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1.
Cell ; 187(14): 3671-3689.e23, 2024 Jul 11.
Article in English | MEDLINE | ID: mdl-38866017

ABSTRACT

Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.


Subject(s)
Cell Survival , Dopaminergic Neurons , NF-kappa B , Tumor Necrosis Factor-alpha , Tumor Suppressor Protein p53 , Dopaminergic Neurons/metabolism , Animals , Humans , NF-kappa B/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Necrosis Factor-alpha/metabolism , Mice , Cell Survival/drug effects , Signal Transduction , Parkinson Disease/metabolism , Pluripotent Stem Cells/metabolism , Apoptosis , Disease Models, Animal , CRISPR-Cas Systems
2.
Cell ; 172(5): 952-965.e18, 2018 02 22.
Article in English | MEDLINE | ID: mdl-29474921

ABSTRACT

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.


Subject(s)
Brain Diseases, Metabolic, Inborn/genetics , Brain Stem/metabolism , Brain Stem/virology , RNA/chemistry , RNA/metabolism , Alleles , Amino Acid Sequence , Animals , Brain Diseases, Metabolic, Inborn/pathology , Brain Stem/pathology , Encephalitis, Viral/genetics , Escherichia coli/metabolism , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Fibroblasts/virology , Herpesvirus 1, Human , Humans , Interferons/metabolism , Introns/genetics , Male , Mice , Mutant Proteins/metabolism , Mutation/genetics , Open Reading Frames/genetics , Pedigree , RNA Nucleotidyltransferases/chemistry , RNA Nucleotidyltransferases/deficiency , RNA Nucleotidyltransferases/genetics , Toll-Like Receptor 3/metabolism , Virus Replication
4.
Nature ; 626(8000): 881-890, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38297124

ABSTRACT

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.


Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Developmental , Human Embryonic Stem Cells , Neural Stem Cells , Neurogenesis , Neurons , Adult , Animals , Humans , Mice , Histocompatibility Antigens/metabolism , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurogenesis/genetics , Neurons/cytology , Neurons/metabolism , Time Factors , Transcription, Genetic
5.
Nature ; 632(8024): 390-400, 2024 Aug.
Article in English | MEDLINE | ID: mdl-39048830

ABSTRACT

Most cases of herpes simplex virus 1 (HSV-1) encephalitis (HSE) remain unexplained1,2. Here, we report on two unrelated people who had HSE as children and are homozygous for rare deleterious variants of TMEFF1, which encodes a cell membrane protein that is preferentially expressed by brain cortical neurons. TMEFF1 interacts with the cell-surface HSV-1 receptor NECTIN-1, impairing HSV-1 glycoprotein D- and NECTIN-1-mediated fusion of the virus and the cell membrane, blocking viral entry. Genetic TMEFF1 deficiency allows HSV-1 to rapidly enter cortical neurons that are either patient specific or derived from CRISPR-Cas9-engineered human pluripotent stem cells, thereby enhancing HSV-1 translocation to the nucleus and subsequent replication. This cellular phenotype can be rescued by pretreatment with type I interferon (IFN) or the expression of exogenous wild-type TMEFF1. Moreover, ectopic expression of full-length TMEFF1 or its amino-terminal extracellular domain, but not its carboxy-terminal intracellular domain, impairs HSV-1 entry into NECTIN-1-expressing cells other than neurons, increasing their resistance to HSV-1 infection. Human TMEFF1 is therefore a host restriction factor for HSV-1 entry into cortical neurons. Its constitutively high abundance in cortical neurons protects these cells from HSV-1 infection, whereas inherited TMEFF1 deficiency renders them susceptible to this virus and can therefore underlie HSE.


Subject(s)
Brain , Encephalitis, Herpes Simplex , Herpesvirus 1, Human , Membrane Proteins , Virus Internalization , Animals , Female , Humans , Male , Brain/cytology , Brain/metabolism , Brain/virology , Encephalitis, Herpes Simplex/virology , Encephalitis, Herpes Simplex/metabolism , Herpesvirus 1, Human/pathogenicity , Herpesvirus 1, Human/physiology , Homozygote , Interferon Type I/metabolism , Interferon Type I/immunology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nectins/genetics , Nectins/metabolism , Neurons/cytology , Neurons/metabolism , Neurons/virology , Pluripotent Stem Cells/cytology , Virus Replication , Child, Preschool , Young Adult , Pedigree
6.
Nature ; 609(7929): 907-910, 2022 09.
Article in English | MEDLINE | ID: mdl-36171373

ABSTRACT

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.


