Research ArticleHEALTH AND MEDICINE

Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells

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Science Advances  06 Dec 2017:
Vol. 3, no. 12, e1701211
DOI: 10.1126/sciadv.1701211
  • Fig. 1 Myeloid cell reconstitution in the brain after intracerebroventricular injection of HSPCs.

    (A and B) Frequency of GFP+ cells identified within CD45+ cells of the brain (A) and BM (B) of BU_TX (BU-treated and transplanted) mice at the indicated time points after intracerebroventricular (ICV) injection of HSPCs transduced with GFP-encoding LVs. n ≥ 3 mice each time point; average and SD are shown. Analyzed by one-way analysis of variance (ANOVA) with Bonferroni post hoc test, 4 days at comparison with 1, 3, 6, and 24 hours shows P < 0.001. (C and D) Expression of the indicated hematopoietic (C) and myeloid/microglia (D) markers by GFP+ (donor) and GFP (recipient) CD45+ cells retrieved from the brain of BU_TX mice at different time points after intracerebroventricular injection of transduced HSPCs (input represents the HSPCs at time of infusion). n ≥ 3 mice each time point; average and SD are shown. Two-way ANOVA showed a significant effect of the markers and time (P < 0.0001). (E) Frequency of GFP+ cells in the total myeloid (CD45+CD11b+) brain compartment at different time points after intracerebroventricular and intravenous (IV) HSPC transplantation in BU-treated (BU) and irradiated (IRR) mice. n ≥ 5 mice per time point and group; average and SD are shown. Two-way ANOVA showed a significant effect of the route of cell administration and time in BU_TX and IRR mice (intracerebroventricular versus intravenous and time, P < 0.005). (F) Reconstruction of a sagittal brain section of a representative intracerebroventricularly transplanted BU-treated mouse, showing widespread distribution of GFP+ cells at 90 days from GFP-transduced HSPC intracerebroventricular injection. GFP (green) and Topro III (TPIII; blue) for nuclei are shown. Images were acquired via DeltaVision Olympus at 20× magnification and processed using Soft Work 3.5.0. Reconstruction was performed with Adobe Photoshop CS 8.0 software. (G) Immunofluorescence analysis for GFP (green) and IBA-1 (red) on brain sections from BU_TX mice at 90 days after intracerebroventricular transplantation of GFP-transduced HSPCs. M, merge. Magnifications (20× and 40×) of the relative dashed box are shown. Images were acquired using the confocal microscope Radiance 2100 (Bio-Rad) IX70 and processed using Soft Work 3.5.0.

  • Fig. 2 Posttransplant brain myeloid cells derived from early HSPCs.

    (A) Experimental scheme. Prospectively isolated LT-HSCs and progenitors within the HSPC pool as per c-kit, Sca-1, and lineage staining and use of the SLAM receptor markers CD150 and CD48. The indicated sorted populations were differentially transduced with LVs encoding GFP (KSL) and deleted nerve growth factor receptor (ΔNGFR) (non-KSL), as well as GFP (LT-HSCs), ΔNGFR (MPP), Tag-blue fluorescent protein (BFP) (HPC-1), and Cherry (HPC-2), and subsequently transplanted intravenously (B and D) or intracerebroventricularly (C and E) in competitive fashion at their original ratio into BU-myeloablated mice. Animals transplanted intracerebroventricularly also received unmanipulated (UM) total BM cells for hematopoietic rescue at day 5 after transplant. (B and C) Frequency of cells derived from intravenously (B) or intracerebroventricularly (C) transplanted KSL and non-KSL within total brain myeloid (CD45+CD11b+) cells, μ and TAμ of BU_TX mice at 90 days after transplant. n = 10 mice per group; average and SD are shown. CNSmac, CNS-associated macrophages. (D and E) Frequency of cells derived from each of the transplanted KSL subpopulations within total brain myeloid cells, μ and TAμ of BU-myeloablated mice transplanted intravenously (D) or intracerebroventricularly (E), at different time points after HCT. n = 10 mice per time point and group; average and SD are shown. (F and G) Immunofluorescence analysis of brain slices of BU-treated mice transplanted mice transplanted intravenously (F) or intracerebroventricularly (G) with KSL subpopulations at 90 days after transplant. In (F), the progeny cells of LT-HSC are GFP+ and those of MPPs are ΔNGFR+ (in red). IBA-1 staining is in the blue channel. Magnification, 20×. M, merge. In the right panels, other representative merged pictures at 20× (top) and their 40× magnifications (bottom) are shown. In (G), the progeny cells of HPC-2 are Cherry+ and those of MPPs are ΔNGFR+ (in green). No GFP+ staining was detected in the absence of ΔNGFR immunofluorescence. TPIII (blue) for nuclei is shown. Magnification, 20× (top). In the bottom panels, other representative merged pictures at 20× (top) and their 40× magnifications (bottom) are shown. Images were acquired using the confocal microscope Radiance 2100 (Bio-Rad) and processed using Soft Work 3.5.0.100. (H) Histogram plots showing the differential level of cxcr4 expression in KSL and non-KSL cells, and KSL subpopulations at the time of transplant.

