| Backgroud:As the promise of stem cell-based therapies begins to be realised, a critical issue is monitoring migration and homing of transplanted cells, as well as engraftment efficiency and functional capability in vivo. Because the ability of multi-directional differentiation and strong proliferation ability and involves no ethical problem, mesenchymal stem cells(MSCs) has attracted increasing attention and gradually become a very practical value source of cells in the field of replacement therapy and gene therapy. More researches about MSCs major in bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs). ADSCs are similar to BMSCs on cell growth kinetics and cell aging and efficiency of gene transfection. Compared to bone marrow, adipose tissues can be acquired by minimal risk in routine liposuction. As an abundant and readily accessible source of multipotent stem cells, ADSCs have been extensively investigated for their various therapeutic properties, and are considered as an ethical, practical and biologically appropriate cell population for cell therapy.Although the researches about animals and humans have shown that ADSCs can promote tissue repair and regeneration, however, the mechanisms of cell therapy are unclear and it is crucial to understand their behavior and fate in vivo. There is no efficient way to detect cell survival and migration and differentiation in vivo. Moreover, effective discern and tracing methods to understand the migration and turnover of transplanted cells in injure site are still absence. How to trace and analyze transplanted MSCs are present hot spot and nodes.The general methods of cell labeling technology include chemiluminescence dyes, magnetic tag, and fluorescent protein by gene transfection, and radioactive isotope. The labeling method of chemiluminescence dyes is direct-viewing and simple, which would be reduction and transfer to unlabeled cells with cellular metabolism. Magnetic resonance imaging (MRI) can be effective to track injected cells by method of noninvasive and real time imaging on living body, which used to track cells labeled with super paramagnetic iron oxide nanoparticles (SPIO). However, MRI can’t display the biological characteristics of transplanted cells and distinguish cell regeneration and cell differentiation from endogenous or exogenesis. The labeling advantage of radioactive isotope is low background signals and high signal-to-noise ratio but a very short tracer time and poor spatial resolution is deficiency. Green fluorescent protein (GFP), capable of expressing fluorescence in living cells, has been widely used as a lineage marker for mammalian cells. Although the fluorescent signal emitted from GFP can be easily detected with optical imaging and fluorescence microscopy, but mainly as the pathological tracer after sacrifice animal and sliceA suitable marker should have the feature of non-cytotoxicity, biocompatible, high labeling efficiency and hypersensitivity of detection. All above labeling methods have their pros and cons, ttherefore, in order to reveal the fate of transplanted cells in vivo it is necessary to monitor ADSCs both in vivo and in vitro conditions simultaneously and develop multimodality tracing technology. Imaging of transplanted stem cells is becoming increasingly important to understanding the biology of stem cells in vivo and as a preclinical tool to evaluate novel therapeutic strategies. Molday ION Rhodamine-BTM (MIRB) is a new kind of superparamagnetic iron oxide which has the labeling function of magnetic and fluorescent. With enhancement type green fluorescence protein (EGFP) gene lentivirus transfection method can be integrated foreign gene into the chromosome of the target cells. EGFP can lasting expression and is not affected by cell proliferation metabolism, which is suitable for the function research of transplanted cells in vivo. The co-labeled technology with fluorescent and magnetic labeling can carry out identification by histological analysis, at the same time which make for realize real-time and dynamic track the survival and distribution of injected stem cells in vivo. Therefore, MIRB and EGFP co-labeled ADSCs may be a more effective labeling method than other. It is still unclear that both of labeling impact on the biological characteristics of ADSCs and feasibility of multi-mode tracer. For to monitor the cellular dynamics of the transplanted stem cells has been proposed in vitro, a reliable technique for tracking the grafted stem cells is needed. So how to monitor the transplanted MSCs has always been the research hotspot and difficulty.In this study, we hypothesized that EGFP and SPIO labeled technology can make up for each other, however, whether which will cause adverse effect to cell biology performance need to understand. So we decided to study and further confirmed.OBJECTIVETo access the feasibility and biological characteristics of ADSCs co-labeled with MIRB and EGFP in vitro. To construct experiment basis for the research of ADSCs transplantation, the effectiveness and security of the two labeling of ADSCs need to detect, meanwhile, the tracing time limit of the two labeling in ADSCs need to compare in vitro. In order to investigate whether lentivirus-EGFP and MIRB would cause negative influence to cell biocharacteristics when they labeled ADSCs simultaneously, we need to observe and detect the influence of these two labeling on ADSCs about cytotoxicity, proliferation, vitality and multi-directional differentiation potential in vitro. All these provide foundation for further experiment of the research team.METHODS:1. Obtain the male rat ADSCs by using density gradient centrifugation and cell adherent method. Flow cytometry (CD34ã€CD44ã€CD45ã€CD105) and osteogenic induction and adipogenic differentiation were used to evaluated the character of stem cells.2. Lentiviral vectors encoding a gene for the emerald green fluorescent protein(lentivirus-EGFP) are used to transfect the P3 ADSCs with different MOI(MOI=0,50,100,200,400). After 48h,72h,5d and 7d transduction, EGFP expression and cell transfection rate of different MOI was measured by testing with flow cytometry technology and observing with inverted fluorescence microscope and the available transfection dosage which is optimal MOI. The changes of morphous and ultrastructure of ADSCs labeled with