Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation

Abstract

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Infiltration of DL-HSPCs in the brain of naïve mice. (A and C) Representative ex vivo MRI of the brain of As2−/− mice transplanted with DL-HSPCs (DL-HSPCs→As2−/−) (A) or KSL cells (C) 5 d after HCT (T2* MR sequences by a 1.5 T human scanner). Hypointense spots within the brain are detected. (B) Iron-containing, Prussian blue+ cells on brain sections from the As2−/− mouse shown in (A) [see (*)]. (D) Prussian blue staining (Left) and GFP immunofluorescence (Right) on brain sections from the As2−/− mouse shown in C. Cells positive for both signals [see (*)] are present. (E) EM showing electron-dense lysosomes (Top and Middle) corresponding to intraphagosomal iron on a brain section (MRI-proven site of migration) from a DL-HSPCs→As2−/− mouse, analyzed short term after HCT. The Middle and Bottom panels show colocalization of the intracellular iron particles (Upper) with positive signal on the corresponding iron map (red color; Lower) obtained at 250 eV by ESI. (F) Representative brain ex vivo MRI of the brain of DL-HSPCs→As2−/− (Upper) and As2+/+ (Lower) mice examined 5 d after HCT (T2* MR sequences by a 3 T human scanner). A similar number of hypointense spots (see red *) is detected, but only in the As2−/− mouse the spots are located at the corpus callosum. (G) Quantification of the MRI hypointense spot number (#) in the brain of As2+/+ and As2−/− mice. Mean and SEM are shown; n = 26 for As2+/+ and n = 37 for As2−/− mice. (H) Distribution of the hypointense spots in the indicated brain areas as in G; CB, cerebellum; Cortex, cortical gray matter; H.d.g., hippocampal dentate gyrus; STR, striatum; SVZ, subventricular zone. (I) GFP+ cell frequency within CD45+CD11b+ cells from the BM and brain of GFP+HSPC→mice, analyzed at 5, 14, and 180 d (dd) and 15 mo (mths) after HCT (pool of As2+/+ and As2−/− mice, n = 10 per time-point). (J) CD45+CD11b+ cells (Top) and GFP+ cells within CD45+CD11b+ cells in brains analyzed short- (5dd; Middle) or long-term (180dd; Bottom) after HCT (Right). UT, untransplanted mouse (Left). (K and L) Confocal images from brain sections of DL-HSPC As2−/− mice short- (K) and long-term (L) after HCT. Expression of GFP, Iba-1 (red), and CD11b (blue) is shown. ToPROIII (TpIII) for nuclei. M, merge; cc, corpus callosum; d.g., dentate gyrus; LV, lateral ventricle.

Fig. 2.

Macrophage/microglia replacement in the brain of preconditioned mice. (A–H) Frequency (%) of GFP+ cells within each indicated population, short and long term after HCT. n ≥ 10 (≥5 As2+/+ and ≥5 As2−/−) mice for each group at each time point. Mean and minimum/maximum values are shown; *P < 0.05, **P < 0.01, ***P < 0.001 at one-way ANOVA (with Bonferroni’s post hoc test).

Fig. 3.

Characterization of donor-derived cells in the brain of preconditioned mice. (A and B) Representative pictures showing IF staining for GFP, MHC class II, Iba-1, and CD11b on brain sections from GFP+HSPCs→mice analyzed at 180 d after the HCT. TPIII: nuclei. Cells with small bodies and pronounced and abundant ramifications are present in BU-treated and irradiated mice at 180 d after HCT. (C) Distribution of GFP+ cells in brain sections from representative BU-treated (Upper) and irradiated (Lower) As2−/− mice at 180 d after HCT. (Magnification: A, 20×; B, 50× compared with A.) Images were acquired at confocal microscope Radiance 2100 (Bio-Rad) (A and B), and Delta Vision Olympus Ix70 (C) and processed by the Soft Work 3.5.0; reconstructions were performed with Adobe Photoshop CS 8.0 software.

Fig. 4.

Microglia reconstitution by donor cells associated to depletion of the endogenous microglia compartment. (A and B) Frequency of μ (A) and TAμ (B) cells of pretreated and transplanted mice at different times from HCT is shown. CT, untreated mice (no conditioning, no HCT). n ≥ 10 (≥5 As2+/+ and ≥5 As2−/−) mice per group for each time point. Mean and minimum/maximum values are shown; *P < 0.05, **P < 0.01 at one-way ANOVA (Dunnet’s post hoc test). (C) Representative pictures showing abnormalities in the morphology of Iba-1+CD11b+GFP− host cells in BU-treated mice 3 mo after HCT. (D) Representative dot plots of the brain of BU-treated and irradiated mice 3 mo after HCT showing variations in the overall frequency and donor chimerism of μ and TAμ cells. (E) Correlation between the relative frequency (%) of μ cells and % GFP+ cells within this population in the brain of BU-treated mice long term. The correlation P value is shown. (F) Frequency of Annexin V+ cells within brain CD45+CD11b+ cells of conditioned mice 14 d after treatment. CT, untreated mice. n ≥ 10 (≥5 As2+/+ and ≥5 As2−/−) mice per group for each time point. Mean and minimum/maximum values are shown; **P < 0.01 at one-way ANOVA (Dunnet’s post hoc test). (G) Representative images showing TUNEL+ (red) Iba-1+ (green) cells on brain sections from mice at 14 d after BU (in blue nuclei by TPIII). M, merged panels. (Magnification: C, 80×; G, 100×.)

Fig. 5.

Depletion of endogenous microglia and proliferation of donor cells in the brain for microglia reconstitution. (A) Frequency of transgene+ [GFP (green) and ΔNGFR (red)] cells within BM and liver CD45+CD11b+ cells and the indicated brain populations at 45, 90, and 180 d after second HCT. (B) Distribution of GFP+ and ΔNGFR+ cells in the mouse brain at 45 d after the second HCT. GFP (green) and ΔNGFR (red) IF, and TPIII for nuclei, are shown. (C and D) Representative pictures showing transgene+ cells in the mouse brain at 45 (D, Upper) and 90 (C and D, Lower) d after second HCT, stained for GFP (green), ΔNGFR (red), CD11b, and Iba-1. (E) Representative plots showing Edu+ cells within CD45+CD11b+ cells at 90 d after the second HCT. No Edu CT, negative control of Edu staining; UT, Edu-treated age matched nontransplanted mouse. (F) Frequency of Edu+ cells within CD45+CD11b+ cells in the brain of age-matched UT mice and of transplanted mice at 90 d after second HCT. (G and H) Percentage of transgene+ cells (G) and of μ and TAμ cells (H) within Edu+/−CD45+CD11b+ cells of the mice shown in G. Mean and SEM are shown and n = 6 mice per group for each time point in A and F–H (I) Representative pictures showing Ki67+ (red) GFP+ cells in the brain of BU-treated mice at 90 d after second HCT (Perkin-Elmer Confocal UltraVIEW Ers Spinning Disk Confocal, acquisition of z-stacks, processed with Volocity software) (Magnification: C, 20×; D, 50×; I, 63×.)