A Semi-automated and Scalable 3D Spheroid Assay to Study Neuroblast Migration.

The subventricular zone of the mammalian brain is the major source of adult born neurons. These neuroblasts normally migrate long distances to the olfactory bulbs but can be re-routed to locations of injury and promote neuroregeneration. Mechanistic understanding and pharmacological targets regulating neuroblast migration is sparse. Furthermore, lack of migration assays limits development of pharmaceutical interventions targeting neuroblast recruitment. We therefore developed a physiologically relevant 3D neuroblast spheroid migration assay that permits the investigation of large numbers of interventions. To verify the assay, 1,012 kinase inhibitors were screened for their effects on migration. Several induced significant increases or decreases in migration. MuSK and PIK3CB were selected as putative targets and their knockdown validated increased neuroblast migration. Thus, compounds identified through this assay system could be explored for their potential in augmenting neuroblast recruitment to sites of injury for neuroregeneration, or for decreasing malignant invasion.

Interpenetrating polymer networks of collagen, hyaluronic acid, and chondroitin sulfate as scaffolds for brain tissue engineering.

Stem cells can provide neuro-protection and potentially neuro-replacement to patients suffering from traumatic brain injuries (TBI), with a practical option being delivery via engineered scaffolds. Collagen (Coll) and glycosaminoglycan (GAG) have been used as scaffolds for brain tissue engineering yet they often do not support cell differentiation and survival. In this study, we developed interpenetrating polymer network scaffolds comprising Coll, and incorporating two commonly found GAGs in the brain, chondroitin sulfate (CS) and/or hyaluronic acid (HA). We seeded these scaffolds with mouse neural stem cells from the subventricular zone (SVZ) niche. Compared to Coll-alone, all other substrates decreased the percent of nestin+ stem cells. Coll-CS-HA was more efficient at suppressing nestin expression than the other scaffolds; all SVZ cells lost nestin expression within 7 days of culture. In contrast to nestin, the percentage of microtubule associated protein 2 (MAP2+) neurons was greater in scaffolds containing, CS, HA or CS-HA, compared to Coll alone. Finally, Coll-CS increased the percentage of glial fibrillary acidic protein (GFAP+) astrocytes compared to Coll scaffolds. Overall, this work shows that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for TBI. STATEMENT OF SIGNIFICANCE: Brain injury is devastating yet with few options for repair. Stem cells that reside in the subventricular zone (SVZ) only repair damage inefficiently due to poor control of their cellular progeny and unsuitable extracellular matrix substrates. To solve these problems, we have systematically generated collagen (Coll) scaffolds with interpenetrating polymer networks (IPN) of hyaluronic acid (HA) or chondroitin sulfate proteoglycans (CS) or both. The scaffolds had defined pore sizes, similar mechanical properties and all three stimulated neurogenesis, whereas only CS stimulated astrocyte genesis. Overall, this work suggests that Coll-HA and Coll-CS-HA scaffolds selectively enhance neurogenesis and may be advantageous in tissue engineering therapy for brain repair.

Maternal transmission of an Igf2r domain 11: IGF2 binding mutant allele (Igf2rI1565A) results in partial lethality, overgrowth and intestinal adenoma progression.

The cation-independent mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R or IGF2R) traffics IGF2 and M6P ligands between pre-lysosomal and extra-cellular compartments. Specific IGF2 and M6P high-affinity binding occurs via domain-11 and domains-3-5-9, respectively. Mammalian maternal Igf2r allele expression exceeds the paternal allele due to imprinting (silencing). Igf2r null-allele maternal transmission results in placenta and heart over-growth and perinatal lethality (>90%) due to raised extra-cellular IGF2 secondary to impaired ligand clearance. It remains unknown if the phenotype is due to either ligand alone, or to both ligands. Here, we evaluate Igf2r specific loss-of-function of the domain-11 IGF2 binding site by replacing isoleucine with alanine in the CD loop (exon 34, I1565A), a mutation also detected in cancers. Igf2rI1565A/+p maternal transmission (heterozygote), resulted in placental and embryonic over-growth with reduced neonatal lethality (<60%), and long-term survival. The perinatal mortality (>80%) observed in homozygotes (Igf2rI1565A/I1565A) suggested that wild-type paternal allele expression attenuates the heterozygote phenotype. To evaluate Igf2r tumour suppressor function, we utilised intestinal adenoma models known to be Igf2 dependent. Bi-allelic Igf2r expression suppressed intestinal adenoma (ApcMin). Igf2rI1565A/+p in a conditional model (Lgr5-Cre, Apcloxp/loxp) resulted in worse survival and increased adenoma proliferation. Growth, survival and intestinal adenoma appear dependent on IGF2R-domain-11 IGF2 binding.