Molecular imaging of bone marrow mononuclear cell survival and homing in murine peripheral artery disease

Abstract

Objectives: This study aims to provide insight into cellular kinetics using molecular imaging after different transplantation methods of bone marrow-derived mononuclear cells (MNCs) in a mouse model of peripheral artery disease (PAD).

Background: MNC therapy is a promising treatment for PAD. Although clinical translation has already been established, there is a lack of knowledge about cell behavior after transplantation and about the mechanism whereby MNC therapy might ameliorate complaints of PAD.

Methods: MNCs were isolated from F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). Male FVB and C57Bl6 mice (n = 50) underwent femoral artery ligation and were randomized into 4 groups receiving the following: 1) single intramuscular (IM) injection of 2 × 10(6) MNCs; 2) 4 weekly IM injections of 5 × 10(5) MNCs; 3) 2 × 10(6) MNCs intravenously; and 4) phosphate-buffered saline as control. Cells were characterized by flow cytometry and in vitro bioluminescence imaging (BLI). Cell survival, proliferation, and migration were monitored by in vivo BLI, which was validated by ex vivo BLI, post-mortem immunohistochemistry, and flow cytometry. Paw perfusion and neovascularization was measured with laser Doppler perfusion imaging (LDPI) and histology, respectively.

Results: In vivo BLI revealed near-complete donor cell death 4 weeks after IM transplantation. After intravenous transplantation, BLI revealed that cells migrated to the injured area in the limb, as well as to the liver, spleen, and bone marrow. Ex vivo BLI showed presence of MNCs in the scar tissue and adductor muscle. However, no significant effects on neovascularization were observed, as monitored by LDPI and histology.

Conclusions: This is one of the first studies to assess kinetics of transplanted MNCs in PAD using in vivo molecular imaging. MNC survival is short-lived, MNCs do not preferentially home to injured areas, and MNCs do not significantly stimulate perfusion in this particular model.

Figure 1. Bone marrow mononuclear cell (MNC) characterization

(A) Flow cytometric analysis following Ficoll-selection of MNCs indicates low numbers of stem/endothelial progenitor cells (Sca-1, Flk-1) and high numbers of adult hematopoietic cells of a predominantly inflammatory phenotype (CD45, CD11-b, Gr-1, NK 1.1) (axes present counts). (B) In vitro BLI signals from various numbers of Fluc expressing MNCs show (C) robust correlation with cell numbers (r²=0.97). Scale bars represent BLI signal in photons/s/cm²/sr. (D) In vitro fluorescence microscopy confirms the expression of GFP by the donor cells.

Figure 2. MNC survival following intramuscular injection into the adductor muscles of FVB mice after femoral artery ligation

(A) In vivo BLI pictures of mice that received either a total of 2×10⁶ MNC by single injection (upper panel) or by weekly injections (lower panel) show MNC survival is short-lived as most of the signal intensity died off at 4 weeks post-transplant. (B) Quantification of signals showed a somewhat more stable level of MNC presence following repeated injections although the difference did not reach statistical significance. Scale bars represent BLI signal in photons/s/cm²/sr.

Figure 3. Immunohistochemistry of GFP⁺ MNCs within the post-ischemic adductor muscle

Representative pictures of anti-GFP muscle staining of (A) positive control slide (magnification 20x), (B) adductor muscle of mouse receiving weekly 5×10⁵ MNC-injections, showing GFP⁺ cells near a blood vessel (magnification 40x)

Figure 4. Laser Doppler Perfusion Imaging (LDPI) of ischemic hindlimbs following intramuscular MNC therapy

(A) Graphic representation and (B) quantification of paw perfusion by LDPI show a significantly decreased perfusion in the affected left hindlimbs as compared to the healthy paw. While a dose-dependent trend towards faster recovery can be observed 3 days after ligation, no significant differences were measured over a total time period of 28 days (* indicates P<0.05).

Figure 5. Immunohistochemistry analysis of arteriogenesis within the post-ischemic adductor muscle

Representative pictures of anti-α smooth muscle staining in mice treated with (A) single 2×10⁶ MNC-injection, (B) weekly 5×10⁵ MNC-injection, and (C) PBS injections as a control. Quantification of (D) mean number of collaterals and (E) mean collateral size showed no significant differences between study groups (P=NS).

Figure 6. In vivo visualization of systemically injected MNC by BLI

(A,B) One day after left femoral artery ligation, 2×10⁶ MNCs were injected via tail vein injection and monitored for 10 days. In vivo BLI pictures and signal quantification on multiple time points show that after an initial low signal period due to scattered MNCs throughout the body, cells then travelled to the injured area but also showed preference for the liver, bone marrow, and spleen. Scale bars represent BLI signal in photons/s/cm²/sr (P=NS).

Figure 7. Functional results following systemic MNC injection after severe hindlimb ischemia

(A) Following ligation of both the femoral and iliac arteries, markedly decreased paw perfusion was observed for a prolonged period. (B) Quantification of paw perfusion revealed systemic MNC injection was not capable of restoring paw perfusion significantly better than PBS treatment. (P=NS).