TGF-ß signaling through SMAD2/3 induces the quiescent microglial phenotype within the CNS environment.

Microglia are myeloid-derived cells that colonize the central nervous system (CNS) at early stages of development and constitute up to 20% of the glial populations throughout life. While extensive progress has been recently made in identifying the cellular origin of microglia, the mechanism whereby the cells acquire the unique ramified and quiescent phenotype within the CNS milieu remains unknown. Here, we show that upon co-culturing of either CD117(+) /Lin(-) hematopoietic progenitors or CD11c(+) bone marrow derived cells with organotypic hippocampal slices or primary glia, the cells acquire a ramified morphology concomitant with reduced levels of CD86, MHCII, and CD11c and up-regulation of the microglial cell-surface proteins CX(3) CR1 and Iba-1. We further demonstrate that the transforming growth factor beta (TGF-ß) signaling pathway via SMAD2/3 phosphorylation is essential for both primary microglia and myeloid-derived cells in order to acquire their quiescent phenotype. Our study suggests that the abundant expression of TGF-ß within the CNS during development and various inflammatory processes plays a key role in promoting the quiescent phenotype of microglia and may thus serve as a target for therapeutic strategies aimed at modulating the function of microglia in neurodegenerative diseases such as Alzheimer's and prion.

Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells.

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) have the unique ability to repopulate the entire hematopoietic system and thus are at the center of diverse, therapeutically relevant studies. The recent development of the CRISPR/Cas9 tool made the powerful research technique of genome editing highly accessible. Our previous studies demonstrated that high editing efficiency is reached when the CRISPR/Cas9 is introduced to CD34+ HSPCs as a ribonucleoprotein (RNP) complex with chemically modified guide RNAs (gRNAs). The current protocol details a quick 4-day procedure for ex vivo genome editing in CD34+ HSPCs by RNP complexes that are targeted to a specific locus by either a single gRNA (sgRNA) or a 2-part gRNA. The delivery of RNP complexes is performed by electroporation in the presence of a nonspecific, ssDNA electroporation enhancer, which highly improves editing efficiency under the described conditions. This approach is simple and effective with the potential to accelerate many biotechnological and therapeutic applications of the CRISPR/Cas9 technology.

Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System.

Genome editing of human cluster of differentiation 34+ (CD34+) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome editing using the CRISPR-Cas9 system in CD34+ HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34+ HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34+ HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34+ HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34+ HSPCs for research and therapy and can be adapted for other hematopoietic cells.

Active Aß vaccination fails to enhance amyloid clearance in a mouse model of Alzheimer's disease with Aß42-driven pathology.

Aß vaccination has been shown to induce remarkable clearance of brain amyloid plaques in mouse models of Alzheimer's disease (AD). However, the extent to which antibody-mediated Aß clearance is affected by predominant formation of Aß42 over Aß40 is unclear. Here we demonstrate for the first time that in a mouse model carrying the human APP mutations KM670/671NL and the human PS1 mutation P166L, Aß vaccination does not result in plaque clearance. This was in spite of the strong T- and B-cell immune responses evoked under the DR1501 genetic background and the activation of microglia at sites of Aß plaques. Our findings suggest the existence of antibody-resistant forms of Aß deposits in the brain consisting of primarily Aß42, and shed light on the mechanisms of antibody-dependent amyloid clearance as well as novel therapeutic strategies for AD.

Stromal cell-induced immune regulation in a transplantable lymphoid-like cell constructs.

Engineering of cell-based constructs for treating a variety of immune-related diseases by local transplantation of the cells in a pre-designed matrix is an emerging therapeutic approach, which can potentially reduce the side effects associated with systemic cell injection. Stromal cells have been shown to exert immunosuppressive properties and thus can be exploited for autoimmune regulation and cell transplantation. Here, we demonstrate the fabrication of a stromal cell-based construct, which serves as a lymphoid-like organ with immune regulatory characteristics. In the proposed system, stromal cells are co-seeded with dendritic cells (DC) in a macro-porous alginate scaffold containing the encephalitogenic myelin-derived peptide, proteolipid protein (PLP). We demonstrate that the presence of stromal cells attenuates DC maturation upon lipopolysaccharide stimulus. In vitro, we show that while the migration of pathogenic PLP-specific T cells to construct cultivated with or without stromal cells does not differ, their activation and proliferation are significantly suppressed in the presence of stromal cells. Upon in vivo transplantation, under the kidney capsule of mice, the pathogenic activation and proliferation of T cells which were drawn into the construct were suppressed in the co-seeded constructs. This system thus serves as a lymphoid-like organ with regulatory characteristics, which can be applied for local tolerance induction, for application in cell transplantations as well as autoimmune diseases.