphisms, CCT and ATC, of the cattle GPR54 affect the activity of transcription factors, therefore, have an important effect on production of GPR54 mRNA transcript.

Glucosamine potentiates the differentiation of adipose-derived stem cells into glucose-responsive insulin-producing cells

BackgroundIslet transplantation might be a logical strategy to restore insulin secretion for the treatment of diabetes, however, the scarcity of donors poses an obstacle for such a treatment. As an alternative islet source, differentiation of stem cells into insulin-producing cells (IPCs) has been tried. Many protocols have been developed to improve the efficiency of differentiation of stem cells into IPCs. In this study, we investigated whether glucosamine supplementation during differentiation of human adipose-derived stem cells (hADSCs) into IPCs can improve the insulin secretory function.MethodsGlucosamine was added to the original differentiation medium at different stages of differentiation of hADSCs into IPCs for 12 days and insulin secretion was analyzed.ResultsAddition of glucosamine alone to the growth medium of hADSCs did not affect the differentiation of hADSCs to IPCs. Supplementation of the differentiation medium with glucosamine at a later stage (protocol G3) proved to have the greatest effect on IPC differentiation. Basal and glucose-stimulated insulin secretion (GSIS) was significantly increased and the expression of insulin and C-peptide was increased in differentiated IPCs as compared with that in differentiated IPCs using the conventional protocol (protocol C). In addition, the expression of beta-cell specific transcription factors such as pancreatic and duodenal homeobox1 (PDX1) and neurogenin 3 (NGN3) was also increased. Furthermore, the expression of genes related to insulin secretion, including synaptotagmin 4 (Syt4), glucokinase (Gck) and glucose transporter 2 (Glut2), was also increased.ConclusionsWe conclude that glucosamine supplementation potentiates the differentiation of hADSCs into IPCs.

Glucagon-like peptide-1 and gastrin synergistically promote the differentiation of insulin-producing cells

Objective Glucagon-like peptide-1(GLP-1) and gastrin synergistically promote the differentiation of insulin-producing cells which differentiated from rat bone marrow mesenchymal stem cells (BMSCs). Methods (1)Prepare IPCs model: pancreatic duodenal homeobox 1 (Pdx-1), neurogenin 3 (Ngn3) combined with V-type tendon fibrosarcoma oncogene homolog A (MafA) co-transfected BMSCs differentiation into IPCs; (2)IPCs were divided into 4 groups: Group A(uninduced group), group B(GLP-1 induction group), group C(gastrin induction group), and group D(GLP-1 combined with gastrin induction group). Cultured in high glucose medium for 7 days, the expression levels of insulin2, Pdx-1, GK, nestin, and glucagon mRNA were detected by RT-PCR. The insulin secretion of each group was detected by ELISA. Results After cultured for 7 days under high glucose conditions, the morphology of IPCs in each induction group changed significantly, gradually aggregated and formed scattered cell masses, and the combined induction group formed large cell masses. The staining of disulfide brown was reddish brown; The levels of insulin secretion increased gradually on the 0, 3rd, 5th, 7th, and 9th day after induction, and the increase was the most significant in the combined induction group (P<0.05). Compared with group A, the expression of insulin2 and GK in group B and D was significantly up-regulated, the expression of glucagon was down-regulated in group D, the expression of Pdx-1 was down-regulated in group C, and the expression of glucagon was up-regulated (P<0.05). Compared with group B, The expression of insulin2 was down-regulated in group C, and the expression level of glucagon was up-regulated. The expression levels of Pdx-1 and Insulin2 were significantly up-regulated in group D, and the expression level of glucagon was down-regulated (P<0.05). Compared with group C, the expression level of Pdx-1, insulin2 and GK was significantly up-regulated in group D, and the expression level of glucagon was down-regulated (P<0.05). Conclusion GLP-1 and gastrin synergistically promote the differentiation of IPCs into islet β cells by up-regulating GK and insulin2 and down-regulating glucagon. Key words: Glucagon-like peptide 1; Gastrin; Pancreatic duodenal homeobox 1; Neurogenin 3; V-type tendon fibrosarcoma oncogene homologous gene A; Insulin-producing cells

Null mutations of NEUROG3 are associated with delayed-onset diabetes mellitus.

Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding β cell mass sufficient to maintain euglycemia until early childhood.

In Vitro Direct Reprogramming of Mouse and Human Astrocytes to Induced Neurons.

