We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm2) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms. This i-BEC human BBB model discriminates species-selective antibody- mediated transcytosis mechanisms, is predictive of in vivo CNS exposure of rodent cross-reactive antibodies and can be implemented into pre-clinical CNS drug discovery and development processes.

Human NT2 cells, which differentiate into neurons and astrocytes, initially express and then permanently down-regulate Nanog and Oct-4 (POU5F1). We investigated the relationship between the expression of these genes and the methylation state of their 5-flanking regions. Gene expression and DNA methylation were assayed with quantitative polymerase chain reaction and bisulfite genomic sequencing, respectively. Retinoic acid-induced differentiation of NT2 cells to neurons is accompanied by a sequential decrease in the expression of both genes, paralleled by sequential epigenetic modification of their upstream regions. This is the first report demonstrating changes in DNA methylation in the promoter regions of Nanog and Oct-4 in a human cell line.

Regulatory mechanisms pertaining to the self-renewal of stem cells remain incompletely understood. Here, we show that functional interactions between small GTPase Rap1 and the adhesion molecule E-cadherin uniquely regulate the self-renewal of human embryonic stem cells (hESCs). Inhibition of Rap1 suppresses colony formation and self-renewal of hESCs, whereas overexpression of Rap1 augments hESC clonogenicity. Rap1 does not directly influence the expression of the pluripotency genes Oct4 and Nanog. Instead, it affects the endocytic recycling pathway involved in the formation and maintenance of E-cadherin-mediated cell-cell cohesion, which is essential for the colony formation and self-renewal of hESCs. Conversely, distinct from epithelial cells, disruption of E-cadherin mediated cell-cell adhesions induces lysosome delivery and degradation of Rap1. This in turn leads to a further downregulation of E-cadherin function and a subsequent reduction in hESC clonogenic capacity. These findings provide the first demonstration that the interplay between Rap1 and E-cadherin along the endocytic recycling pathway serves as a timely and efficient mechanism to regulate hESC self-renewal. Given the availability of specific activators for Rap1, this work provides a new perspective to enable better maintenance of human pluripotent stem cells. STEM CELLS 2010;28:247-257 Disclosure of potential conflicts of interest is found at the end of this article.

The blood–brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re‐directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB. However, these studies have been performed with antibodies that recognize different TfR epitopes and have different binding characteristics, preventing inter‐study comparisons. In this study, the efficiency of transcytosis in vitro and intracellular trafficking in endosomal compartments were evaluated in an in vitro BBB model for affinity variants (K d from 5 to174 nM) of the rat TfR‐binding antibody, OX26. Distribution in subcellular fractions of the rat brain endothelial cells was determined using both targeted quantitative proteomics‐selected reaction monitoring and fluorescent imaging with markers of early‐ and late endosomes. The OX26 variants with affinities of 76 and 108 nM showed improved trancytosis (Papp values) across the in vitro BBB model compared with a 5 nM OX26. Although ~40% of the 5 nM OX26 and ~35% of TfR co‐localized with late‐endosome/lysosome compartment, 76 and 108 nM affinity variants showed lower amounts in lysosomes and a predominant co‐localization with early endosome markers. The study links bivalent TfR antibody affinity to mechanisms of sorting and trafficking away from late endosomes and lysosomes, resulting in improvement in their transcytosis efficiency.Open Practices Open Science: This manuscript was awarded with the Open Materials Badge.For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14193.

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell-type-specific epigenomes and suggest mechanisms by which they may be created and maintained.

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs). AXL, a putative ZIKV cellular entry receptor, was also highly expressed in ZIKV-susceptible i-BEC and i-NPs. This iPSC-derived BBB model can help elucidate the mechanism by which ZIKV can infect BECs, cross the BBB and gain access to the CNS.Electronic supplementary materialThe online version of this article (10.1186/s12987-018-0100-y) contains supplementary material, which is available to authorized users.

Recently, human amniotic fluid (AF) cells have attracted a great deal of attention as an alternative cell source for transplantation and tissue engineering. AF contains a variety of cell types derived from fetal tissues, of which a small percentage is believed to represent stem cell sub-population(s). In contrast to human embryonic stem (ES) cells, AF cells are not subject to extensive legal or ethical considerations; nor are they limited by lineage commitment characteristic of adult stem cells. However, to become therapeutically valuable, better protocols for the isolation of AF stem cell sub-populations need to be developed. This study was designed to examine the molecular components involved in self-renewal, neural commitment and differentiation of AF cells obtained at different gestational ages. Our results showed that, although morphologically heterogeneous, AF cells derived from early gestational periods ubiquitously expressed KERATIN 8 (K8), suggesting that the majority of these cells may have an epithelial origin. In addition, AF cells expressed various components of NOTCH signaling (ligands, receptors and target genes), a pathway involved in stem cell maintenance, determination and differentiation. A sub-population of K8 positive cells (<10%) co-expressed NESTIN, a marker detected in the neuroepithelium, neural stem cells and neural progenitors. Throughout the gestational periods, a much smaller AF cell sub-population (<1%) expressed pluripotency markers, OCT4a, NANOG and SOX2, from which SOX2 positive AF cells could be isolated through single cell cloning. The SOX2 expressing AF clones showed the capacity to give rise to a neuron-like phenotype in culture, expressing neuronal markers such as MAP2, NFL and NSE. Taken together, our findings demonstrated the presence of fetal cells with stem cell characteristics in the amniotic fluid, highlighting the need for further research on their biology and clinical applications.

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