Molecular Imaging Of Adipose Derived Stromal Cells In Hindlimb Ischemia Mice

Background and objectivePeripheral arterial disease (PAD), which is characterized by the arterial stenosis of lower extremity, is highly prevalent and extremely in people with age over 70 years, smoking, diabetic complications or systemic atherosclerosis. Apart from the surgery and endovascular intervention, which have disappointing long-term follow-up visit results, cell therapy offers a promising approach for more angiogenesis and collateral circulation for PAD patients. However, harvesting a large amount of bone marrow, as the most common source of cells, has a certain limitation, while it's still a challenge to track cells in vivo.In this study, we try to use the adipose derived stromal cells (ADSCs), which were harvested from adipose tissue ofβ-Actin-luc transgenic mice and have stable Fluc expression, to treat the murine hindlimb ischemia, as an animal model of PAD. Moreover, we aim to monitor the ADSCs, as well as tissue perfusion and therapeutic angiogenesis in ischemic hindlimb by multiple molecular imaging strategies. MethodsFVB transgenic mice were selected by bioluminescence imaging, and then propagated until stably carryingβ-Actin-luc gene. Stromal vascular fraction (SVF) was harvested from adipose tissue, which was anatomized from the transgenic mice by modified collagenase digestion method under sterile conditions. SVF was cultured in conventional medium for mesenchymal stromal cell (MSC) in vitro. Then adherent cells were digested and collected as the primary passage of ADSCs. For further characterization, cell surface antigen phenotyping was performed at passage 3 on ADSCs, which were incubated with CD44-PE,CD34-PE,CD31-PE,CD45-PE and CD90-APC monoclonal antibody and identified by flow cytometry. Bioluminescence imaging of the ADSCs in vitro and in vivo was performed by the In Vivo Optical Imaging System, as to confirm Fluc expression.Models of PAD were created in male athymic BALB/c nude mice by ligating and transecting the unilateral femoral artery and all the side branches. ADSCs (1×10⁶) or PBS were injected intramuscularly at random, then the models of PAD were naturally classified as ADSCs group (n=20) and PBS group (n=20). Control (n=10) and sham operation (n=10) group were set as well. After intraperitoneal injection of the D-luciferin, animals were subjected to bioluminescence imaging for tracking the ADSCs in vivo. Laser Doppler perfusion imager (LDPI) was used for assessing the microcirculation of the ischemic hindlimbs, which is quantified by relative LDPI index and self-control. Immunohistological analysis was used to display the therapeutic angiogenesis. Rate of limp and amputation were calculated meanwhile. ResultsInbred strain of the transgenic mice stably carriedβ-Actin-luc reporter gene after imaging selection and propagation. Immunophenotypic analyses showed that ADSCs were positive for CD90 (98.9%) and CD44 (89.7%), while negative for CD45, CD34 and CD31(<5.0%), providing the evidence for the mesenchymal differentiation potency of ADSCs. Bioluminescence imaging displayed that ADSCs stably expressed the Fluc reporter gene, and a robust correlation existed between different cell numbers and Fluc average radiance (correlation coefficient: r²=0.94). After 24 hours the engrafted ADSCs survived and displayed BLI signal, which increased rapidly and then decreased smoothly during 0-42 minutes after peritoneal injection of D-luciferin, the peak being (6.92×10⁶±4.11×10⁵) P·s^-1·cm^-2·sr^-1 at 21 min.The color-coded LDPI imaging showed that there was a statistical dicrease of hindlimb perfusion after unilateral femoral artery ligation, compared to the control and sham group, confirming the efficacy of PAD model. The engrafted ADSCs were found survived in the ischemic hindlimb with consistent bioluminescence average radiance, which decreased gradually from (6.59×10⁶±1.02×10⁶) P·s^-1·cm^-2·sr^-1 on day 0 to (2.50×10⁵±1.66×10⁵) P·s^-1·cm^-2·sr^-1 on day 35, displaying a finite survival capcity of the ADSCs in the host. The relative LDPI index was significantly higher in the ADSCs-transplanted group than that in the PBS group, which suggested ADSCs could further improve peripheral perfusion in the hindlimb (P<0.05) on the basis of collateral compensation. Immunohistochemisty also demonstrated more angiogenesis in the hindlimb of the ADSCs treated animal.C onclusions1. ADSCs fromβ-Actin-luc transgenic mice could highly express the markers of MSC, stably carry the Fluc reporter gene and facilitate the tracking and quantifying by reporter gene optical imaging in vitro and in vivo.2. Bioluminescence imaging is an exciting approach for long-term and consecutive monitoring ADSCs in vivo, which proved a finite survival capcity of the ADSCs after transplantation.3. ADSCs transplantation might be a promising cellular strategy for hindlimb ischemia with enhanced peripheral perfusion, more angiogenesis and better prognosis.