Expression Profiles of Subtracted Genes During Neural Differentiation from Human Embryonic Stem Cells using Suppression Subtractive Hybridization

Human embryonic stem cells (human ES cells) are pluripotent and self-renewing cells that can be isolated from the inner cell mass at the blastocyst stage. Human ES cells differentiate into specific cell lineages according to the expression of related genes. During neural development, specific gene expressions induced human ES cells some morphological changes. We used suppression subtractive hybridization to identify genes which shows altered expression during neural differentiation from human ES cells. We identified 90 genes as downregulated and 64 genes as upregulated in neural precursor (NP) cells derived from human ES cells compared with human ES cells. To obtain further information about differentiation, we performed expression profiling of subtracted genes between human ES cells and NP cells. Of the subtracted genes in human ES cells, the 19 genes showed decreased expression in NP cells. Meanwhile, of the subtracted genes in NP cells, the 20 genes were upregulated. Among them, COPZ1, CPSF6, MATR3 and TOMD3 were specific gene expressions that they significantly expressed up and down-regulation during neural development derived from ES cells. These genes have not previously been associated with neural differentiation, but they may be potentially participated in neurogenesis.

Modeling Genetic Epileptic Encephalopathies using Brain Organoids

SummaryEpileptic encephalopathies (EEs) are a group of disorders associated with intractable seizures, brain development and functional abnormalities, and in some cases, premature death. Pathogenic human germline biallelic mutations in tumor suppressor WW domain-containing oxidoreductase (WWOX) are associated with a relatively mild autosomal-recessive spinocerebellar ataxia-12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE). In this study, we generated an in-vitro model for EEs, using the devastating WOREE syndrome as a prototype, by establishing brain organoids from CRISPR-engineered human ES cells and from patient-derived iPSCs. Using these models, we discovered dramatic cellular and molecular CNS abnormalities, including neural population changes, cortical differentiation malfunctions, and Wnt-pathway and DNA-damage response impairment. Furthermore, we provide a proof-of-concept that ectopic WWOX expression could potentially rescue these phenotypes. Our findings underscore the utility of modeling childhood epileptic encephalopathies using brain organoids and their use as a unique platform to test possible therapeutic intervention strategies.

SSEA3 and Sialyl Lewis a Glycan Expression Is Controlled by B3GALT5 LTR through Lamin A-NFYA and SIRT1-STAT3 Signaling in Human ES Cells

B3GALT5 is involved in the synthesis of embryonic stem (ES) cell marker glycan, stage-specific embryonic antigen-3 (SSEA3). This gene has three native promoters and an integrated retroviral long terminal repeat (LTR) promoter. We found that B3GALT5-LTR is expressed at high levels in human ES cells. B3GALT5-LTR is also involved in the synthesis of the cancer-associated glycan, sialyl Lewis a. Sialyl Lewis a is expressed in ES cells and its expression decreases upon differentiation. Retinoic acid induced differentiation of ES cells, decreased the short form of NFYA (NFYAs), increased phosphorylation of STAT3, and decreased B3GALT5-LTR expression. NFYAs activated, and constitutively-active STAT3 (STAT3C) repressed B3GALT5-LTR promoter. The NFYAs and STAT3C effects were eliminated when their binding sites were deleted. Retinoic acid decreased the binding of NFYA to B3GALT5-LTR promoter and increased phospho-STAT3 binding. Lamin A repressed NFYAs and SSEA3 expression. SSEA3 repression mediated by a SIRT1 inhibitor was reversed by a STAT3 inhibitor. Repression of SSEA3 and sialyl Lewis a synthesis mediated by retinoic acid was partially reversed by lamin A short interfering RNA (siRNA) and a STAT3 inhibitor. In conclusion, B3GALT5-LTR is regulated by lamin A-NFYA and SIRT1-STAT3 signaling that regulates SSEA3 and sialyl Lewis a synthesis in ES cells, and sialyl Lewis a is also a ES cell marker.

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library

The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.