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. 2016 Jun;23(6):500-9.
doi: 10.1038/gt.2016.21. Epub 2016 Mar 2.

Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse

K-H W Lau  1 , S-T Chen  1 , X Wang  1 , S Mohan  1 , J E Wergedal  1 , C Kesavan  1 , A K Srivastava  2 , D S Gridley  3 , S L Hall  1
Affiliations

Opposing effects of Sca-1(+) cell-based systemic FGF2 gene transfer strategy on lumbar versus caudal vertebrae in the mouse

K-H W Lau et al. Gene Ther. 2016 Jun.

Abstract

Our previous work showed that a Sca-1(+) cell-based FGF2 therapy was capable of promoting robust increases in trabecular bone formation and connectivity on the endosteum of long bones. Past work reported that administration of FGF2 protein promoted bone formation in red marrow but not in yellow marrow. The issue as to whether the Sca-1(+) cell-based FGF2 therapy is effective in yellow marrow is highly relevant to its clinical potential for osteoporosis, as most red marrows in a person of an advanced age are converted to yellow marrows. Accordingly, this study sought to compare the osteogenic effects of this stem cell-based FGF2 therapy on red marrow-filled lumbar vertebrae with those on yellow marrow-filled caudal vertebrae of young adult W(41)/W(41) mice. The Sca-1(+) cell-based FGF2 therapy drastically increased trabecular bone formation in lumbar vertebrae, but the therapy not only did not promote bone formation but instead caused substantial loss of trabecular bone in caudal vertebrae. The lack of an osteogenic response was not due to insufficient engraftment of FGF2-expressing Sca-1(+) cells or inadequate FGF2 expression in caudal vertebrae. Previous studies have demonstrated that recipient mice of this stem cell-based FGF2 therapy developed secondary hyperparathyroidism and increased bone resorption. Thus, the loss of bone mass in caudal vertebrae might in part be due to an increase in resorption without a corresponding increase in bone formation. In conclusion, the Sca-1(+) cell-based FGF2 therapy is osteogenic in red marrow but not in yellow marrow.

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Conflict of interest statement

Conflict-of-Interest: There is no conflict-of-interest to disclose.

Figures

Figure 1
Figure 1
Effects of marrow transplantation of MLV-FGF2- or MLV-gfp-transduced Sca-1+ cells on relative levels of engraftment (A), as well as serum levels of FGF2 (B), and serum PTH (C) in recipient mice. In A, engraftment of FGF2-expressing Sca-1+ cells was assessed by measuring the relative level of human FGF2 genomic DNA content in peripheral blood cells of recipient mice of MLV-FGF2-transduced cells (FGF2) or control MLV-gfp-transduced cells (GFP) at 6 or 14 weeks post-transplantation, respectively. N = 7 per group. In B, serum FGF2 levels of recipient mice of MLV-FGF2-transduced cells (FGF-2 mice) or the control MLV-gfp-transduced cells (GFP mice) at 14 weeks post-transplantation were assayed with a commercial ELISA kit. N = 7 per group. In C, serum PTH levels of recipient mice of MLV-FGF2- or MLV-gfp-transduced Sca-1+ cells at 14 weeks post-transplantation were measured with a commercial ELISA kit. N = 7 per group.
Figure 2
Figure 2
Sca-1+ cell-based systemic FGF2 gene therapy promoted massive trabecular bone formation but also caused osteomalacia in recipient mice. In A, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative control mice receiving the marrow transplantation of MLV-gfp-transduced Sca-1+ cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. In B, a cross-sectional slice (5 µm in thickness) of L3 lumbar vertebrae of a representative mice receiving the marrow transplantation of MLV-FGF2-transduced Sca-1+ cells at 14 weeks post-transplantation stained with the Goldner’s trichrome dye for mineralized bone. Mineralized bone is stained dark blue in color, whereas un-mineralized bone matrix is stained in red.
Figure 3
Figure 3
Static bone histomorphometric parameters of L3 vertebrae of recipient mice of MLV-gfp-transduced Sca-1+ cells (green bars) or MLV-FGF2-transduced Sca-1+ cells (yellow bars) at 14 weeks post-transplantation. %Md.Ar, % mineralized bone area; %O.Ar, % osteoid area; %L.Pm, % bone forming surface; Tb.Wi, trabecular width; and Tb.N, trabecular number. N = 7 per group. N.S. = not significant.
Figure 4
Figure 4
Serum FGF2 (A), Serum PTH (B), Serum alkaline phosphatase (ALP) activity (C), and bone ALP activity (D) of recipient mice receiving either MLV-FGF2-transduced Sca-1+ cells (FGF-2) or MLV-gfp-transduced Sca-1+ cells (GFP) after 14 weeks post-transplantation in the second marrow transplantation experiment. FGF2 and PTH were measured with respective commercial ELISA kits. ALP activity was assayed as described in Materials and Methods. N = 7 for the GFP group, and N = 4 for the FGF2 group.
Figure 5
Figure 5
Micro-CT analyses at the secondary spongiosa of the distal femur (A) or at S3 caudal vertebra (B) of recipient mice receiving either MLV-FGF2-transduced Sca-1+ cells (FGF-2) or MLV-gfp-transduced Sca-1+ cells (GFP) after 14 weeks post-transplantation. Top panels show a representative three-dimensional reconstruction of the trabecular structure each at the femur metaphysis (A) or at S3 caudal vertebrae (B) for each treatment group. Bottom bar graphs show the quantitative analyses of the three-dimensional bone parameters at each both site. N = 7 for the GFP group, and N = 4 for the FGF2 group.
Figure 6
Figure 6
A. Relative engraftment of transplanted β-galactosidase (βgal)-expressing cells at the marrow cavity of femurs (left columns) or at the entire caudal vertebra (right columns) of recipient mice receiving MLV-FGF2-transduced Sca-1+ cells (FGF-2, N = 20) after 14 weeks post-transplantation compared to that of control recipient mice receiving MLV-gfp-transduced Sca-1+ cells (GFP, N = 4). N.S. = not significant. B. Relative engraftment of gfp-expressing cells in the marrow cavity compared to that in the entire caudal vertebra of recipient mice receiving MLV-FGF2-transduced Sca-1+ cells. N = 20 per group. Engraftment in each panel was assessed by measuring the relative gfp mRNA levels (normalized against Ppia mRNA).
Figure 7
Figure 7
Relative expression levels of FGF2 mRNA in the marrow cavity or in the entire caudal vertebra of recipient mice receiving MLV-FGF2-transduced Sca-1+ cells (N = 20) compared to respective FGF2 mRNA expression levels at each bone site of control recipient mice receiving MLV-gfp-transduced cells (N = 4). *** P < 0.001 compared to respective GFP control group. N.S. = not significant.
Figure 8
Figure 8
A proposed model to account for the contrasting effects on the Sca-1+ cell-based systemic FGF2 gene therapy on skeletal sites with red marrows as opposed to those on skeletal sites with yellow marrows. Please see text for detailed description of the model.
See this image and copyright information in PMC

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