Subject(s)
Consensus , Nervous System , Organoids , Terminology as Topic , Humans , Models, Biological , Nervous System/cytology , Nervous System/pathology , Organoids/cytology , Organoids/pathology , Pluripotent Stem Cells/cytology
7.
Nature ; 604(7905): 354-361, 2022 04.
Article in English | MEDLINE | ID: mdl-35355015

ABSTRACT

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.


Subject(s)
Melanoma , Skin Neoplasms , Animals , Animals, Genetically Modified , Carcinogenesis/genetics , Foot , Hand , Humans , Melanoma/pathology , Nails , Oncogenes/genetics , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Transcription, Genetic , Zebrafish/genetics , Melanoma, Cutaneous Malignant
8.
Cell ; 145(6): 827-30, 2011 Jun 10.
Article in English | MEDLINE | ID: mdl-21663788

ABSTRACT

Building on the discovery that MyoD expression reprograms fibroblasts into muscle, three papers (Vierbuchen et al., 2010; Ieda et al., 2010; Szabo et al., 2010) recently reported the reprogramming of fibroblasts into neurons, cardiomyocytes, and blood cell progenitors without first passing the cells through a pluripotent state. Here we discuss the advantages and challenges of harnessing this direct reprogramming method for regenerative medicine.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells/cytology , Regenerative Medicine , Animals , Cell Differentiation , Fibroblasts/cytology , Humans , Transcription Factors/metabolism
9.
Cell ; 147(7): 1511-24, 2011 Dec 23.
Article in English | MEDLINE | ID: mdl-22196728

ABSTRACT

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-ß signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.


Subject(s)
Chromatin Assembly and Disassembly , Embryonic Stem Cells/metabolism , Histones/metabolism , Smad Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , Goosecoid Protein/genetics , Homeodomain Proteins/genetics , Humans , Mice , Models, Molecular , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Alignment
10.
Genes Dev ; 32(17-18): 1161-1174, 2018 09 01.
Article in English | MEDLINE | ID: mdl-30115631

ABSTRACT

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.


Subject(s)
Alternative Splicing , Basic Helix-Loop-Helix Transcription Factors/genetics , Cadherins/metabolism , Embryonic Stem Cells/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Antigens, CD , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cadherins/genetics , Cell Differentiation/genetics , Cell Line , Embryonic Stem Cells/cytology , Exons , Gene Expression Regulation , Humans , RNA Precursors/chemistry , RNA, Messenger/chemistry , Regulatory Sequences, Ribonucleic Acid
11.
Mol Psychiatry ; 29(7): 2084-2094, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38383769

ABSTRACT

CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of ß-amyloid 42 (Aß42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aß42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.


Subject(s)
Alzheimer Disease , Amyloid beta-Peptides , Microglia , Monocytes , Phagocytosis , Sialic Acid Binding Ig-like Lectin 3 , Signal Transduction , Single-Chain Antibodies , Microglia/metabolism , Microglia/drug effects , Humans , Sialic Acid Binding Ig-like Lectin 3/metabolism , Amyloid beta-Peptides/metabolism , Phagocytosis/drug effects , Phagocytosis/physiology , Single-Chain Antibodies/pharmacology , Single-Chain Antibodies/metabolism , Signal Transduction/drug effects , Alzheimer Disease/metabolism , Monocytes/metabolism , Monocytes/drug effects , Antibodies, Monoclonal, Humanized/pharmacology , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Phosphorylation/drug effects
12.
Proc Natl Acad Sci U S A ; 118(8)2021 02 23.
Article in English | MEDLINE | ID: mdl-33542154

ABSTRACT

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (ETV2, ERG, and FLI1) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.


