Migration of neural progenitors in the complex tissue environment of the central nervous system is not well understood. Progress in this area has the potential to drive breakthroughs in neuroregenerative therapies, brain cancer treatments, and neurodevelopmental studies. To a large extent, advances have been limited due to a lack of controlled environments recapitulating characteristics of the central nervous system milieu.Reductionist cell culture models are frequently too simplistic, and physiologically more relevant approaches such as ex vivo brain slices or in situ experiments provide little control and make information extraction difficult. Here, we present a brain-on-chip model that bridges the gap between cell culture and ex vivo/ in vivo conditions through recapitulation of self-organized neural differentiation. We use a new multi-layer silicone elastomer device, over the course of four weeks to differentiate pluripotent human (NTERA2) cells into neuronal clusters interconnected with thick axonal bundles and interspersed with astrocytes, resembling the brain parenchyma. Neurons within the device express the neurofilament heavy (NF200) mature axonal marker and the microtubule-associated protein (MAP2ab) mature dendritic marker, demonstrating that the devices are sufficiently biocompatible to allow neuronal maturation. This neuronal-glial environment is interfaced with a layer of human brain microvascular endothelial cells showing characteristics of the blood-brain barrier including the expression of zonula occludens (ZO1) tight junctions and increased trans-endothelial electrical resistance. We used this device to model migration of human neural progenitors in response to chemotactic cues within a brain-tissue setting. We show that in the presence of an environment mimicking brain conditions, neural progenitor cells show a significantly enhanced chemotactic response towards shallow gradients of CXCL12, a key chemokine expressed during embryonic brain development and in pathological tissue regions of the central nervous system. Our brain-on-chip model thus provides a convenient and scalable model of neural differentiation and maturation extensible to analysis of complex cell and tissue behaviors.

Glioblastoma (GBM) remains the most aggressive primary brain cancer in adults. Similar to other cancers, GBM cells undergo metabolic reprogramming to promote proliferation and survival. Glycolytic inhibition is widely used to target such reprogramming. However, the stability of glycolytic inhibition in GBM remains unclear especially in a hypoxic tumor microenvironment. In this study, it was determined that glucose-6-phosphatase-α (G6PC/G6Pase) expression is elevated in GBM when compared to normal brain. Human-derived brain tumor initiating cells (BTICs) utilize this enzyme to counteract glycolytic inhibition induced by 2-Deoxy-D-glucose (2DG) and sustain malignant progression. Down-regulation of G6PC renders the majority of these cells unable to survive glycolytic inhibition, and promotes glycogen accumulation through the activation of glycogen synthase (GYS1) and inhibition of glycogen phosphorylase (PYGL). Moreover, BTICs that survive G6PC knockdown are less aggressive (reduced migration, invasion, proliferation, and increased astrocytic differentiation). Collectively, these findings establish G6PC as a key enzyme with pro-malignant functional consequences that has not been previously reported in GBM and identify it as a potential therapeutic target.

The subventricular zone of the lateral ventricles (SVZ) is the largest source of neural stem cells (NSCs) in the adult mammalian brain. Newly generated neuroblasts from the SVZ form cellular chains that migrate through the rostral migratory stream (RMS) into the olfactory bulb (OB), where they become mature neurons. Migration through the RMS is a highly regulated process of intrinsic and extrinsic factors, orchestrated to achieve direction and integration of neuroblasts into OB circuitry. These factors include internal cytoskeletal and volume regulators, extracellular matrix proteins, and chemoattractant and chemorepellent proteins. All these molecules direct the cells away from the SVZ, through the RMS, and into the OB guaranteeing their correct integration. Following brain injury, some neuroblasts escape the RMS and migrate into the lesion site to participate in regeneration, a phenomenon that is also observed with brain tumors. This review focuses on factors that regulate the migration of SVZ precursor cells in the healthy and pathologic brain. A better understanding of the factors that control the movement of newly generated cells may be crucial for improving the use of NSC-replacement therapy for specific neurological diseases.

Endogenous electric fields modulate many physiological processes by promoting directional migration, a process known as galvanotaxis. Despite the importance of galvanotaxis in development and disease, the mechanism by which cells sense and migrate directionally in an electric field remains unknown. Here, we show that electrophoresis of cell surface heparan sulfate (HS) critically regulates this process. HS was found to be localized at the anode-facing side in fetal neural progenitor cells (fNPCs), fNPCderived astrocytes and brain tumor-initiating cells (BTICs), regardless of their direction of galvanotaxis. Enzymatic removal of HS and other sulfated glycosaminoglycans significantly abolished or reversed the cathodic response seen in fNPCs and BTICs. Furthermore, Slit2, a chemorepulsive ligand, was identified to be colocalized with HS in forming a ligand gradient across cellular membranes. Using both imaging and genetic modification, we propose a novel mechanism for galvanotaxis in which electrophoretic localization of HS establishes cell polarity by functioning as a co-receptor and provides repulsive guidance through Slit-Robo signaling.

SUMMARY Human neural progenitor cell (NPC) migration within the subventricular zone (SVZ) of the lateral ganglionic eminence is an active process throughout early brain development. The migration of human NPCs from the SVZ to the olfactory bulb during fetal stages resembles what occurs in adult rodents. As the human brain develops during infancy, this migratory stream is drastically reduced in cell number and becomes barely evident in adults. The mechanisms regulating human NPC migration are unknown. The Slit-Robo signaling pathway has been defined as a chemorepulsive cue involved in axon guidance and neuroblast migration in rodents. Slit and Robo proteins expressed in the rodent brain help guide neuroblast migration from the subventricular zone (SVZ) through the rostral migratory stream to the olfactory bulb. Here, we present the first study on the role that Slit and Robo proteins play in human-derived fetal neural progenitor cell migration (hfNPC). We describe that Robo1 and Robo2 isoforms are expressed in the human fetal SVZ. Furthermore, we demonstrate that Slit2 is able to induce a chemorepellent effect on the migration of hfNPCs derived from the human fetal SVZ. In addition, when Robo1 expression is inhibited, hfNPCs are unable to migrate to the olfactory bulb of mice when injected in the anterior SVZ. Our findings indicate that the migration of human NPCs from the SVZ is partially regulated by the Slit-Robo axis. This pathway could be regulated to direct the migration of NPCs in human endogenous neural cell therapy.

<p>Supplementary Movie S2, related to Supplementary Figure 3: G6PC is required in brain tumor initiating cells (BTICs) to regain their aggressive migratory behavior.</p>

<p>Supplementary Figure S1, related to Experimental Procedures: Molecular characterization of primary BTICs. Supplementary Figure S2, related to Supplementary Figure 1: BTICs recover their proliferative and invasive capacities in vitro. Supplementary Figure S3, related to Supplementary Figure 3: BTICs express G6PC and its inhibition decreases cell migration, invasion and proliferation. Supplementary Figure S4, related to Supplementary Figure 3: G6PC knockdowns exhibit decreased cell invasion and migration. Supplementary Figure S5, related to Supplementary Figure 4: Pharmacological inhibition of G6PC induces an increase in the expression of the astrocytic marker GFAP in the recovery group. Supplementary Figure S6, related to Supplementary Figure 5: G6PC knockdown activates glycogen synthase (GYS1) and inhibits glycogen phosphorylase (PYGL). Supplementary Figure S7, related to Supplementary Figure 6: Invasion and proliferation capacity of BTICs in vivo.</p>

<p>Supplementary Movie S1, related to Supplementary Figure 1: Brain tumor initiating cells (BTICs) escape glycolytic inhibition with an aggressive phenotype.</p>