  • Fig. 3 Fgd5+ HSCs generate a microglia-like progeny in the brain upon both intracerebroventricular and intravenous transplantation.

    (A) Experimental scheme. Fgd5+ HSCs (Lin ckit+ Sca-1+ Flk2 CD34) were isolated from CD45.2 Fdg5-green donor mice. Five hundred Fgd5+ HSCs were transplanted intravenously or intracerebroventricularly into BU-myeloablated or lethally IRR CD45.1 recipient mice. Transplanted animals also received UM CD45.1 total BM cells for hematopoietic rescue at day 5 after transplant. FSC, forward side scatter. (B and C) Frequency of donor cells (CD45.2+) within brain myeloid CD11b+ cells of mice transplanted intravenously (B) and intracerebroventricularly (C) with Fgd5 cells after BU and irradiation conditioning. n ≥ 4 per group. (D and E) Frequency of μ, TAμ, and CNSmac populations within donor-derived cells in intravenously (D) and intracerebroventricularly (E) transplanted BU-conditioned mice. n ≥ 4 per group; average and SD are shown. Student’s t test, P < 0.001 for BU versus irradiation in (B) and (C).

  • Fig. 4 Microglia signature is present in myeloid cells retrieved from the brain of transplanted mice.

    (A) Frequency of μ and TAμ cells within GFP+ cells in the brain of mice at 90 days after transplantation of GFP+ HSPCs intravenously or intracerebroventricularly. n = 5 per group; average and SD are shown. Two-way ANOVA showed a significant effect of the populations analyzed (P < 0.0001). (B) Fold change expression (calculated as 2−DDCT) of selected microglia genes, obtained by real-time polymerase chain reaction (PCR) in each indicated population retrieved from the brain of BU-treated, intravenously and intracerebroventricularly transplanted mice or from P10 mice, calculated on expression of the same genes in ADULT_CT_μ cells. Mean values are shown. For statistical tests, refer to table S1. (C and D) Principal components analysis (PCA) (C) and heat map (D) showing expression analysis of the genes identified as microglia signature by Butovsky et al. within our samples (μ and TAμ retrieved from naïve P10 and ADULT_CT, and HCT animals) and samples reported in Gosselin et al. (30), including microglia and macrophages. LPM, large peritoneal macrophages; SPM, small peritoneal macrophages; BMDM, BM-derived macrophages; TGEM, thioglycollate-elicited peritoneal macrophages.

  • Fig. 5 Myeloid cells from the brain of transplanted mice display functions of maturing microglia.

    Functional enrichment of differentially up-regulated (A) and down-regulated (B) genes in μCT cells versus μ transplanted cells. Functional enrichment of differentially up-regulated (C) and down-regulated (D) genes in μCT cells versus TAμ transplanted cells. GSEA preranked analysis was performed using our RNA-seq differential gene expression data on GO biological processes (http://software.broadinstitute.org/gsea/msigdb/collections.jsp) with default parameters. Semantic similarity of GOs (GOSemSim) was used to cluster significantly enriched GOs [GOs with false discovery rate (FDR) < 0.05 for up-regulation and FDR < 0.001 for down-regulation were chosen to enhance representation clarity]. A complete list of GO enrichments is presented in table S3. (E and F). Fold change of RNA-seq normalized expression values of genes selected from Matcovitch-Natan et al. (35) in the indicated populations retrieved from the brain of BU_TX mice or P10 mice versus ADULT_CT_μ cells. In (E), genes whose expression is up-regulated in adult mice and, in (F), genes whose expression is up-regulated in p10 mice are shown, as for Matcovitch-Natan et al. (35) For statistical tests, refer to table S4.

