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Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling,axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development,the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks,gene expression and unique peak profiles associated with dysregulated genes,suggesting a coordinated epigenetic program. Treatment with isoxazole-9,which targets key dysregulated pathways,led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. View Publication

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The mammalian embryonic zeta-globin genes,including that of humans,are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif,ZF2,contributes to the repression of the HS-40-regulated human zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)(2) knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization,our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies. View Publication

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here,we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistochemistry,live imaging,and single-cell transcriptomics. We find that the cytoarchitecture,cell type composition,and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably,however,live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this,the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity,and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. View Publication

The amyloid-beta precursor protein (AbetaPP) is a ubiquitously expressed transmembrane protein whose cleavage product,the amyloid-beta (Abeta) protein,is deposited in amyloid plaques in neurodegenerative conditions such as Alzheimer disease,Down syndrome,and head injury. We recently reported that this protein,normally associated with neurodegenerative conditions,is expressed by human embryonic stem cells (hESCs). We now report that the differential processing of AbetaPP via secretase enzymes regulates the proliferation and differentiation of hESCs. hESCs endogenously produce amyloid-beta,which when added exogenously in soluble and fibrillar forms but not oligomeric forms markedly increased hESC proliferation. The inhibition of AbetaPP cleavage by beta-secretase inhibitors significantly suppressed hESC proliferation and promoted nestin expression,an early marker of neural precursor cell (NPC) formation. The induction of NPC differentiation via the non-amyloidogenic pathway was confirmed by the addition of secreted AbetaPPalpha,which suppressed hESC proliferation and promoted the formation of NPCs. Together these data suggest that differential processing of AbetaPP is normally required for embryonic neurogenesis. View Publication

Primary dilated cardiomyopathy (DCM) is a non-ischemic heart disease with impaired pumping function of the heart. In this study,we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a healthy volunteer and a primary DCM patient to investigate the impact of DCM on iPSC-CMs' responses to different types of anisotropic strain. A bioreactor system was established that generates cardiac-mimetic forces of 150 kPa at 5% anisotropic cyclic strain and 1. Hz frequency. After confirming cardiac induction of the iPSCs,it was determined that fibronectin was favorable to other extracellular matrix protein coatings (gelatin,laminin,vitronectin) in terms of viable cell area and density,and was therefore selected as the coating for further study. When iPSC-CMs were exposed to three strain conditions (no strain,5% static strain,and 5% cyclic strain),the static strain elicited significant induction of sarcomere components in comparison to other strain conditions. However,this induction occurred only in iPSC-CMs from a healthy volunteer (control iPSC-CMs")� View Publication

Bipolar disorder is a complex neuropsychiatric disorder that is characterized by intermittent episodes of mania and depression; without treatment,15% of patients commit suicide. Hence,it has been ranked by the World Health Organization as a top disorder of morbidity and lost productivity. Previous neuropathological studies have revealed a series of alterations in the brains of patients with bipolar disorder or animal models,such as reduced glial cell number in the prefrontal cortex of patients,upregulated activities of the protein kinase A and C pathways and changes in neurotransmission. However,the roles and causation of these changes in bipolar disorder have been too complex to exactly determine the pathology of the disease. Furthermore,although some patients show remarkable improvement with lithium treatment for yet unknown reasons,others are refractory to lithium treatment. Therefore,developing an accurate and powerful biological model for bipolar disorder has been a challenge. The introduction of induced pluripotent stem-cell (iPSC) technology has provided a new approach. Here we have developed an iPSC model for human bipolar disorder and investigated the cellular phenotypes of hippocampal dentate gyrus-like neurons derived from iPSCs of patients with bipolar disorder. Guided by RNA sequencing expression profiling,we have detected mitochondrial abnormalities in young neurons from patients with bipolar disorder by using mitochondrial assays; in addition,using both patch-clamp recording and somatic Ca(2+) imaging,we have observed hyperactive action-potential firing. This hyperexcitability phenotype of young neurons in bipolar disorder was selectively reversed by lithium treatment only in neurons derived from patients who also responded to lithium treatment. Therefore,hyperexcitability is one early endophenotype of bipolar disorder,and our model of iPSCs in this disease might be useful in developing new therapies and drugs aimed at its clinical treatment. View Publication

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation,microcephaly,and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4),Artemis,and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders,we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity,but not Artemis deficiency,were associated with markedly reduced reprogramming efficiency,which could be partially rescued by genetic complementation. Moreover,we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest,particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-,but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming,with a major role for LIG4 and DNA-PKcs and a minor,if any,for Artemis. View Publication

Human U1 small nuclear (sn)RNA,required for splicing of pre-mRNA,is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes,also located on chromosome 1 (1q12-21),were thought to be pseudogenes. However,many of these variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells� View Publication

BackgroundX-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome,the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates,triggering silencing of the chromosome. In mouse,an alternative Xist promoter,P2 is also the site of YY1 binding,which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation,including absence of a functional antisense regulator Tsix,and absence of strictly paternal inactivation in extraembryonic tissues,prompting us to examine regulatory regions for the human XIST gene.ResultsWe demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However,YY1 binding is insufficient to drive P2 expression or establish the DHS,which may require a development-specific factor. Furthermore,reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.ConclusionsThe differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter,P2,that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition,this region binds YY1 on the unmethylated inactive X chromosome,and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST. View Publication

The blood-brain barrier (BBB) is a critical component of the central nervous system (CNS) that regulates the flux of material between the blood and the brain. Because of its barrier properties,the BBB creates a bottleneck to CNS drug delivery. Human in vitro BBB models offer a potential tool to screen pharmaceutical libraries for CNS penetration as well as for BBB modulators in development and disease,yet primary and immortalized models respectively lack scalability and robust phenotypes. Recently,in vitro BBB models derived from human pluripotent stem cells (hPSCs) have helped overcome these challenges by providing a scalable and renewable source of human brain microvascular endothelial cells (BMECs). We have demonstrated that hPSC-derived BMECs exhibit robust structural and functional characteristics reminiscent of the in vivo BBB. Here,we provide a detailed description of the methods required to differentiate and functionally characterize hPSC-derived BMECs to facilitate their widespread use in downstream applications. View Publication

It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular,the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory,the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel,provides a robust model of human development and in the future,may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally,we demonstrate that following continued cell culture,stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore,also offer an in vitro model of disease. View Publication