Subject(s)
Endothelium, Vascular/cytology , Pluripotent Stem Cells/cytology , Transcription Factors/genetics , Animals , Blood-Brain Barrier , Brain/blood supply , Brain/cytology , Cell Differentiation , Cell Line , Cellular Reprogramming/physiology , Endothelium, Vascular/physiology , Gene Expression , Humans , Mice, Inbred Strains , Pluripotent Stem Cells/physiology , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , Single-Cell Analysis , Transcription Factors/metabolism , Transcriptional Regulator ERG/genetics , Transcriptional Regulator ERG/metabolism
13.
Nature ; 531(7592): 105-9, 2016 Mar 03.
Article in English | MEDLINE | ID: mdl-26863197

ABSTRACT

The enteric nervous system (ENS) is the largest component of the autonomic nervous system, with neuron numbers surpassing those present in the spinal cord. The ENS has been called the 'second brain' given its autonomy, remarkable neurotransmitter diversity and complex cytoarchitecture. Defects in ENS development are responsible for many human disorders including Hirschsprung disease (HSCR). HSCR is caused by the developmental failure of ENS progenitors to migrate into the gastrointestinal tract, particularly the distal colon. Human ENS development remains poorly understood owing to the lack of an easily accessible model system. Here we demonstrate the efficient derivation and isolation of ENS progenitors from human pluripotent stem (PS) cells, and their further differentiation into functional enteric neurons. ENS precursors derived in vitro are capable of targeted migration in the developing chick embryo and extensive colonization of the adult mouse colon. The in vivo engraftment and migration of human PS-cell-derived ENS precursors rescue disease-related mortality in HSCR mice (Ednrb(s-l/s-l)), although the mechanism of action remains unclear. Finally, EDNRB-null mutant ENS precursors enable modelling of HSCR-related migration defects, and the identification of pepstatin A as a candidate therapeutic target. Our study establishes the first, to our knowledge, human PS-cell-based platform for the study of human ENS development, and presents cell- and drug-based strategies for the treatment of HSCR.


Subject(s)
Cell Lineage , Cell- and Tissue-Based Therapy , Drug Discovery/methods , Enteric Nervous System/pathology , Hirschsprung Disease/drug therapy , Hirschsprung Disease/pathology , Neurons/pathology , Aging , Animals , Cell Differentiation , Cell Line , Cell Movement , Cell Separation , Cell- and Tissue-Based Therapy/methods , Chick Embryo , Colon/drug effects , Colon/pathology , Disease Models, Animal , Female , Gastrointestinal Tract/drug effects , Gastrointestinal Tract/pathology , Hirschsprung Disease/therapy , Humans , Male , Mice , Neurons/drug effects , Pepstatins/metabolism , Pluripotent Stem Cells/pathology , Receptor, Endothelin B/metabolism , Signal Transduction
14.
Nature ; 538(7625): 397-401, 2016 Oct 20.
Article in English | MEDLINE | ID: mdl-27706135

ABSTRACT

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.


Subject(s)
Molecular Chaperones/metabolism , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Drug Discovery , Female , Genes, myc/genetics , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Humans , Mice , Molecular Chaperones/antagonists & inhibitors , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/chemistry , Neoplasms/drug therapy , Neoplasms/genetics , Organ Specificity
16.
Genes Chromosomes Cancer ; 60(4): 272-281, 2021 04.
Article in English | MEDLINE | ID: mdl-33336840

ABSTRACT

Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.


Subject(s)
Cell Division , Chromosome Aberrations , Human Embryonic Stem Cells/cytology , Selection, Genetic , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation , Chromosomes, Human, Pair 17 , DNA Copy Number Variations , Humans , Hydroxyurea , Stress, Physiological , Transcriptome
18.
Proc Natl Acad Sci U S A ; 115(37): E8775-E8782, 2018 09 11.
Article in English | MEDLINE | ID: mdl-30154162

