Marker-free coselection for CRISPR-driven genome editing in human cells.

Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36ß, IL-36γ, IL-36R) in EAE. METHODS: EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE. RESULTS: Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1ß, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls. CONCLUSION: This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling.

New neurons are constantly being generated in the postnatal subventricular zone. They have to migrate long distances via the rostral migratory stream (RMS) to reach their final destination in the olfactory bulb (OB). In adults, these neuronal precursors migrate in chains, ensheathed by astrocytic processes, and travel toward the OB along blood vessels (BVs) that topographically outline the RMS. The molecular and cellular mechanisms leading to the development of the RMS and the formation of the migration-promoting vasculature scaffold in the adult mice remain unclear. We now reveal that astrocytes orchestrate the formation and structural reorganization of the vasculature scaffold in the RMS and, during early developmental stages, the RMS contains only a few BVs oriented randomly with respect to the migrating neuroblasts. The first parallel BVs appeared at the outer border of the RMS, where vascular endothelial growth factor (VEGF)-expressing astrocytes are located. Gain-of-function and loss-of-function experiments revealed that astrocyte-derived VEGF plays a crucial role in the formation and growth of new BVs. Real-time videoimaging also showed that the migration of neuronal precursors in the developing RMS differs substantially from neuronal displacement in the adult migratory stream partially because of not yet fully developed vasculature scaffold. The downregulation of VEGF in vivo, specifically in the astrocytes of the developing RMS, affected the development of the vasculature scaffold and led to alterations in neuroblast migration. Altogether, our results demonstrate that astrocytes orchestrate the formation and growth of parallel BVs, crucial migration-promoting scaffolds in the adult migratory stream, via VEGF signaling.