An analogue of the Prolactin Releasing Peptide reduces obesity and promotes adult neurogenesis.

Hypothalamic Adult Neurogenesis (hAN) has been implicated in regulating energy homeostasis. Adult-generated neurons and adult Neural Stem Cells (aNSCs) in the hypothalamus control food intake and body weight. Conversely, diet-induced obesity (DIO) by high fat diets (HFD) exerts adverse influence on hAN. However, the effects of anti-obesity compounds on hAN are not known. To address this, we administered a lipidized analogue of an anti-obesity neuropeptide, Prolactin Releasing Peptide (PrRP), so-called LiPR, to mice. In the HFD context, LiPR rescued the survival of adult-born hypothalamic neurons and increased the number of aNSCs by reducing their activation. LiPR also rescued the reduction of immature hippocampal neurons and modulated calcium dynamics in iPSC-derived human neurons. In addition, some of these neurogenic effects were exerted by another anti-obesity compound, Liraglutide. These results show for the first time that anti-obesity neuropeptides influence adult neurogenesis and suggest that the neurogenic process can serve as a target of anti-obesity pharmacotherapy.

Science shines in a new virtual SY-Stem.

The third 'Symposium for the Next Generation of Stem Cell Research' (SY-Stem) was held virtually on 3-5 March 2021, having been cancelled in 2020 due to the COVID-19 pandemic. As in previous years, the meeting highlighted the work of early career researchers, ranging from postgraduate students to young group leaders working in developmental and stem cell biology. Here, we summarize the excellent work presented at the Symposium, which covered topics ranging from pluripotency, species-specific aspects of development and emerging technologies, through to organoids, single-cell technology and clinical applications.

Feasibility and Safety of Oral Risperidone to Treat Prehospital Agitation.

OBJECTIVE: Agitation is a common prehospital problem and frequently presents without a clear etiology. Given the dynamic environment of the prehospital setting, there has historically been a varied approach to treating agitation with a heavy reliance on parenteral medications. Newer best practice guidelines recommend the incorporation of oral medications to treat patients experiencing agitation. Therefore, we evaluated the use of oral risperidone in a single system after a change in protocol occurred. METHODS: This was conducted as a retrospective chart review of an urban/suburban Emergency Medical Services system over the period of 8 months. The first day this medication was implemented throughout the service was included. Charts were included for selection if they included risperidone oral dissolving tablet (ODT) as a charted medication. The primary outcome was administration of additional medications to treat agitation. Exploratory outcome measures included acceptance of medication, documented injury to paramedics, documented injuries to patients, scene times, and adverse events that could possibly be linked to the medication. RESULTS: A total of 552 records were screened for inclusion. Risperidone was offered to 530 patients and accepted by 512 (96.6%). Of these 512 patients, the median age of included patients was 39 years old (IQR 29-52 years old) with a range of 18-89 years old. Rescue or additional medications for agitation were required in 9 (1.8%) cases. There were a total of 4 (0.8%) potential complications following administration of risperidone. There were no reported assaults with subsequent injuries to prehospital personnel or injuries sustained by patients reported in this study. CONCLUSIONS: Risperidone ODT was found to be a safe and effective medication to treat mild agitation in a large urban and suburban EMS system. The need for additional medications to treat agitation was rare, and there were no documented injuries to either patients or paramedics.

Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations.

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

A comparative transcriptomic analysis of glucagon-like peptide-1 receptor- and glucose-dependent insulinotropic polypeptide receptor-expressing cells in the hypothalamus