  • Fig. 6 Intracerebroventricular delivery of HSPCs has therapeutic relevance.

    (A) Frequency of human CD45+CD11b+ cells retrieved from the brain of NSG and Rag−/−γ−chain−/−As2−/− (RagMLD) mice transplanted intravenously or intracerebroventricularly with umbilical cord blood (CB)–derived CD34+ cells after BU treatment or sublethal irradiation (Rag−/−γ−chain−/−As2−/−), 12 (NSG) and 5 (Rag−/−γ-chain−/−As2−/−) weeks after transplant. Values are expressed as fold to intravenous transplantation, with intravenous transplantation equal to 3 ± 1.3 in NSG mice, and to 2.9 ± 0.7 in RagMLD. n ≥ 5 mice per group; average and SD are shown. P < 0.001 by Student’s t test in NSG mice; P < 0.05 by one-way ANOVA with Bonferroni post hoc test in RagMLD mice. (B) Representative box plots showing human cell engraftment (human CD45) in the brain of NSG mice that received human CD34+ HSPCs, and expression of the human CX3CR1 marker in the human cells identified in the transplanted mice brains. (C) Immunofluorescence analysis for GFP (green) and IBA-1 (red) on brain sections from NSG mice at 90 days after intracerebroventricular transplantation of GFP-transduced CD34+ cells. In blue, nuclei stained by TPIII. Magnifications (20× and 40×) of the relative dashed box are shown. M, merge. (D) ARSA activity (expressed as fold to the value measured in Rag−/−γ−chain−/−As2+/+ wild-type mice tissues) measured in the brain and BM of Rag−/−γ−chain−/−As2−/− mice transplanted with ARSA-transduced cells intracerebroventricularly or intravenously, as indicated. n = 3 mice per group; average and SD are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Bonferroni post hoc test.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/e1701211/DC1

    Supplementary Materials and Methods

    fig. S1. Experimental schemes for the different transplantation settings described in the paper, using both mouse and human HSPCs.

    fig. S2. Analysis of the engraftment of murine HSPC subpopulations in BU_TX or irradiated mice.

    fig. S3. FACS plots of cell populations sorted for gene expression analysis.

    fig. S4. Evaluation of hCD34+ cell engraftment in BM and brain of transplanted NSG mice.

    table S1. ANOVA P values with Tukey’s post hoc test of contrasts between cells in Fig. 4B.

    table S2. Differential expression of RNA-seq profiles of μ and TAμ cell populations.

    table S3A. Genes up-regulated in μCT versus μBU_TX as per Fig. 5A.

    table S3B. Genes down-regulated in μCT versus μBU_TX as per Fig. 5B.

    table S3C. Genes up-regulated in μCT versus TAμBU_TX as per Fig. 5C.

    table S3D. Genes down-regulated in μCT versus TAμBU_TX as per Fig. 5D.

    table S4. Moderated t test (limma) after FDR (46) adjustment for the different contrast of interest.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Experimental schemes for the different transplantation settings described in the paper, using both mouse and human HSPCs.
    • fig. S2. Analysis of the engraftment of murine HSPC subpopulations in BU_TX or irradiated mice.
    • fig. S3. FACS plots of cell populations sorted for gene expression analysis.
    • fig. S4. Evaluation of hCD34+ cell engraftment in BM and brain of transplanted NSG mice.
    • table S1. ANOVA P values with Tukey’s post hoc test of contrasts between cells in Fig. 4B.
    • table S4. Moderated t test (limma) after FDR (46) adjustment for the different contrast of interest.
    • Legends for tables S2 and S3A to S3D

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • table S2(Microsoft Excel format). Differential expression of RNA-seq profiles ofμ and TAμ cell populations.
    • table S3A (Microsoft Excel format). Genes up-regulated in μCT versus μBU_TX as per Fig. 5A.
    • table S3B (Microsoft Excel format). Genes down-regulated in μCT versusμBU_TX as per Fig. 5B.
    • table S3C (Microsoft Excel format). Genes up-regulated in μCT versus TAμBU_TX as per Fig. 5C.
    • table S3D (Microsoft Excel format). Genes down-regulated in μCT versus TAμBU_TX as per Fig. 5D.

    Files in this Data Supplement: