A dynamic in vitro model of Down syndrome neurogenesis with trisomy 21 gene dosage correction.

Excess gene dosage from chromosome 21 (chr21) causes Down syndrome (DS), spanning developmental and acute phenotypes in terminal cell types. Which phenotypes remain amenable to intervention after development is unknown. To address this question in a model of DS neurogenesis, we derived trisomy 21 (T21) human induced pluripotent stem cells (iPSCs) alongside, otherwise, isogenic euploid controls from mosaic DS fibroblasts and equipped one chr21 copy with an inducible XIST transgene. Monoallelic chr21 silencing by XIST is near-complete and irreversible in iPSCs. Differential expression reveals that T21 neural lineages and iPSCs share suppressed translation and mitochondrial pathways and activate cellular stress responses. When XIST is induced before the neural progenitor stage, T21 dosage correction suppresses a pronounced skew toward astrogenesis in neural differentiation. Because our transgene remains inducible in postmitotic T21 neurons and astrocytes, we demonstrate that XIST efficiently represses genes even after terminal differentiation, which will empower exploration of cell type-specific T21 phenotypes that remain responsive to chr21 dosage.

Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network.

Modeling the developmental etiology of viable human aneuploidy can be challenging in rodents due to syntenic boundaries, or primate-specific biology. In humans, monosomy-X (45,X) causes Turner syndrome (TS), altering craniofacial, skeletal, endocrine, and cardiovascular development, which in contrast remain unaffected in 39,X-mice. To learn how human monosomy-X may impact early embryonic development, we turned to human 45,X and isogenic euploid induced pluripotent stem cells (hiPSCs) from male and female mosaic donors. Because neural crest (NC) derived cell types are hypothesized to underpin craniofacial and cardiovascular changes in TS, we performed a highly-powered differential expression study on hiPSC-derived anterior neural crest cells (NCCs). Across three independent isogenic panels, 45,X NCCs show impaired acquisition of PAX7+SOX10+ markers, and disrupted expression of other NCC-specific genes, relative to their isogenic euploid controls. In particular, 45,X NCCs increase cholesterol biosynthesis genes while reducing transcripts that feature 5' terminal oligopyrimidine (TOP) motifs, including those of ribosomal protein and nuclear-encoded mitochondrial genes. Such metabolic pathways are also over-represented in weighted co-expression gene modules that are preserved in monogenic neurocristopathy. Importantly, these gene modules are also significantly enriched in 28% of all TS-associated terms of the human phenotype ontology. Our analysis identifies specific sex-linked genes that are expressed from two copies in euploid males and females alike and qualify as candidate haploinsufficient drivers of TS phenotypes in NC-derived lineages. This study demonstrates that isogenic hiPSC-derived NCC panels representing monosomy-X can serve as a powerful model of early NC development in TS and inform new hypotheses towards its etiology.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta.

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the Y chromosome in males and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants in these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using in vitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely used model for human syncytiotrophoblast development. While these isogenic panels trigger a GATA2/3- and TFAP2A/C-driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene coexpression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition and function, and that the combined haploinsufficiency of the pseudoautosomal region likely plays a key role in these changes.

Zinc finger protein 274 regulates imprinted expression of transcripts in Prader-Willi syndrome neurons.

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity and is caused by the absence of paternal contribution to chromosome 15q11-q13. Using induced pluripotent stem cell (iPSC) models of PWS, we previously discovered an epigenetic complex that is comprised of the zinc-finger protein ZNF274 and the SET domain bifurcated 1 (SETDB1) histone H3 lysine 9 (H3K9) methyltransferase and that silences the maternal alleles at the PWS locus. Here, we have knocked out ZNF274 and rescued the expression of silent maternal alleles in neurons derived from PWS iPSC lines, without affecting DNA methylation at the PWS-Imprinting Center (PWS-IC). This suggests that the ZNF274 complex is a separate imprinting mark that represses maternal PWS gene expression in neurons and is a potential target for future therapeutic applications to rescue the PWS phenotype.

iPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response.

Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) currently lacking any effective treatment. Promoting endogenous brain repair offers a potential strategy to halt and possibly restore neurologic function in PPMS. To understand how the microenvironment within white matter lesions plays a role in repair we have focused on neural progenitor cells (NPCs) since these are found in lesions in PPMS and have been found to influence oligodendrocyte progenitor cell maturation (OPCs). To better understand the cellular nature of NPCs in PPMS we developed iPS cells from blood samples of PPMS patients and age matched non-disease spouse or blood relative controls. Using these iPS cell lines we determined that the NPCs from PPMS cases provided no neuroprotection against active CNS demyelination compared to NPCs from control iPS lines which were capable of completely preventing injury. Conditioned media (CM) from PPMS NPCs provides no protection to OPCs and prevents maturation of OPCs into oligodendrocytes in vitro. We also found that CM from PPMS iPS NPCs elicited patient-specific differences in the response to compounds that should foster oligodendrocyte (OL) maturation. Together, these data establish a new model for understanding the nature of myelination defects in PPMS which may lead to novel targeted approaches for preventing demyelination in these patients.

Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

Cell polarity and neurogenesis in embryonic stem cell-derived neural rosettes.

Embryonic stem cells (ESCs) undergoing neural differentiation form radial arrays of neural stem cells, termed neural rosettes. These structures manifest many of the properties associated with embryonic and adult neurogenesis, including cell polarization, interkinetic nuclear migration (INM), and a gradient of neuronal differentiation. We now identify novel rosette structural features that serve to localize key regulators of neurogenesis. Cells within neural rosettes have specialized basal as well as apical surfaces, based on localization of the extracellular matrix receptor ß1 integrin. Apical processes of cells in mature rosettes terminate at the lumen, where adherens junctions are apparent. Primary cilia are randomly distributed in immature rosettes and tightly associated with the neural stem cell's apical domain as rosettes mature. Components of two signaling pathways known to regulate neurogenesis in vivo and in rosettes, Hedgehog and Notch, are apically localized, with the Hedgehog effector Smoothened (Smo) associated with primary cilia and the Notch pathway γ-secretase subunit Presenilin 2 associated with the adherens junction. Increased neuron production upon treatment with the Notch inhibitor DAPT suggests a major role for Notch signaling in maintaining the neural stem cell state, as previously described. A less robust outcome was observed with manipulation of Hedgehog levels, though consistent with a role in neural stem cell survival or proliferation. Inhibition of both pathways resulted in an additive effect. These data support a model by which cells extending a process to the rosette lumen maintain neural stem cell identity whereas release from this association, either through asymmetric cell division or apical abscission, promotes neuronal differentiation.

Derivation and isolation of NKX2.1-positive basal forebrain progenitors from human embryonic stem cells.

Gamma aminobutyric acid (GABA)-expressing interneurons are the major inhibitory cells of the cerebral cortex and hippocampus. These interneurons originate in the medial ganglionic eminence (MGE) and lateral ganglionic eminence of the ventral forebrain during embryonic development and show reduced survival and function in a variety of neurological disorders, including temporal lobe epilepsy. We and others have proposed that embryonic stem cell (ESC)-derived ventral forebrain progenitors might provide a source of new GABAergic interneurons for cell-based therapies. While human ESCs (hESCs) are readily differentiated in vitro into dorsal telencephalic neural progenitors, standard protocols for generating ventral subtypes of telencephalic progenitors are less effective. We now report efficient derivation of GABAergic progenitors using an established hESC reporter line that expresses green fluorescent protein (GFP) under the control of an endogenous NKX2.1 promoter. GABAergic progenitors were derived from this hESC line by a modified monolayer neural differentiation protocol. Consistent with sonic hedgehog (SHH)-dependent specification of NKX2.1-positive progenitors in the embryonic MGE, we show a dose-dependent increase in the generation of NKX2.1:GFP-positive progenitors after SHH treatment in vitro. Characterization of NKX2.1:GFP-positive cells confirms their identity as MGE-like neural progenitors, based on gene expression profiles and their ability to differentiate into GABAergic interneurons. We are also able to generate highly enriched populations of NKX2.1:GFP-positive progenitors, including cells with telencephalic identity, by fluorescence-activated cell sorting. These hESC-derived ventral forebrain progenitors are suitable candidates for cell-based therapies that aim at replacing dysfunctional or damaged cortical or hippocampal GABAergic interneurons.

Embryonic stem cell neurogenesis and neural specification.

The prospect of using embryonic stem cell (ESC)-derived neural progenitors and neurons to treat neurological disorders has led to great interest in defining the conditions that guide the differentiation of ESCs, and more recently induced pluripotent stem cells (iPSCs), into neural stem cells (NSCs) and a variety of neuronal and glial subtypes. Over the past decade, researchers have looked to the embryo to guide these studies, applying what we know about the signaling events that direct neural specification during development. This has led to the design of a number of protocols that successfully promote ESC neurogenesis, terminating with the production of neurons and glia with diverse regional addresses and functional properties. These protocols demonstrate that ESCs undergo neural specification in two, three, and four dimensions, mimicking the cell-cell interactions, patterning, and timing that characterizes the in vivo process. We therefore propose that these in vitro systems can be used to examine the molecular regulation of neural specification.