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-β) 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-β 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.

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 DNAfree 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.

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