Direct neuronal reprogramming, rewiring the epigenetic and transcriptional network of a differentiated cell type to neuron, apart from being a very promising approach for the treatment of brain injury and neurodegeneration, also offers a prime opportunity to investigate the molecular underpinnings of neuronal cell fate determination, as the precise molecular mechanisms that establish neuronal fate and diversity at the transcriptional and epigenetic level are incompletely understood. Recent studies from a number of groups, including ours, have shown that astrocytes can be directly reprogrammed into functional neurons in vitro and in vivo following ectopic overexpression of combinations of transcription factors, neurogenic proteins, miRNAs, and small chemical molecules.In this chapter we describe the protocols for in vitro converting primary cortical astrocytes of mouse and human origin to induced neurons, through forced expression of two neurogenic molecules, either each one alone or in combination: the master regulatory bHLH proneural transcription factor NEUROGENIN-2 (NEUROG2) and the neurogenic protein CEND1. Forced expression of each one of the two neurogenic proteins in primary astrocytes via retroviral gene transfer results in their direct conversion to subtype-specific induced neurons, while simultaneous coexpression of both molecules drives them predominantly toward acquisition of a neural precursor cell (NPC) state. Although mouse and human astrocytes exhibit differences in their reprogramming rate and particular characteristics, they can both get efficiently in vitro transdifferentiated to NPCs and induced neurons upon NEUROG2 or/and CEND1 forced expression using the reprogramming protocols described in the chapter, presenting valuable cellular platforms for mechanistic studies and in vivo applications to restore neurodegeneration.

The cellular regulators PTEN and BMI1 help mediate NEUROGENIN-3–induced cell cycle arrest

Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.

Direct neuronal reprogramming of olfactory ensheathing cells for CNS repair

Direct conversion of readily available non-neural cells from patients into induced neurons holds great promise for neurological disease modeling and cell-based therapy. Olfactory ensheathing cells (OECs) is a unique population of glia in olfactory nervous system. Based on the regeneration-promoting properties and the relative clinical accessibility, OECs are attracting increasing attention from neuroscientists as potential therapeutic agents for use in neural repair. Here, we report that OECs can be directly, rapidly and efficiently reprogrammed into neuronal cells by the single transcription factor Neurogenin 2 (NGN2). These induced cells exhibit typical neuronal morphologies, express multiple neuron-specific markers, produce action potentials, and form functional synapses. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of OECs is effectively reprogrammed towards that of neuronal lineage. Importantly, these OEC-derived induced neurons survive and mature after transplantation into adult mouse spinal cords. Taken together, our study provides a direct and efficient strategy to quickly obtain neuronal cells from adult OECs, suggestive of promising potential for personalized disease modeling and cell replacement-mediated therapeutic approaches to neurological disorders.

Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells

BackgroundCell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window.MethodsWe performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 (MSI1), Neurogenin 2 (NGN2), and Methyl-CpG-Binding Domain 2 (MBD2). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry.ResultsHuman somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes.ConclusionsHuman drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination.

A Comprehensive Structure-Function Study of Neurogenin3 Disease-Causing Alleles during Human Pancreas and Intestinal Organoid Development

Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells, whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3-/- pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability, DNA binding, and gene transcription. Moreover, NEUROG3 was highly unstable in the intestinal epithelium, explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships.

Transcriptome Analysis of Small Molecule-Mediated Astrocyte-to-Neuron Reprogramming.

Chemical reprogramming of astrocytes into neurons represents a promising approach to regenerate new neurons for brain repair, but the underlying mechanisms driving this trans-differentiation process are not well understood. We have recently identified four small molecules – CHIR99021, DAPT, LDN193189, and SB431542 – that can efficiently reprogram cultured human fetal astrocytes into functional neurons. Here we employ the next generation of RNA-sequencing technology to investigate the transcriptome changes during the astrocyte-to-neuron (AtN) conversion process. We found that the four small molecules can rapidly activate the hedgehog signaling pathway while downregulating many glial genes such as FN1 and MYL9 within 24 h of treatment. Chemical reprogramming is mediated by several waves of differential gene expression, including upregulation of hedgehog, Wnt/β-catenin, and Notch signaling pathways, together with downregulation of TGF-β and JAK/STAT signaling pathways. Our gene network analyses reveal many well-connected hub genes such as repulsive guidance molecule A (RGMA), neuronatin (NNAT), neurogenin 2 (NEUROG2), NPTX2, MOXD1, JAG1, and GAP43, which may coordinate the chemical reprogramming process. Together, these findings provide critical insights into the molecular cascades triggered by a combination of small molecules that eventually leads to chemical conversion of astrocytes into neurons.