lentivirus-EGFP were observed by fluorescence microscope and laser confocal microscopy. The vitality rate of lentivirus-EGFP labeled ADSCs were detected by counting method under optical microscope after trypan blue staining.3. Passage 3 (P3) rat ADSCs were plated in a 6 well plate (1×104 cells cm-2) in 1 mL medium and incubated at 37℃ with 5% CO2. After the cells adhered overnight, MIRB including 2 mg Fe3+mL-1 was added to the medium at the concentrations of 10 ug Fe mL-1 for overnight. After incubation, the MIRB-containing medium was removed by aspiration and ADSCs were washed three times with PBS to remove extracellular MIRB. Then MIRB-labeled ADSCs were incubated at 37℃ with 5% CO2 in CCM for subsequent experiments. The labeling rate and the rat of viable were tested by Prussian blue staining and trypan blue staining.4. P3 rat ADSCs were prepared into cell suspension, which added the lentivirus-EGFP viruses (MOI=400). After 24 hour medium was exchanged into CCM. When lentivirus-EGFP labeled ADSCs growned together on the whole we exchanged the medium at the concentrations of 10 ug Fe mL-1 and overnight we changed the medium into CCM again. Cells collected 3 days later. The fluorescence expressed by ADSCs co-labeled with lentivirus-EGFP and MIRB were observed by fluorescence microscope.5. To access the influence of labeling on the biological characteristics of ADSCs: MIRB effect on the transfection efficiency of EGFP analysised by Flow cytometry. Cell proliferation, cell activity, and cell apoptosis rate detected by MTT, trypan blue staining test, and flow cytometry. JEM 1400 electron microscope to observe the distribution of superparamagnetic iron oxide nanoparticles in cells. Oid red O staining for adipogenesis potential of MIRB-ADSCs and EGFP-MIRB-ADSCs.6. Preparation cell debris (labeled with SPIO) by hypotension dissolve method. Co-culture the debris with unlabeled and EGFP labeled cells. After 7 days culture, cell shape and fluorescence expressing observed by fluorescence microscope and confocal microscope, and further the distribution of SPIO in unlabeled and EGFP ADSCs were confirmed by Prussian blue staining.RESULTS:1. Fbroblast-like rat ADSCs (P3) with slim bodies. Positive ADSCs for P3 homogeneous markers of surface proteins CD44, CD105, as well as the negative ones for hematopoietic lineage marker of CD45 and CD34. Oil red O staining after 14 days adipogenic induction, transparent lipid droplet appeared in kytoplasm. Alizarin red staining after 28 days osteogenic induction, red calcium salt deposition shaped in aggregated cells.2. With an MOI of 400:1, after 48 hours green fluorescent can be observed, more than 81.4% of ADSCs infected with lentivirus-EGFP showed strong fluorescent signals expressed by GFP at day 5.3. ADSCs were labeled with 20 g/mL"1 Fe3+MIRB successfully, and the percentage of rhodamine B-positive cells was more than 95%. The labeling rate of ADSCs was approximated to 100%, according to the observation of blue-stained iron particles. A similar proportion was observed with rhodamine B staining. TEM results revealed the SPIO particles were located in the endosomal vesicles of labeled ADSCs.4. When MIRB labeled EGFP-ADSCs 3 days later, the transfection efficiency of lentivirus-EGFP labeled cells (MOI=400) is 82.3% analysised by flow cytometry. MTT proliferation assays demonstrated no difference in ADSC number at 1,3,5 and 7 days between control, lentivirus-EGFP infected, MIRB labeled and co-labeled groups. Flowcytometric analysis at 1,2,3,4 weeks using annexin V-PE and 7-AAD as markers for apoptosis and necrosis, respectively, disclosed similar low rates of apoptosis and necrosis between co-labeled and control groups. Compared with the unlabeled ADSCs, no significant differences were detected in cell viability, proliferation, membranous antigen and multiple differentiation potential in the co-labeled samples (P> 0.05).5. The ADSCs can be labeled with GFP and MIRB successfully, showing different colored fluorescence for marker identification. The fluorescence emitted by GFP gene was sustained and stably expressed, while fluorescence with MIRB attenuated with time.6. The group of GFP labeled ADSCs co-cultured with the SPIO+debris emit red and green fluorescence simultaneously; The red fluorescent was in the cytoplasm and the green fluorescent was in the cell nucleus. The group of unlabeled ADSCs showed red fluorescence significantly.CONCLUSION:1. The transfection efficiency of lentivirus-EGFP on ADSCs is efficient. With culture, green fluorescence expressed by GFP can maintain stability and constant. Lentivirus-EGFP has no significantly influence on bionomics of ADSCs.2. MRIB labeling method is simply, fast and almost completely. We can observe the MIRB labeled cells by iron particle and red fluorescence. MRIB is a new kind of labeling which coupled SPIO and Rhodamine B and can be intaked by cells.3. The green fluorescence expressed by EGFP in ADSCs co-labeled with lentivirus-EGFP and MIRB can be observed stable and long-term while the red fluorescence emit from MIRB were waning. The two labeling can confirm and make up each other in different period of stem cell transplant research.4. Our research shows that the co-labeled ADSCs technology with lentivirus-EGFP and MIRB is feasible. The two labeling have no obvious toxic and side-effect on the biological characteristics of cells, which are available and safe.5. Co-labeled ADSCs can be used effectively in non-pathological cell trace by MRI in a way of invasiveness and real time and pathological cell trace by fluorescence microscope which can pinpoint transplanted cell in tissue. This multimodality imaging of trace method is hopeful to get more certainty, reliability, and economy research information.6. Because unlabeled cell can uptake the debris from the dead SPIO labeled cells, SPIO+cell detected by us may be not the original cell at the last. SPIO labeled cells can be used in a short-term and invasive trace in the research of transplanted cells. The method of SPIO labeling alone can result in false-positive rate gradually increased with the time and are not available to long-term cell trace. SPIO labeled stem cell cannot be detected accurately in vivo just by MRI. The method of co-labeled with EGFP and SPIO is feasible to distinguish the source of survival SPIO labeled cell in vivo. |