OBJECTIVE: The hypothalamus is a key region of the brain implicated in homeostatic regulation, and is an integral centre for the control of feeding behaviour. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretin hormones with potent glucoregulatory function through engagement of their respective cognate receptors, GLP-1R and GIPR. Recent evidence indicates that there is a synergistic effect of combining GIP- and GLP-1-based pharmacology on appetite and body weight. The mechanisms underlying the enhanced weight loss exhibited by GIPR/GLP-1R co-agonism are unknown. Gipr and Glp1r are expressed in the hypothalamus in both rodents and humans. To better understand incretin receptor-expressing cell populations, we compared the cell types and expression profiles of Gipr- and Glp1r-expressing hypothalamic cells using single-cell RNA sequencing. METHODS: Using Glp1r-Cre or Gipr-Cre transgenic mouse lines, fluorescent reporters were introduced into either Glp1r- or Gipr-expressing cells, respectively, upon crossing with a ROSA26-EYFP reporter strain. From the hypothalami of these mice, fluorescent Glp1rEYFP+ or GiprEYFP+ cells were FACS-purified and sequenced using single-cell RNA sequencing. Transcriptomic analysis provided a survey of both non-neuronal and neuronal cells, and comparisons between Glp1rEYFP+ and GiprEYFP + populations were made. RESULTS: A total of 14,091 Glp1rEYFP+ and GiprEYFP+ cells were isolated, sequenced and taken forward for bioinformatic analysis. Both Glp1rEYFP+ and GiprEYFP+ hypothalamic populations were transcriptomically highly heterogeneous, representing vascular cell types, oligodendrocytes, astrocytes, microglia, and neurons. The majority of GiprEYFP+ cells were non-neuronal, whereas the Glp1rEYFP+ population was evenly split between neuronal and non-neuronal cell types. Both Glp1rEYFP+ and GiprEYFP+ oligodendrocytes express markers for mature, myelin-forming oligodendrocytes. While mural cells are represented in both Glp1rEYFP+ and GiprEYFP+ populations, Glp1rEYFP+ mural cells are largely smooth muscle cells, while the majority of GiprEYFP+ mural cells are pericytes. The co-expression of regional markers indicate that clusters of Glp1rEYFP+ and GiprEYFP+ neurons have been isolated from the arcuate, ventromedial, lateral, tuberal, suprachiasmatic, and premammillary nuclei of the hypothalamus. CONCLUSIONS: We have provided a detailed comparison of Glp1r and Gipr cells of the hypothalamus with single-cell resolution. This resource will provide mechanistic insight into how engaging Gipr- and Glp1r-expressing cells of the hypothalamus may result in changes in feeding behaviour and energy balance.

Proopiomelanocortin Processing in the Hypothalamus Is Directly Regulated by Saturated Fat: Implications for the Development of Obesity.

In outbred mice, susceptibility or resistance to diet-induced obesity is associated with rapid changes in hypothalamic proopiomelanocortin (POMC) levels. Here, we evaluated 3 hypotheses that potentially explain the development of the different obesity phenotypes in outbred Swiss mice. First, rapid and differential changes in the gut microbiota in obesity-prone (OP) and obesity-resistant (OR) mice fed on a high-fat diet (HFD) might cause differential efficiencies in fatty acid harvesting leading to changes in systemic fatty acid concentrations that in turn affect POMC expression and processing. Second, independently of the gut microbiota, OP mice might have increased blood fatty acid levels after the introduction of a HFD, which could affect POMC expression and processing. Third, fatty acids might act directly in the hypothalamus to differentially regulate POMC expression and/or processing in OP and OR mice. We evaluated OP and OR male Swiss mice using 16S rRNA sequencing for the determination of gut microbiota; gas chromatography for blood lipid determination; and immunoblot and real-time polymerase chain reaction for protein and transcript determination and indirect calorimetry. Some experiments were performed with human pluripotent stem cells differentiated into hypothalamic neurons. We did not find evidence supporting the first 2 hypotheses. However, we found that in OP but not in OR mice, palmitate induces a rapid increase in hypothalamic POMC, which is followed by increased expression of proprotein convertase subtilisin/kexin type 1 PC1/3. Lentiviral inhibition of hypothalamic PC1/3 increased caloric intake and body mass in both OP and OR mice. In human stem cell-derived hypothalamic cells, we found that palmitate potently suppressed the production of POMC-derived peptides. Palmitate directly regulates PC1/3 in OP mice and likely has a functional impact on POMC processing.

Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells.

Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases.

Evolutionarily conserved regulation of hypocretin neuron specification by Lhx9.

Loss of neurons that express the neuropeptide hypocretin (Hcrt) has been implicated in narcolepsy, a debilitating disorder characterized by excessive daytime sleepiness and cataplexy. Cell replacement therapy, using Hcrt-expressing neurons generated in vitro, is a potentially useful therapeutic approach, but factors sufficient to specify Hcrt neurons are unknown. Using zebrafish as a high-throughput system to screen for factors that can specify Hcrt neurons in vivo, we identified the LIM homeobox transcription factor Lhx9 as necessary and sufficient to specify Hcrt neurons. We found that Lhx9 can directly induce hcrt expression and we identified two potential Lhx9 binding sites in the zebrafish hcrt promoter. Akin to its function in zebrafish, we found that Lhx9 is sufficient to specify Hcrt-expressing neurons in the developing mouse hypothalamus. Our results elucidate an evolutionarily conserved role for Lhx9 in Hcrt neuron specification that improves our understanding of Hcrt neuron development.