ABSTRACT

Herpes simplex virus type 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Some HSE children carry inborn errors of the Toll-like receptor 3 (TLR3)-dependent IFN-α/ß- and -λ-inducing pathway. Induced pluripotent stem cell (iPSC)-derived cortical neurons with TLR3 pathway mutations are highly susceptible to HSV-1, due to impairment of cell-intrinsic TLR3-IFN immunity. In contrast, the contribution of cell-intrinsic immunity of human trigeminal ganglion (TG) neurons remains unclear. Here, we describe efficient in vitro derivation and purification of TG neurons from human iPSCs via a cranial placode intermediate. The resulting TG neurons are of sensory identity and exhibit robust responses to heat (capsaicin), cold (icilin), and inflammatory pain (ATP). Unlike control cortical neurons, both control and TLR3-deficient TG neurons were highly susceptible to HSV-1. However, pretreatment of control TG neurons with poly(I:C) induced the cells into an anti-HSV-1 state. Moreover, both control and TLR3-deficient TG neurons developed resistance to HSV-1 following pretreatment with IFN-ß but not IFN-λ. These data indicate that TG neurons are vulnerable to HSV-1 because they require preemptive stimulation of the TLR3 or IFN-α/ß receptors to induce antiviral immunity, whereas cortical neurons possess a TLR3-dependent constitutive resistance that is sufficient to block incoming HSV-1 in the absence of prior antiviral signals. The lack of constitutive resistance in TG neurons in vitro is consistent with their exploitation as a latent virus reservoir in vivo. Our results incriminate deficiencies in the constitutive TLR3-dependent response of cortical neurons in the pathogenesis of HSE.


Subject(s)
Immunity/immunology , Induced Pluripotent Stem Cells/metabolism , Neurons/metabolism , Toll-Like Receptor 3/metabolism , Antiviral Agents/pharmacology , Cell Differentiation/genetics , Cells, Cultured , Cerebral Cortex/cytology , Child , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/physiology , Humans , Immunity/genetics , Induced Pluripotent Stem Cells/cytology , Interferon-beta/pharmacology , Mutation , Neurons/drug effects , Neurons/virology , Poly I-C/pharmacology , Toll-Like Receptor 3/genetics , Trigeminal Ganglion/cytology
19.
Neurobiol Dis ; 144: 105025, 2020 10.
Article in English | MEDLINE | ID: mdl-32745521

ABSTRACT

Amyotrophic lateral sclerosis is a disease characterized by progressive paralysis and death. Most ALS-cases are sporadic (sALS) and patient heterogeneity poses challenges for effective therapies. Applying metabolite profiling on 77-sALS patient-derived-fibroblasts and 43-controls, we found ~25% of sALS cases (termed sALS-1) are characterized by transsulfuration pathway upregulation, where methionine-derived-homocysteine is channeled into cysteine for glutathione synthesis. sALS-1 fibroblasts selectively exhibited a growth defect under oxidative conditions, fully-rescued by N-acetylcysteine (NAC). [U13C]-glucose tracing showed transsulfuration pathway activation with accelerated glucose flux into the Krebs cycle. We established a four-metabolite support vector machine model predicting sALS-1 metabotype with 97.5% accuracy. Both sALS-1 metabotype and growth phenotype were validated in an independent cohort of sALS cases. Importantly, plasma metabolite profiling identified a system-wide cysteine metabolism perturbation as a hallmark of sALS-1. Findings reveal that sALS patients can be stratified into distinct metabotypes with differential sensitivity to metabolic stress, providing novel insights for personalized therapy.


Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Cysteine/metabolism , Fibroblasts/metabolism , Glucose/metabolism , Glutathione/metabolism , Metabolome , Aged , Case-Control Studies , Cells, Cultured , Female , Humans , Male , Metabolic Networks and Pathways , Metabolomics , Middle Aged , Serine/metabolism , Skin/cytology
20.
Nat Rev Genet ; 15(2): 82-92, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24434846

ABSTRACT

After years of incremental progress, several recent studies have succeeded in deriving disease-relevant cell types from human pluripotent stem cell (hPSC) sources. The prospect of an unlimited cell source, combined with promising preclinical data, indicates that hPSC technology may be on the verge of clinical translation. In this Review, we discuss recent progress in directed differentiation, some of the new technologies that have facilitated the success of hPSC therapies and the remaining hurdles on the road towards developing hPSC-based cell therapies.


Subject(s)
Cell Differentiation , Germ Layers/cytology , Pluripotent Stem Cells/cytology , Regenerative Medicine/methods , Cell Culture Techniques/methods , Cell Culture Techniques/trends , Gene Expression Regulation, Developmental , Germ Layers/metabolism , Humans , Models, Biological , Pluripotent Stem Cells/metabolism , Regenerative Medicine/legislation & jurisprudence , Regenerative Medicine/trends , Translational Research, Biomedical/legislation & jurisprudence , Translational Research, Biomedical/methods , Translational Research, Biomedical/trends
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