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. 2020 Jan 7:12:100242.
doi: 10.1016/j.bonr.2020.100242. eCollection 2020 Jun.

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis

Eleonora Palagano  1   2 , Sharon Muggeo  1   2 , Laura Crisafulli  1   2 , Irina L Tourkova  3 , Dario Strina  1   2 , Stefano Mantero  1   2 , Elena Fontana  1   2 , Silvia L Locatelli  4 , Marta Monari  5 , Emanuela Morenghi  6 , Carmelo Carlo-Stella  4   7 , John B Barnett  8 , Harry C Blair  3 , Paolo Vezzoni  1   2 , Anna Villa  1   2 , Cristina Sobacchi  1   2 , Francesca Ficara  1   2
Affiliations

Generation of an immunodeficient mouse model of tcirg1-deficient autosomal recessive osteopetrosis

Eleonora Palagano et al. Bone Rep. .

Abstract

Background: Autosomal recessive osteopetrosis is a rare skeletal disorder with increased bone density due to a failure in osteoclast bone resorption. In most cases, the defect is cell-autonomous, and >50% of patients bear mutations in the TCIRG1 gene, encoding for a subunit of the vacuolar proton pump essential for osteoclast resorptive activity. The only cure is hematopoietic stem cell transplantation, which corrects the bone pathology by allowing the formation of donor-derived functional osteoclasts. Therapeutic approaches using patient-derived cells corrected ex vivo through viral transduction or gene editing can be considered, but to date functional rescue cannot be demonstrated in vivo because a relevant animal model for xenotransplant is missing.

Methods: We generated a new mouse model, which we named NSG oc/oc, presenting severe autosomal recessive osteopetrosis owing to the Tcirg1 oc mutation, and profound immunodeficiency caused by the NSG background. We performed neonatal murine bone marrow transplantation and xenotransplantation with human CD34+ cells.

Results: We demonstrated that neonatal murine bone marrow transplantation rescued NSG oc/oc mice, in line with previous findings in the oc/oc parental strain and with evidence from clinical practice in humans. Importantly, we also demonstrated human cell chimerism in the bone marrow of NSG oc/oc mice transplanted with human CD34+ cells. The severity and rapid progression of the disease in the mouse model prevented amelioration of the bone pathology; nevertheless, we cannot completely exclude that minor early modifications of the bone tissue might have occurred.

Conclusion: Our work paves the way to generating an improved xenograft model for in vivo evaluation of functional rescue of patient-derived corrected cells. Further refinement of the newly generated mouse model will allow capitalizing on it for an optimized exploitation in the path to novel cell therapies.

Keywords: Mouse model; Osteopetrosis; Transplantation.