Large structural variants in KOLF2.1J are unlikely to compromise neurological disease modelling.

Gracia-Diaz and colleagues analysed high-density DNA microarray and whole genome sequencing (WGS) data from the KOLF2.1J 'reference' human induced pluripotent stem cell (hiPSC) line1, and report the presence of five high-confidence heterozygous copy number variants (CNVs) at least 100kbp in length2. Since three of these CNVs span coding genes, some of which have been associated with neurodevelopmental disease, the authors raise the concern that these CNVs may compromise the utility of KOLF2.1J for neurological disease modelling. We appreciate their thorough analysis and thoughtful interpretation, and agree that potential users of this line should be made aware of all cases where KOLF2.1J differs from the reference genome. However, we believe that the benefits from the widespread use of KOLF2.1J outweigh the potential risks that might arise from the identified CNVs.

Protein-truncating variants in BSN are associated with severe adult-onset obesity, type 2 diabetes and fatty liver disease.

Obesity is a major risk factor for many common diseases and has a substantial heritable component. To identify new genetic determinants, we performed exome-sequence analyses for adult body mass index (BMI) in up to 587,027 individuals. We identified rare loss-of-function variants in two genes (BSN and APBA1) with effects substantially larger than those of well-established obesity genes such as MC4R. In contrast to most other obesity-related genes, rare variants in BSN and APBA1 were not associated with normal variation in childhood adiposity. Furthermore, BSN protein-truncating variants (PTVs) magnified the influence of common genetic variants associated with BMI, with a common variant polygenic score exhibiting an effect twice as large in BSN PTV carriers than in noncarriers. Finally, we explored the plasma proteomic signatures of BSN PTV carriers as well as the functional consequences of BSN deletion in human induced pluripotent stem cell-derived hypothalamic neurons. Collectively, our findings implicate degenerative processes in synaptic function in the etiology of adult-onset obesity.

Neural stem cells in mammalian development.

Neural stem cells (NSCs) are primary progenitors that give rise to neurons and glia in the embryonic, neonatal and adult brain. In recent years, we have learned three important things about these cells. First, NSCs correspond to cells previously thought to be committed glial cells. Second, embryonic and adult NSCs are lineally related: they transform from neuroepithelial cells into radial glia, then into cells with astroglial characteristics. Third, NSCs divide asymmetrically and often amplify the number of progeny they generate via symmetrically dividing intermediate progenitors. These advances challenge our traditional perceptions of glia and stem cells, and provide the foundation for understanding the molecular basis of mammalian NSC behavior.

Distinctive neurons of the anterior cingulate and frontoinsular cortex: a historical perspective.

Human anterior cingulate and frontoinsular cortices participate in healthy social-emotional processing. These regions feature 2 related layer 5 neuronal morphotypes, the von Economo neurons and fork cells. In this paper, we review the historical accounts of these neurons and provide a German-to-English translation of von Economo's seminal paper describing the neurons which have come to bear his name. We close with a brief discussion regarding the functional and clinical relevance of these neurons and their home regions.

Regulation of the length of neuronal primary cilia and its potential effects on signalling.

Primary cilia protrude from most vertebrate cell bodies and act as specialized 'signalling antennae' that can substantially lengthen or retract in minutes to hours in response to specific stimuli. Here, we review the conditions and mechanisms responsible for regulating primary cilia length (PCL) in mammalian nonsensory neurons, and propose four models of how they could affect ciliary signalling and alter cell state and suggest experiments to distinguish between them. These models include (i) the passive indicator model, where changes in PCL have no consequence; (ii) the rheostat model, in which a longer cilium enhances signalling; (iii) the local concentration model, where ciliary shortening increases the local protein concentration to facilitate signalling; and (iv) the altered composition model where changes in PCL skew signalling.

Differentiation, Transcriptomic Profiling, and Calcium Imaging of Human Hypothalamic Neurons.