Bromodomain and Extra Terminal Protein Inhibitors Promote Pancreatic Endocrine Cell Fate.

Bromodomain and extraterminal (BET) proteins are epigenetic readers that interact with acetylated lysines of histone tails. Recent studies have demonstrated their role in cancer progression because they recruit key components of the transcriptional machinery to modulate gene expression. However, their role during embryonic development of the pancreas has never been studied. Using mouse embryonic pancreatic explants and human induced pluripotent stem cells (hiPSCs), we show that BET protein inhibition with I-BET151 or JQ1 enhances the number of neurogenin3 (NEUROG3) endocrine progenitors. In mouse explants, BET protein inhibition further led to increased expression of β-cell markers but in the meantime, strongly downregulated Ins1 expression. Similarly, although acinar markers, such as Cpa1 and CelA, were upregulated, Amy expression was repressed. In hiPSCs, BET inhibitors strongly repressed C-peptide and glucagon during endocrine differentiation. Explants and hiPSCs were then pulsed with BET inhibitors to increase NEUROG3 expression and further chased without inhibitors. Endocrine development was enhanced in explants with higher expression of insulin and maturation markers, such as UCN3 and MAFA. In hiPSCs, the outcome was different because C-peptide expression remained lower than in controls, but ghrelin expression was increased. Altogether, by using two independent models of pancreatic development, we show that BET proteins regulate multiple aspects of pancreatic development.

Essential Role of Protein Arginine Methyltransferase 1 in Pancreas Development by Regulating Protein Stability of Neurogenin 3.

BackgroundProtein arginine methyltransferase 1 (PRMT1) is a major enzyme responsible for the formation of methylarginine in mammalian cells. Recent studies have revealed that PRMT1 plays important roles in the development of various tissues. However, its role in pancreas development has not yet been elucidated.MethodsPancreatic progenitor cell-specific Prmt1 knock-out (Prmt1 PKO) mice were generated and characterized for their metabolic and histological phenotypes and their levels of Neurog3 gene expression and neurogenin 3 (NGN3) protein expression. Protein degradation assays were performed in mPAC cells.ResultsPrmt1 PKO mice showed growth retardation and a severely diabetic phenotype. The pancreatic size and β-cell mass were significantly reduced in Prmt1 PKO mice. Proliferation of progenitor cells during the secondary transition was decreased and endocrine cell differentiation was impaired. These defects in pancreas development could be attributed to the sustained expression of NGN3 in progenitor cells. Protein degradation assays in mPAC cells revealed that PRMT1 was required for the rapid degradation of NGN3.ConclusionPRMT1 critically contributes to pancreas development by destabilizing the NGN3 protein.

Evidence of Müller Glia Conversion Into Retina Ganglion Cells Using Neurogenin2.

Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.

Complete Disruption of Autism-Susceptibility Genes by Gene Editing Predominantly Reduces Functional Connectivity of Isogenic Human Neurons.

SummaryAutism spectrum disorder (ASD) is phenotypically and genetically heterogeneous. We present a CRISPR gene editing strategy to insert a protein tag and premature termination sites creating an induced pluripotent stem cell (iPSC) knockout resource for functional studies of ten ASD-relevant genes (AFF2/FMR2, ANOS1, ASTN2, ATRX, CACNA1C, CHD8, DLGAP2, KCNQ2, SCN2A, TENM1). Neurogenin 2 (NGN2)-directed induction of iPSCs allowed production of excitatory neurons, and mutant proteins were not detectable. RNA sequencing revealed convergence of several neuronal networks. Using both patch-clamp and multi-electrode array approaches, the electrophysiological deficits measured were distinct for different mutations. However, they culminated in a consistent reduction in synaptic activity, including reduced spontaneous excitatory postsynaptic current frequencies in AFF2/FMR2-, ASTN2-, ATRX-, KCNQ2-, and SCN2A-null neurons. Despite ASD susceptibility genes belonging to different gene ontologies, isogenic stem cell resources can reveal common functional phenotypes, such as reduced functional connectivity.

Transcription factor programming of human ES cells generates functional neurons expressing both upper and deep layer cortical markers.