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Figures

Fig. 1
Fig. 1
The NSG oc/oc mouse model presents failure to thrive, lack of tooth eruption and reduced life span. a Left: body weight of NSG, NSG oc/+ and NSG oc/oc mice from post-natal day 11 (P11) to P20. NSG oc/+: n ≥ 3; NSG and NSG oc/oc: n ≥ 6. *p < 0.05; **p < 0.01. Right: representative photographs of NSG and NSG oc/oc littermates showing apparent reduced size in the latter. b Representative photographs of NSG and NSG oc/oc littermates showing lack of tooth eruption in the latter. c Kaplan-Mayer survival curves of NSG and NSG oc/oc mice (Log-rank test, ***p < 0.0001).
Fig. 2
Fig. 2
The NSG oc/oc mouse model displays marked splenomegaly and severe immunodeficiency. a Spleen weight, body weight and spleen weight over body weight ratio in NSG and NSG oc/oc mice (n ≥ 7 per genotype). b Representative images of formalin-fixed paraffin-embedded spleen sections from NSG and NSG oc/oc mice, stained with Hematoxylin/Eosin (H/E), anti-mouse CD3 or anti-mouse B220 antibody; positive controls are shown in Supplementary Fig. 1. Scale bar: 500 μm in the low magnification images, 100 μm in the inset. c Left: number of total splenocytes in NSG and NSG oc/oc mice (n ≥ 8 per genotype). Right: number of cells of all main splenic cell subpopulations (CD11b+ myeloid cells, TER119+ erythroid cells, B220+ B cells, CD4+ and CD8+ T lymphocytes, NKp46+ NK cells) as determined by FACS analysis, following the gating strategy depicted in Supplementary Fig. 2 (n ≥ 5 per genotype). d Number of double negative (DN), double positive (DP) and CD8, CD4 single-positive (SP8 and SP4) thymocytes after gating on CD45+ hematopoietic cells, as described in Supplementary Fig. 3 (n ≥ 3 per genotype). **p <0.01; ***p < 0.001. The NSG oc/oc mouse model displays marked splenomegaly and severe immunodeficiency. a Spleen weight, body weight and spleen weight over body weight ratio in NSG and NSG oc/oc mice (n ≥ 7 per genotype). b Representative images of formalin-fixed paraffin-embedded spleen sections from NSG and NSG oc/oc mice, stained with Hematoxylin/Eosin (H/E), anti-mouse CD3 or anti-mouse B220 antibody; positive controls are shown in Supplementary Fig. 1. Scale bar: 500 μm in the low magnification images, 100 μm in the inset. c Left: number of total splenocytes in NSG and NSG oc/oc mice (n ≥ 8 per genotype). Right: number of cells of all main splenic cell subpopulations (CD11b+ myeloid cells, TER119+ erythroid cells, B220+ B cells, CD4+ and CD8+ T lymphocytes, NKp46+ NK cells) as determined by FACS analysis, following the gating strategy depicted in Supplementary Fig. 2 (n ≥ 5 per genotype). d Number of double negative (DN), double positive (DP) and CD8, CD4 single-positive (SP8 and SP4) thymocytes after gating on CD45+ hematopoietic cells, as described in Supplementary Fig. 3 (n ≥ 3 per genotype). **p <0.01; ***p < 0.001.
Fig. 3
Fig. 3
NSG oc/oc mice present osteoclast-rich osteopetrosis. a Calcium and phosphorus serum levels in NSG and NSG oc/oc mice (n ≥ 8 per genotype). **p < 0.01, ****p < 0.0001. b Representative images of decalcified formalin-fixed paraffin-embedded vertebral sections of NSG and NSG oc/oc mice stained with H/E (upper panels) and TRAP (middle panels); undecalcified formalin-fixed resin-embedded vertebral sections stained with von Kossa/van Gieson staining (lower panels). Scale bar: 500 μm. c In vitro osteoclastogenesis from splenic precursors of NSG and NSG oc/oc mice: representative images of TRAP staining on plastic (upper panels) and of Toluidine blue staining of resorption pits on dentin discs (lower panels). Scale bars: 400 μm.
Fig. 4
Fig. 4
Neonatal murine bone marrow transplantation corrects the pathology in NSG oc/oc mice. a MicroCT analysis of the femur of untreated NSG and NSG oc/oc mice, and murine BM-transplanted NSG and NSG oc/oc mice (n = 3 in all groups but transplanted NSG, where n = 2). b Representative images of decalcified formalin-fixed paraffin-embedded vertebral sections of transplanted (tx) NSG and NSG oc/oc mice 3 weeks after transplantation, stained with H/E. Scale bar: 500 μm. c Calcium and phosphorus serum levels in NSG and NSG oc/oc mice 3 weeks after transplantation (n = 2 per genotype; bars indicate the range). d Representative images of spleen of tx NSG and tx NSG oc/oc mice 3 weeks after transplantation, stained with H/E, anti-mouse CD3 or anti-mouse B220 antibody. Scale bar: 500 μm. e Growth curve of NSG and NSG oc/oc mice after transplantation, during the 3-month follow-up. f Representative images of decalcified formalin-fixed paraffin-embedded vertebral sections of transplanted (tx) NSG and NSG oc/oc mice 3 months after transplantation, stained with H/E. Scale bar: 500 μm. g Representative images of spleen of tx NSG and tx NSG oc/oc mice 3 months after transplantation, stained with H/E, anti-mouse CD3 or anti-mouse B220 antibody. Scale bar: 500 μm. h Histograms from FACS analysis of bone marrow (BM) and spleen (SP) cell suspensions from NSG and NSG oc/oc mice 3 months after transplantation (n = 3 per genotype).
Fig. 5
Fig. 5
Analysis of mice transplanted with human CD34+ cells. a FACS analysis of the BM of a representative NSG oc/oc mouse 2 weeks after transplantation. Human CD45+ leukocytes derived from transplanted CD34+ cells were gated (left pseudocolor plot) and analyzed for the expression of the human CD14 monocyte marker (histogram on the right, blue line; gray line represents the Fluorescent-Minus-One control). b Scatter plots showing (left) the percentage of human CD45+ leukocytes within the BM and the spleen of NSG and NSG oc/oc mice, and (right) the percentage of human CD14+ cells within the BM. c Representative immunohistochemical staining of vertebral bone of transplanted mice two weeks after transplantation, showing the presence of human CD45+ cells. Scale bar: 200 μm. d MicroCT analysis of the femur of NSG and NSG oc/oc mice (n = 3 per group) and NSG oc/oc mice transplanted with human cells (n = 5).
Supplementary Fig 1
Supplementary Fig 1
Representative images of spleen of C57BL/6J mice stained with H/E, anti-mouse CD3 or anti-mouse B220 antibody, as technical control of the same stainings in NSG and NSG oc/oc mice. Scale bar: 500 μm.
Supplementary Fig 2
Supplementary Fig 2
Immunophenotypic analysis of splenocytes from NSG oc/oc and control mice. a Representative FACS analysis of splenocytes from C57BL/6J adult WT mice, serving as technical control (top panels), from NSG mice (middle panels) and from NSG oc/oc mice (bottom panels), after red blood cells lysis. Erythroid cells are defined as TER119+NKp46-CD11b-B220-CD4-CD8- (highlighted in red); NK cells as TER119-NKp46+CD11b-B220-CD4-CD8- (light blue); myeloid cells as TER119-NKp46-CD11b+B220-CD4-CD8- (green); B cells as TER119-NKp46-CD11b-B220+CD4-CD8- (orange); CD4 and CD8 T lymphocytes as TER119-NKp46-CD11b-B220-, CD4+CD8- or CD4-CD8+, respectively (dark blue). b Histogram showing mean percentage of splenic subpopulations depicted in a in the spleen of NSG and NSG-oc/oc mice. *p < 0.05; **p < 0.01.
Supplementary Fig 3
Supplementary Fig 3
Immunophenotypic analysis of thymocytes from NSG oc/oc and control mice. Representative FACS analysis of thymocytes from C57BL/6J adult WT mice, serving as technical control (top panels), from NSG mice (middle panels) and from NSG oc/oc mice (bottom panels). After gating on the hematopoietic cell fraction based on the expression of the pan-leukocyte marker CD45, the four main thymus subpopulation are defined as follows: DP: double positive CD4+CD8+; SP4: single positive CD4+CD8-; SP8: single positive CD4-CD8+; DN: double negative CD4-CD8-.
Supplementary Fig 4
Supplementary Fig 4
Immunohistochemical staining with anti-human CD45 antibody. Left: negative control. Representative section of formalin-fixed paraffin-embedded decalcified bone from a wild type untreated C57BL/6J mouse stained with anti-human CD45 antibody; as expected, no positive cells are found. Right: positive control. Representative section of a formalin-fixed paraffin-embedded decalcified human osteomedullary biopsy. Scale bars: 200 μm.
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