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Human pluripotent stem cells (hPSCs) can be differentiated into many types of hypothalamic neurons, progenitors, and glia. This updated unit includes published studies and protocols with new advances in the differentiation, maturation, and interrogation by transcriptomic profiling and calcium imaging of human hypothalamic cell populations. Specifically, new methods to freeze and thaw hypothalamic progenitors after they have been patterned and before substantial neurogenesis has occurred are provided that will facilitate experimental flexibility and planning. Also included are updated recipes and protocols for neuronal maturation, with details on the equipment and methods for examining their transcriptomic response and cell-autonomous properties in culture in the presence of synaptic blockers. Together, these protocols facilitate the adoption and use of this model system for fundamental biological discovery and therapeutic translation to human diseases such as obesity, diabetes, sleep disorders, infertility, and chronic stress. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hPSC maintenance Basic Protocol 2: Hypothalamic neuron differentiation Support Protocol 1: Cortical neuron (control) differentiation Basic Protocol 3: Neuronal maturation Support Protocol 2: Cryopreservation and thawing of neuronal progenitors Support Protocol 3: Quality control: Confirmation of hypothalamic patterning and neurogenesis Support Protocol 4: Bulk RNA sequencing of hypothalamic cultures Basic Protocol 4: Calcium imaging of hypothalamic neurons using Fura-2 AM Alternate Protocol: Calcium imaging of green fluorescent hypothalamic neurons using Rhod-3 AM.

MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

Whole-genome analysis of human embryonic stem cells enables rational line selection based on genetic variation.

Despite their widespread use in research, there has not yet been a systematic genomic analysis of human embryonic stem cell (hESC) lines at a single-nucleotide resolution. We therefore performed whole-genome sequencing (WGS) of 143 hESC lines and annotated their single-nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer-scale structural and single-nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESC genomes and human blood-derived genomes at similar frequencies. Moreover, WGS allowed us to identify SNVs associated with cancer and other diseases that could alter cellular phenotypes and compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection.

The consequences of recurrent genetic and epigenetic variants in human pluripotent stem cells.

It is well established that human pluripotent stem cells (hPSCs) can acquire genetic and epigenetic changes during culture in vitro. Given the increasing use of hPSCs in research and therapy and the vast expansion in the number of hPSC lines available for researchers, the International Society for Stem Cell Research has recognized the need to reassess quality control standards for ensuring the genetic integrity of hPSCs. Here, we summarize current knowledge of the nature of recurrent genetic and epigenetic variants in hPSC culture, the methods for their detection, and what is known concerning their effects on cell behavior in vitro or in vivo. We argue that the potential consequences of low-level contamination of cell therapy products with cells bearing oncogenic variants are essentially unknown at present. We highlight the key challenges facing the field with particular reference to safety assessment of hPSC-derived cellular therapeutics.

Nitric Oxide Synthase Neurons in the Preoptic Hypothalamus Are NREM and REM Sleep-Active and Lower Body Temperature.

When mice are exposed to external warmth, nitric oxide synthase (NOS1) neurons in the median and medial preoptic (MnPO/MPO) hypothalamus induce sleep and concomitant body cooling. However, how these neurons regulate baseline sleep and body temperature is unknown. Using calcium photometry, we show that NOS1 neurons in MnPO/MPO are predominantly NREM and REM active, especially at the boundary of wake to NREM transitions, and in the later parts of REM bouts, with lower activity during wakefulness. In addition to releasing nitric oxide, NOS1 neurons in MnPO/MPO can release GABA, glutamate and peptides. We expressed tetanus-toxin light-chain in MnPO/MPO NOS1 cells to reduce vesicular release of transmitters. This induced changes in sleep structure: over 24 h, mice had less NREM sleep in their dark (active) phase, and more NREM sleep in their light (sleep) phase. REM sleep episodes in the dark phase were longer, and there were fewer REM transitions between other vigilance states. REM sleep had less theta power. Mice with synaptically blocked MnPO/MPO NOS1 neurons were also warmer than control mice at the dark-light transition (ZT0), as well as during the dark phase siesta (ZT16-20), where there is usually a body temperature dip. Also, at this siesta point of cooled body temperature, mice usually have more NREM, but mice with synaptically blocked MnPO/MPO NOS1 cells showed reduced NREM sleep at this time. Overall, MnPO/MPO NOS1 neurons promote both NREM and REM sleep and contribute to chronically lowering body temperature, particularly at transitions where the mice normally enter NREM sleep.

The genetic architecture of DNA replication timing in human pluripotent stem cells.

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure but has a poorly understood genetic basis. To systematically identify genetic regulators of replication timing, we exploited inter-individual variation in human pluripotent stem cells from 349 individuals. We show that the human genome's replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs) - sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, and are enriched at sites of histone H3 trimethylation of lysines 4, 9, and 36 together with histone hyperacetylation. H3 trimethylation marks are individually repressive yet synergistically associate with early replication. We identify pluripotency-related transcription factors and boundary elements as positive and negative regulators of replication timing, respectively. Taken together, human replication timing is controlled by a multi-layered mechanism with dozens of effectors working combinatorially and following principles analogous to transcription regulation.

Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation.

Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.