Journal: Nature Communications

Article Title: Therapeutic efficacy of intracerebral hematopoietic stem cell gene therapy in an Alzheimer’s disease mouse model

doi: 10.1038/s41467-024-52301-w

Figure Lengend Snippet: Histograms showing the donor-cell chimerism within the CD45 + /CD11b + cell population in the Brain [BR], Spinal Cord [SC] and Bone marrow [BM] at 45 ( a ), 90 ( b ), and 240 ( c ) days post transplant (pTX). The progeny of intra-CNS transplanted cells (CNS Inj.) either ICV or ITL (as indicated), is shown in green, whereas the progeny of IV administered cells (IV inj.) are shown in blue. On the x-axis different hematopoietic subpopulations used in each combinatorial setting are shown: BM = Bone Marrow; Lin = Lineage negative cells; KSL = c-kit + , Sca-1 + Lin - cells. d Frequency of ICV (green) and IV delivered cells (blue) within the indicated brain subpopulations (μ and TAμ) at 90 days pTX. Representative dot plots showing the gating strategy used to identify μ and TAμ subpopulations are shown on the left. Histograms ( a – d ) show the mean ± SEM of mice at 45 days pTX [ n = 8 IV Lin, n = 9 ICV Lin IV Lin, n = 7 ICV Lin IV BM, n = 9 ITL Lin IV BM mice, n = 3 experimental replicates], 90 days [ n = 11 IV Lin, n = 10 ICV Lin IV Lin and ICV Lin IV BM, n = 7 ICV Lin IV KSL, n = 5 ITL Lin IV BM mice, n = 4 experimental replicates] and 240 days [ n = 4 mice/group, n = 2 experimental replicates]. Statistics for a – d : 1-way ANOVA followed by Tukey post-hoc test for comparisons between the contribution of all donor-derived cells (CNS inj. + IV inj.) among the transplant settings; for additional statistical information refer to Tables and . Source data ( a – d ) are provided as a Source Data file. e , f Representative laser scanning confocal microscope microphotographs of IF-stained brain sections from mice co-transplanted with Lin- both ICV and IV, at 90 days pTX [ n = 5 slices from n = 3 mice]. ICV-derived cells (green signal) are stained with ( e ) anti-GFP antibody, IV-derived cells (red signal) with anti- ΔNGFR antibody, DAPI for nuclei (blue), and ( f ) Iba1 for microglia cells (arrows indicate representative co-localizations of Iba1 and GFP/ΔNGFR signals). CTX cortex, OB Olfactory Bulb, STR striatum, HPC hippocampus, CB Cerebellum. Magnification 40x. M= merge. Images were acquired at Leika SP2 confocal microscope. Scale bar ( e , f ) = 50 µm.

Article Snippet: Cells from BM and CNS tissues (brain and spinal cord) were analyzed by FC upon re-suspension in blocking solution (PBS 5%FBS, 1%BSA) and labeling at 4 °C for 15 min with the following specific antibodies: rat PE anti-mouse CD45 (BD Pharmingen) 1:100; rat APC anti-mouse CD45 (BD Biosciences) 1:150; rat Brilliant Violet 510 anti-mouse CD45, (BioLegend) 1:150; rat Pacific Blue antimouse CD45.2 (Bio Legend) 1:100; mouse PE anti-Mouse CD45.1 (BD bioscience) 1:100; rat APC anti-mouse CD11b (eBioscience) 1:100; mouse Alexa Fluor 647 anti-Human CD271 (NGF receptor) (BD Pharmingen) 1:30; rat APC 780 anti-mouse CD11b (eBioscience) 1:100; rat APC 780 anti-mouse CD117(c-kit) (eBioscience) 1:100; rat PE-Cy7 anti- mouse Ly-6A/E Sca-1 (Sca-1) (BD Bioscence) 1:150.

Techniques: Derivative Assay, Microscopy, Staining