Canine Bone Marrow Mesenchymal Cells Research Articles

Osteogenic and Angiogenic Potency of VEGF165-Transfected Canine Bone Marrow Mesenchymal Cells Combined with Coral Hydroxyapatite in Vitro.

To explore the osteogenic and angiogenic potential of human vascular endothelial growth factor 165 (hVEGF165) gene-transfected canine bone marrow mesenchymal stem cells (BMSCs) combined with coral hydroxyapatite (CHA) scaffold. We constructed a lentiviral vector and transfected canine BMSCs with the best multiplicity of infection. Osteogenesis was induced in the transfected groups (GFP-BMSCs group and hVEGF-BMSCs group) and non-transfected group (BMSCs group), followed by the evaluation of alkaline phosphatase (ALP) activity and alizarin red S staining. Cells from the three groups were co-cultured with CHA granules, respectively to obtain the tissue-engineered bone. MTT assay and fluorescence microscopy were employed to assess cell proliferation and adhesion. The expression of osteogenic and angiogenic related genes and proteins were evaluated at 7, 14, 21, and 28days post osteoinduction in cell culture alone and cell co-culture with CHA, respectively using RT-PCR and ELISA. The hVEGF165 gene was transfected into BMSCs successfully. Higher ALP activity and more calcified nodules were found in the hVEGF-BMSCs group than in the control groups (p < 0.001). Cells attached and proliferated in CHA particles. Both cells cultured alone and cells co-culture with CHA expressed more osteogenic and angiogenic related genes and proteins in the hVEGF-BMSCs group compared to the GFP-BMSCs and BMSCs groups (p < 0.05). High expression of hVEGF165 in BMSCs potentially promote the osteogenic potential of BMSCs, and synergically drive the expression of other osteogenic and angiogenic factors. hVEGF-BMSCs co-cultured with CHA expressed more osteogenic and angiogenic related factors, creating a favorable microenvironment for osteogenesis and angiogenesis. Also, the findings have allowed for the construction of a CHA-hVEGF-BMSCs tissue-engineered bone.

A method for quantitative cell tracking using SPECT for the evaluation of myocardial stem cell therapy

A promising SPECT-based method for evaluating stem cells therapy uses (111)In-labelled cells, transfected with a reporter gene. Cells are first transplanted to the infarct, and subsequently interrogated for transgenic expression using a systemic injection of an (131)I-labelled reporter probe. The method is impeded by the physical effects of scatter, (131)I/(111)In cross-talk, and attenuation. We hypothesize that correcting for physical effects improves detection of transgenic expression in transplanted cells when (111)In localization is available. Canine bone marrow mesenchymal cells (BMMCs), radiolabelled and transfected, were injected into infarcted myocardium. Next, a reporter probe was injected systemically, and 22 SPECT scans were acquired over 20 h. Finally, (99m)Tc-sestamibi was injected and imaged. The animal was killed, the heart sectioned, and counted for (131)I and (111)In in a well-counter ('gold standard'). Canine SPECTs were reconstructed in two ways: with corrections for physical effects and without corrections. The first (111)In reconstruction and the (99m)Tc reconstruction were used to define volumes-of-interest over the transplanted BMMC (VBMMC) and normal myocardium (VNM), respectively. (131)I reconstructions without corrections for physical effects had negligible differential uptake. With corrections, VBMMC was consistently higher than VNM, demonstrating transgene expression. (131)I had the following VBMMC:VNM activity ratio: without correction for physical effects=0.869; with corrections=1.23; and well-counter=1.21. VNM showed the following (131)I:(111)In activity ratio: without corrections=3.07; with corrections=1.38; and well-counter=1.58. In dual-isotope SPECT, corrections for physical effects were required to detect transgene expression in cells transplanted into an infarction when localization information was available.

Determining the minimum number of detectable cardiac-transplanted 111In-tropolone-labelled bone-marrow-derived mesenchymal stem cells by SPECT

In this work, we determined the minimum number of detectable 111In-tropolone-labelled bone-marrow-derived stem cells from the maximum activity per cell which did not affect viability, proliferation and differentiation, and the minimum detectable activity (MDA) of 111In by SPECT. Canine bone marrow mesenchymal cells were isolated, cultured and expanded. A number of samples, each containing 5 × 106 cells, were labelled with 111In-tropolone from 0.1 to 18 MBq, and cell viability was measured afterwards for each sample for 2 weeks. To determine the MDA, the anthropomorphic torso phantom (DataSpectrum Corporation, Hillsborough, NC) was used. A point source of 202 kBq 111In was placed on the surface of the heart compartment, and the phantom and all compartments were then filled with water. Three 111In SPECT scans (duration: 16, 32 and 64 min; parameters: 128 × 128 matrix with 128 projections over 360°) were acquired every three days until the 111In radioactivity decayed to undetectable quantities. 111In SPECT images were reconstructed using OSEM with and without background, scatter or attenuation corrections. Contrast-to-noise ratio (CNR) in the reconstructed image was calculated, and MDA was set equal to the 111In activity corresponding to a CNR of 4. The cells had 100% viability when incubated with no more than 0.9 MBq of 111In (80% labelling efficiency), which corresponded to 0.14 Bq per cell. Background correction improved the detection limits for 111In-tropolone-labelled cells. The MDAs for 16, 32 and 64 min scans with background correction were observed to be 1.4 kBq, 700 Bq and 400 Bq, which implies that, in the case where the location of the transplantation is known and fixed, as few as 10 000, 5000 and 2900 cells respectively can be detected.