NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Autophagy Dysfunction as a Phenotypic Readout in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Diseases.

Autophagy is an evolutionarily conserved catabolic pathway for the degradation of cytoplasmic constituents in eukaryotic cells. It is the primary disposal route for selective removal of undesirable cellular materials like aggregation-prone proteins and damaged organelles for maintaining cellular homeostasis, and for bulk degradation of intracellular macromolecules and recycling the breakdown products for providing energy homeostasis during starvation. These functions of autophagy are attributed to cellular survival and thus pertinent for human health; however, malfunction of this process is detrimental to the cells, particularly for post-mitotic neurons. Thus, basal autophagy is vital for maintaining neuronal homeostasis, whereas autophagy dysfunction contributes to neurodegeneration. Defective autophagy has been demonstrated in several neurodegenerative diseases wherein pharmacological induction of autophagy is beneficial in many of these disease models. Elucidating the mechanisms underlying defective autophagy is imperative for the development of therapies targeting this process. Disease-affected human neuronal cells can be established from patient-derived human induced pluripotent stem cells (hiPSCs) that provide a clinically relevant platform for studying disease mechanisms and drug discovery. Thus, modeling autophagy dysfunction as a phenotypic readout in patient-derived neurons provides a more direct platform for investigating the mechanisms underlying defective autophagy and evaluating the therapeutic efficacy of autophagy inducers. Toward this, several hiPSC-derived neuronal cell models of neurodegenerative diseases have been employed. In this review, we highlight the key methodologies pertaining to hiPSC maintenance and neuronal differentiation, and studying autophagy at an endogenous level in hiPSC-derived neuronal cells.

Intravital imaging of mouse embryos.

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.

Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras.

Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.

Human Induced Pluripotent Stem Cell Models of Neurodegenerative Disorders for Studying the Biomedical Implications of Autophagy.

Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis, and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases, including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers, as well as the nature of the autophagy defects, may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell types, such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modeling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation. It outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.

Microcephaly Modeling of Kinetochore Mutation Reveals a Brain-Specific Phenotype.

Most genes mutated in microcephaly patients are expressed ubiquitously, and yet the brain is the only major organ compromised in most patients. Why the phenotype remains brain specific is poorly understood. In this study, we used in vitro differentiation of human embryonic stem cells to monitor the effect of a point mutation in kinetochore null protein 1 (KNL1; CASC5), identified in microcephaly patients, during in vitro brain development. We found that neural progenitors bearing a patient mutation showed reduced KNL1 levels, aneuploidy, and an abrogated spindle assembly checkpoint. By contrast, no reduction of KNL1 levels or abnormalities was observed in fibroblasts and neural crest cells. We established that the KNL1 patient mutation generates an exonic splicing silencer site, which mainly affects neural progenitors because of their higher levels of splicing proteins. Our results provide insight into the brain-specific phenomenon, consistent with microcephaly being the only major phenotype of patients bearing KNL1 mutation.

Matched Developmental Timing of Donor Cells with the Host Is Crucial for Chimera Formation.

Chimeric mice have been generated by injecting pluripotent stem cells into morula-to-blastocyst stage mouse embryo or by introducing more mature cells into later stage embryos that correspond to the differentiation stage of the donor cells. It has not been rigorously tested, however, whether successful chimera formation requires the developmental stage of host embryo and donor cell to be matched. Here, we compared the success of chimera formation following injection of primary neural crest cells (NCCs) into blastocysts or of embryonic stem cells (ESCs) into E8.5 embryos (heterochronic injection) with that of injecting ESCs cells into the blastocyst or NCCs into the E8.5 embryos (isochronic injection). Chimera formation was efficient when donor and host were matched, but no functional chimeric contribution was found in heterochronic injections. This suggests that matching the developmental stage of donor cells with the host embryo is crucial for functional engraftment of donor cells into the developing embryo.

YY1 Is a Structural Regulator of Enhancer-Promoter Loops.

There is considerable evidence that chromosome structure plays important roles in gene control, but we have limited understanding of the proteins that contribute to structural interactions between gene promoters and their enhancer elements. Large DNA loops that encompass genes and their regulatory elements depend on CTCF-CTCF interactions, but most enhancer-promoter interactions do not employ this structural protein. Here, we show that the ubiquitously expressed transcription factor Yin Yang 1 (YY1) contributes to enhancer-promoter structural interactions in a manner analogous to DNA interactions mediated by CTCF. YY1 binds to active enhancers and promoter-proximal elements and forms dimers that facilitate the interaction of these DNA elements. Deletion of YY1 binding sites or depletion of YY1 protein disrupts enhancer-promoter looping and gene expression. We propose that YY1-mediated enhancer-promoter interactions are a general feature of mammalian gene control.

Alternative SET/TAFI Promoters Regulate Embryonic Stem Cell Differentiation.

Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETß, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage.

SMARCE1 is required for the invasive progression of in situ cancers.

Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.

Molecular Criteria for Defining the Naive Human Pluripotent State.

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.

Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos.