Human neurodegenerative disorders affect specific types of cortical neurons. Efficient protocols for the generation of such neurons for cell replacement, disease modeling and drug screening are highly warranted. Current methods for the production of cortical neurons from human embryonic stem (ES) cells are often time-consuming and inefficient, and the functional properties of the generated cells have been incompletely characterized. Here we have used transcription factor (TF) programming with the aim to induce rapid differentiation of human ES cells to layer-specific cortical neurons (hES-iNs). Three different combinations of TFs, NEUROGENIN 2 (NGN2) only, NGN2 plus Forebrain Embryonic Zinc Finger-Like Protein 2 (FEZF2), and NGN2 plus Special AT-Rich Sequence-Binding Protein 2 (SATB2), were delivered to human ES cells by lentiviral vectors. We observed only subtle differences between the TF combinations, which all gave rise to the formation of pyramidal-shaped cells, morphologically resembling adult human cortical neurons expressing cortical projection neuron (PN) markers and with mature electrophysiological properties. Using ex vivo transplantation to human organotypic cultures, we found that the hES-iNs could integrate into adult human cortical networks. We obtained no evidence that the hES-iNs had acquired a distinct cortical layer phenotype. Instead, our single-cell data showed that the hES-iNs, similar to fetal human cortical neurons, expressed both upper and deep layer cortical neuronal markers. Taken together, our findings provide evidence that TF programming can direct human ES cells towards cortical neurons but that the generated cells are transcriptionally profiled to generate both upper and deep layer cortical neurons. Therefore, most likely additional cues will be needed if these cells should adopt a specific cortical layer and area identity.

CTCF Regulates Otic Neurogenesis via Histone Modification in the Neurog1 Locus.

The inner ear is a complex sensory organ responsible for hearing and balance. Formation of the inner ear is dependent on tight regulation of spatial and temporal expression of genes that direct a series of developmental processes. Recently, epigenetic regulation has emerged as a crucial regulator of the development of various organs. However, what roles higher-order chromatin organization and its regulator molecules play in inner ear development are unclear. CCCTC-binding factor (CTCF) is a highly conserved 11-zinc finger protein that regulates the three-dimensional architecture of chromatin, and is involved in various gene regulation processes. To delineate the role of CTCF in inner ear development, the present study investigated inner ear-specific Ctcf knockout mouse embryos (Pax2-Cre; Ctcffl/fl). The loss of Ctcf resulted in multiple defects of inner ear development and severely compromised otic neurogenesis, which was partly due to a loss of Neurog1 expression. Furthermore, reduced Neurog1 gene expression by CTCF knockdown was found to be associated with changes in histone modification at the gene’s promoter, as well as its upstream enhancer. The results of the present study demonstrate that CTCF plays an essential role in otic neurogenesis by modulating histone modification in the Neurog1 locus.

EphrinB2 signalling modulates the neural differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) originate from the embryonic neural crest and have neurogenic potential. The present study investigated the roles of the forward and reverse EphrinB2 signalling pathways during DPSC neurogenesis. Treatment of DPSCs with recombinant EphrinB2-Fc protein over 7 days in a neural induction culture resulted in significant downregulation of the following neural markers: βIII-Tubulin, neural cell adhesion molecule (NCAM), nestin, neurogenin 2 (NGN2), neurofilament medium polypeptide and Musashi1. Immunocytochemistry revealed that EphrinB2-Fc-treated DPSCs exhibited more rounded morphologies with fewer neurite outgrowths as well as reduced protein expression of βIII-tubulin and NGN2. Treatment of DPSCs with a peptide inhibitor specific to the EphB4 receptor significantly upregulated expression of the neural markers microtubule-associated protein 2, Musashi1, NGN2 and neuron-specific enolase, whereas treatment with a peptide inhibitor specific to the EphB2 receptor exerted negligible effects on neurogenesis. Transgenic expression of EphrinB2 in DPSCs resulted in significant upregulation of Musashi1 and NCAM gene expression, while treatment of DPSCs with recombinant EphB4-Fc protein led to significant upregulation of only Musashi1. Thus, it may be concluded that stimulation of forward EphrinB2-EphB4 signalling markedly inhibited neurogenesis in DPSCs, whereas suppression of this forward signalling pathway with peptide inhibitor specific to EphB4 promoted neurogenesis. Meanwhile, stimulation of reverse EphB4-EphrinB2 signalling only marginally enhanced the neural differentiation of DPSCs. The present findings indicate the potential application of peptide or small molecule inhibitors of EphrinB2 forward signalling in neural tissue engineering with DPSCs.