A patterned human neural tube model using microfluidic gradients.

The human nervous system is a highly complex but organized organ. The foundation of its complexity and organization is laid down during regional patterning of the neural tube, the embryonic precursor to the human nervous system. Historically, studies of neural tube patterning have relied on animal models to uncover underlying principles. Recently, models of neurodevelopment based on human pluripotent stem cells, including neural organoids1-5 and bioengineered neural tube development models6-10, have emerged. However, such models fail to recapitulate neural patterning along both rostral-caudal and dorsal-ventral axes in a three-dimensional tubular geometry, a hallmark of neural tube development. Here we report a human pluripotent stem cell-based, microfluidic neural tube-like structure, the development of which recapitulates several crucial aspects of neural patterning in brain and spinal cord regions and along rostral-caudal and dorsal-ventral axes. This structure was utilized for studying neuronal lineage development, which revealed pre-patterning of axial identities of neural crest progenitors and functional roles of neuromesodermal progenitors and the caudal gene CDX2 in spinal cord and trunk neural crest development. We further developed dorsal-ventral patterned microfluidic forebrain-like structures with spatially segregated dorsal and ventral regions and layered apicobasal cellular organizations that mimic development of the human forebrain pallium and subpallium, respectively. Together, these microfluidics-based neurodevelopment models provide three-dimensional lumenal tissue architectures with in vivo-like spatiotemporal cell differentiation and organization, which will facilitate the study of human neurodevelopment and disease.

ORAI1-dependent synaptic plasticity in rat hippocampal neurons.

It is well established that neurons are plastic and can change the strength of their connections with other neurons depending on their recent history. While many molecular entities involved in plastic processes were already described, the role of a store-operated calcium channel ORAI1 in neuronal plasticity is not known as yet. Using dominant negative form of ORAI1, we were able to show that ORAI1 is needed for formation of new dendritic spines following chemical induction of long term potentiation (cLTP), and that this is due to the release of Ca+2 from ryanodine receptor-associated endoplasmic reticulum stores. We propose that when ORAI1 is deficient, there is less Ca+2 in the stores, less releasable Ca+2 and consequently less LTP and spine formation.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

PRDM16 co-operates with LHX2 to shape the human brain.

PRDM16 is a dynamic transcriptional regulator of various stem cell niches, including adipocytic, hematopoietic, cardiac progenitors, and neural stem cells. PRDM16 has been suggested to contribute to 1p36 deletion syndrome, one of the most prevalent subtelomeric microdeletion syndromes. We report a patient with a de novo nonsense mutation in the PRDM16 coding sequence, accompanied by lissencephaly and microcephaly features. Human stem cells were genetically modified to mimic this mutation, generating cortical organoids that exhibited altered cell cycle dynamics. RNA sequencing of cortical organoids at day 32 unveiled changes in cell adhesion and WNT-signaling pathways. ChIP-seq of PRDM16 identified binding sites in postmortem human fetal cortex, indicating the conservation of PRDM16 binding to developmental genes in mice and humans, potentially at enhancer sites. A shared motif between PRDM16 and LHX2 was identified and further examined through comparison with LHX2 ChIP-seq data from mice. These results suggested a collaborative partnership between PRDM16 and LHX2 in regulating a common set of genes and pathways in cortical radial glia cells, possibly via their synergistic involvement in cortical development.

An On-Chip Method for Long-Term Growth and Real-Time Imaging of Brain Organoids.

Brain organoids are an emerging technique for studying human neurodevelopment in vitro, with biomedical implications. However, three-dimensional tissue culture poses several challenges, including lack of nutrient exchange at the organoid core and limited imaging accessibility of whole organoids. Here we present a method for culturing organoids in a micro-fabricated device that enables in situ real-time imaging over weeks with efficient nutrient exchange by diffusion. Our on-chip approach offers a means for studying the dynamics of organoid development, cell differentiation, cell cycle, and motion. © 2018 by John Wiley & Sons, Inc.

The Interplay Between Epithelial-Mesenchymal Transition (EMT) and the Thyroid Hormones-αvß3 Axis in Ovarian Cancer.

Ovarian cancer is a highly metastatic disease. The metastatic potential is enhanced by epithelial to mesenchymal transition (EMT) in which αvß3 integrin plays a role. Thyroid hormones (L-thyroxine, T4, and 3,5,3'-triiodo-L-thyronine, T3) bind this integrin, and we hypothesized that the thyroid hormone-αvß3 axis may be involved in EMT activity in ovarian cancer. The transcription (mRNA), protein abundance (westerns), and protein localization (fluorescence microscopy) of several EMT markers were studied in ovarian cancer cells (OVCAR-3, A2780, and SKOV-3) treated with 1 nM T3 or 100 nM T4 for 1-24 h. The protein levels of ß-catenin, and its downstream targets, zeb-1, slug, and vimentin, were significantly induced by both hormones, while the effect on transcription was limited. The pre-incubation of the cells overnight with two integrin inhibitors, RGD (0.1-10 µM) or αvß3 blocking antibody (1-100 ng/mL), prevented the induction of ß-catenin by T3 and zeb-1 by T4, indicating direct integrin involvement. The transcription of the mesenchymal markers, ß-catenin, zeb-1, slug/snail, vimentin, and n-cadherin was hardly affected by T3 and T4, while that of the epithelial markers, e-cadherin and zo-1, was inhibited. Our results suggest a novel role for the thyroid hormone-αvß3 axis in EMT, with possible implications for ovarian cancer metastasis.