The neural crest (NC) represents multipotent cells that arise at the interphase between ectoderm and prospective epidermis of the neurulating embryo. The NC has major clinical relevance because it is involved in both inherited and acquired developmental abnormalities. The aim of this study was to establish an experimental platform that would allow for the integration of human NC cells (hNCCs) into the gastrulating mouse embryo. NCCs were derived from pluripotent mouse, rat, and human cells and microinjected into embryonic-day-8.5 embryos. To facilitate integration of the NCCs, we used recipient embryos that carried a c-Kit mutation (W(sh)/W(sh)), which leads to a loss of melanoblasts and thus eliminates competition from the endogenous host cells. The donor NCCs migrated along the dorsolateral migration routes in the recipient embryos. Postnatal mice derived from injected embryos displayed pigmented hair, demonstrating differentiation of the NCCs into functional melanocytes. Although the contribution of human cells to pigmentation in the host was lower than that of mouse or rat donor cells, our results indicate that hNCCs, injected in utero, can integrate into the embryo and form mature functional cells in the animal. This mouse-human chimeric platform allows for a new approach to study NC development and diseases.

A Systematic Approach to Identify Candidate Transcription Factors that Control Cell Identity.

Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

Impaired autophagy in the lipid-storage disorder Niemann-Pick type C1 disease.

Autophagy dysfunction has been implicated in misfolded protein accumulation and cellular toxicity in several diseases. Whether alterations in autophagy also contribute to the pathology of lipid-storage disorders is not clear. Here, we show defective autophagy in Niemann-Pick type C1 (NPC1) disease associated with cholesterol accumulation, where the maturation of autophagosomes is impaired because of defective amphisome formation caused by failure in SNARE machinery, whereas the lysosomal proteolytic function remains unaffected. Expression of functional NPC1 protein rescues this defect. Inhibition of autophagy also causes cholesterol accumulation. Compromised autophagy was seen in disease-affected organs of Npc1 mutant mice. Of potential therapeutic relevance is that HP-ß-cyclodextrin, which is used for cholesterol-depletion treatment, impedes autophagy, whereas stimulating autophagy restores its function independent of amphisome formation. Our data suggest that a low dose of HP-ß-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may provide a rational treatment strategy for NPC1 disease.

The lamina propria of adult human oral mucosa harbors a novel stem cell population.

The highly regenerative capacity of the human adult oral mucosa suggests the existence of a robust stem cell (SC) population in its lamina propria (OMLP). The purpose of this study was to characterize the availability, growth, immunophenotype, and potency of this presumable SC population. Cells positive for the embryonic stem cell transcription factors Oct4 and Sox2 and for p75 formed distinct cord-like structure in the OMLP. Regardless of donor age, trillions of cells, termed human oral mucosa stem cells (hOMSC), 95% of which express mesenchymal stromal cell markers, were simply, and reproducibly produced from a biopsy of 3-4 x 2 x 1 mm(3). A total of 40-60% of these cells was positive for Oct4, Sox2, and Nanog and 60-80% expressed constitutively neural and neural crest SC markers. hOMSC differentiated in culture into mesodermal (osteoblastic, chondroblastic, and adipocytic), definitive endoderm and ectodermal (neuronal) lineages. Unexpectedly, hOMSC treated with dexamethasone formed tumors consisting of two germ layer-derived tissues when transplanted in severe combined immune deficiency mice. The tumors consisted of tissues produced by neural crest cells during embryogenesis-cartilage, bone, fat, striated muscle, and neural tissue. These results show that the adult OMLP harbors a primitive SC population with a distinct primitive neural-crest like phenotype and identifies the in vivo localization of putative ancestors for this population. This is the first report on ectodermal- and mesodermal-derived mixed tumors formation by a SC population derived from a nonmalignant somatic adult human tissue.

Gliomas display a microRNA expression profile reminiscent of neural precursor cells.

Gliomas express many genes that play a role in neural precursor cells (NPCs), but no direct comparison between glioma and stem cell (SC) gene expression profiles has been performed. To investigate the similarities and differences between gliomas and SCs, we compared the microRNA (miRNA) expression signatures of glial tumors, embryonic SCs (ESCs), NPCs, and normal adult brains from both human and mouse tissues. We demonstrated that both human gliomas (regardless of their grade) and methylcholanthrene-induced mouse glioma shared an miRNA expression profile that is reminiscent of NPCs. About half of the miRNAs expressed in the shared profile clustered in seven genomic regions susceptible to genetic/epigenetic alterations in various cancers. These clusters comprised the miR17 family, mir183-182, and the SC-specific clusters mir367-302 and mir371-373, which are upregulated in gliomas, ESCs, and NPCs. The bipartite cluster of 7 + 46 miRNAs on chromosome 14q32.31, which might represent the largest tumor suppressor miRNA cluster, was downregulated in the shared expression profile. This study provides the first evidence for association between these clusters and gliomas. Despite the broad similarity in the miRNA expression profiles, 15 miRNAs showed disparate expression between SC and gliomas. Ten miRNAs belong to the 2 SC-specific clusters and the remaining (mir135b, mir141, mir205, mir200C, and mir301a) have been previously shown to associate with malignancies. Our finding showed that all gliomas displayed NPC-like miRNA signatures, which may have implications for studies of glioma origins. Furthermore, careful study of the 15 miRNAs that differ in expression between SCs and gliomas, particularly those 5 that are not SC-specific, may enhance our understanding of gliomagenesis.

Enriched population of PNS neurons derived from human embryonic stem cells as a platform for studying peripheral neuropathies.

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases.