ABSTRACT
In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
Subject(s)
Central Nervous System/pathology , Herpesvirus 1, Human/immunology , Induced Pluripotent Stem Cells/cytology , Toll-Like Receptor 3/deficiency , Astrocytes/immunology , Astrocytes/virology , Biomarkers , Cell Differentiation , Cell Lineage , Cell Separation , Cells, Cultured , Central Nervous System/cytology , Central Nervous System/immunology , Central Nervous System/virology , Child , Disease Susceptibility , Encephalitis, Herpes Simplex/immunology , Encephalitis, Herpes Simplex/metabolism , Encephalitis, Herpes Simplex/pathology , Encephalitis, Herpes Simplex/virology , Herpesvirus 1, Human/pathogenicity , Humans , Immunity, Innate , Induced Pluripotent Stem Cells/virology , Interferons/immunology , Membrane Transport Proteins/deficiency , Membrane Transport Proteins/genetics , Neural Stem Cells/immunology , Neural Stem Cells/virology , Neurons/immunology , Neurons/pathology , Neurons/virology , Oligodendroglia/immunology , Oligodendroglia/pathology , Oligodendroglia/virology , Toll-Like Receptor 3/geneticsABSTRACT
The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT: Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.
Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Hedgehog Proteins/pharmacology , Kruppel-Like Transcription Factors/metabolism , Motor Neurons/cytology , Nerve Tissue Proteins/metabolism , Tretinoin/metabolism , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/drug effects , Female , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/physiology , Humans , Male , Motor Neurons/drug effects , Motor Neurons/metabolism , Tretinoin/pharmacology , Zinc Finger Protein Gli3ABSTRACT
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.
Subject(s)
Aging/pathology , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Adult , Age of Onset , Aged , Aged, 80 and over , Animals , Biomarkers/metabolism , Cell Differentiation , Cellular Reprogramming , Cellular Senescence , Child , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Dopaminergic Neurons/transplantation , Dopaminergic Neurons/ultrastructure , Fibroblasts/metabolism , Humans , Lamin Type A , Mesencephalon/pathology , Mice , Middle Aged , Parkinson Disease/pathology , Phenotype , Tissue DonorsABSTRACT
Endothelial cells line blood vessels and coordinate many aspects of vascular biology. More recent work has shown that endothelial cells provide a key niche in vivo for neural stem cells. In vitro, endothelial cells secrete a factor that expands neural stem cells while inhibiting their differentiation. Here, we show that a transformed mouse endothelial cell line (bEnd.3) maintains human pluripotent stem cells in an undifferentiated state. bEnd.3 cells have a practical advantage over mouse embryonic fibroblasts for pluripotent stem cell maintenance since they can be expanded in vitro and engineered to express genes of interest. We demonstrate this capability by producing fluorescent and drug-resistant feeder cells. Further, we show that bEnd.3 secretes an activity that maintains human embryonic stem cells without direct contact.
Subject(s)
Endothelial Cells/physiology , Feeder Cells/physiology , Induced Pluripotent Stem Cells/physiology , Animals , Cell Line , Cell Proliferation , Cell Shape , Cells, Cultured , Coculture Techniques , Embryonic Stem Cells/cytology , Embryonic Stem Cells/physiology , Endothelial Cells/cytology , Endothelial Cells/metabolism , Feeder Cells/cytology , Genetic Engineering , Humans , Induced Pluripotent Stem Cells/cytology , Mice , NeurogenesisABSTRACT
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer great promise in regenerative medicine and disease modeling due to their unlimited self-renewal and broad differentiation capacity. There is evidence that the growth properties and critical signaling pathways differ between murine and human ESCs; therefore, it is essential to perform functional studies to test the putatively conserved mechanisms of pluripotent stem cell self-renewal between species. Previously, we identified the transcription factor Zfx as a key regulator of self-renewal in murine ESCs. Here we extend those findings to human ESCs. ZFX knockdown in hESCs hindered clonal growth and decreased colony size after serial replating. ZFX overexpression enhanced clone formation in the presence of Y-27632, increased colony size at low density and decreased expression of differentiation-related genes in human ESCs. ZFX-overexpressing hESCs resisted spontaneous differentiation but could be directed to differentiate into endodermal and neural cell fates when provided with the appropriate cues. Thus, ZFX acts as a molecular rheostat regulating the balance between self-renewal and differentiation in hESCs, revealing the close evolutionary conservation of the self-renewal mechanisms in murine and human ESCs.
Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Kruppel-Like Transcription Factors/metabolism , Animals , Cell Differentiation/genetics , Cell Line , Cell Proliferation , Cell Size , Chromosomes, Artificial, Bacterial/genetics , Clone Cells , Endoderm/cytology , Endoderm/metabolism , Gene Expression Profiling , Gene Expression Regulation , Gene Knockdown Techniques , Humans , Kruppel-Like Transcription Factors/genetics , Mice , Transgenes/geneticsABSTRACT
Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.