The Study Of Inducing Mouse Embryonic Stem Cells To Differentiate Into Endothelial Cells And In Vivo Magnetic Resonance Tracking After CLIO-Tat Labeling

Part One Differentiation of Murine Embryonic Stem Cells into Endothelial Cells using 3D and 2D MethodsObjective The objective of this study was to differentate murine embryonic stem cells (mESCs) into endothelial lineage, both in two-dimensional (2D) cultures and three-dimensional (3D), multicellular embryoid bodies (EBs).Materials and Methods To differentiate murine embryonic stem cells (mESCs) cells into endothelial cells (ECs) in 3D cultures, the hanging drop method was used to generate EBs, which were transfered into suspension culture on day 3 and subsequent plated on gelatin-coated plates on day 5. In order to enhance the differentiation into endothelial cells, mESCs were fed endothelial cell EGM-2 medium supplemented with an extra 50ng vascular endothelial growth factor (VEGF) per ml from day 3. The murine embryonic stem cell-derived endothelial cells (mESDECs) were stained for Flk-1, flurescence-activated cell sorting (FACS) sorted, and re-plated at day 9. In 2D differention system, mESCs were differentiated on collagen type IV-coated plates without leukemia inhibitory factor (LIF) supplementation, cultured with EGM-2 medium for 4 d. On day 4, the cells were FCAS sorted using Flk-1, re-plated in fibronectin (FN)-coated dishes and incubated in EGM-2 medium with VEGF (50ng/ml) supplementation. After culturing for approximately 1 w, the endothelial-like cells with cobblestone morphology were manually isolated and re-plate for further purification. The behaviors of mESDECs were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) uptake assay, matrigel assay and flow cytometry. The mRNA expressions of platelet-endothelial cell adhesion molecule (PECAM-1), endothelial-specific vascular endothelial cadherin (VE-Cadherin), angiopoietin receptor Tie-2, VEGF receptors 1 and 2 (Flt-1 and Flk-1, respectively), von Willebrand factor (vWF) and Octamer-4 (Oct-4) were observed using RT-PCR. The ESDECs were incubated with 10μg/mL of Dil-Ac-LDL before detection with fluorescence microscopy. In addition, the formation of endothelial tubes was assessed by seeding cells in 6-well plates coated with Matrigel. The kinetics of the expressions of PECAM-1, CD34 and VE-cadherin during the differentiation were also studied according to flow cytometric analysis.Results 1. The mESCs show an undifferentiated state when were cultured on inactivated mouse embryonic fibroblast cells (MEF), in the presence of LIF. The undifferentiated ESCs colonies are constituted of tightly packed round or oval cells with no visible individual cell boundaries.2. The hanging drop method presents the advantage of obtaining well-calibrated EBs almost identical in size, and shows higher efficiency of EB formation (>90%) than suspension method. When EBs are seeded on a 3D culture plate, vessel-like tube structures are observed on the outskirts of EB colonies. In addition, PECAM, an endothelial marker, is detected in the tubes using immunfluorescence staining.3. In 2D differentitate cultures, the Flk-1+ cell outgrowths exhibit predominantly two different morphologies. These include endothelial-like cells with cobblestone morphology and more striated smooth muscle-like cell populations. After further purified using manual selection, the cells acquire relatively uniform endothelial cell morphology and are able to propagate and expand in culture.4. The mESDECs obtained by two different methods display characteristics similar to murine aortic endothelial cells (MAECs). Immunocytochemistry and RT-PCR analyses revealed that MESDECs express endothelial cell-specific marker proteins such as VE-Cadherin, PECAM-1, Flk-1, Flt-1, Tie-2, and vWF, in which the expressions persist for long periods of time after differentiation. The cells have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to form tube-like structures when cultured on matrugel.5. The proportion of Flk-1+ cells obtained by 2D method (23%) is higher than which of the 3D (13%). After purification, about 98% ESDECs express VE-Cadherin, compare with the latter (95%). The 2D derivation sysoem is simple and efficient in vitro model.Conclusion Both 3D and 2D differentiation methods are efficient to derive a nearly pure population of endothelial cells from a murine ESC line in vitro. The mESDECs derived from the different systems show similar behavior and functional property with MAECs. However, the efficiency of 2D method is better than the other's. Part TwoEffects of CLIO-Tat Labeling on Endothelial Differentiation of Embryonic Stem Cells and in vitro Magnetic Resonance Tracking of CLIO-Tat Labeled Embryonic Stem CellsObjective The objectives of this study were to evaluate the feasibility of HIV transduction domain cross-linked iron oxide (CLIO-Tat) labeling technique and its labeling efficiency, and to determine whether CLIO-Tat affects viability, proliferation, and cell behavior of murine embryonic stem cell (ESCs) and murine embryonic stem cell-derived endothelial cells (mESDECs).Materials and Methods To determine the beat labeling efficiency of CLIO-Tat, various doses of CLIO-Tat were used to incubate with mESCs for different times. Cellular labeling was evaluated with Prussian blue staining, MR imaging and ICP atomic emission spectrometer. Proliferation and viability were evaluated using MTT assay and Trypan blue rejection method. Then, the effects of SPIO nanoparticles on the growth and differentiation of mESCs in vitro were investigated. The growth ability was evaluated through MTT, and the cells viability was tested through Trypan blue rejection method. Cell cycle and apoptosis were assessed using FCS. After differentiation, mESDECs were confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, Dil-Ac-LDL uptake assay, matrigel assay and flow cytometry.Results After labeling of mESCs with CLIO-Tat (50μg/ml) complexes, There were intracytoplasmatic blue particles in nearly every cell in the Prussian blue staining. Iron content of labeled mESCs is (25.3±2.1)pg per cell. CLIO-Tat has no adverse effects on the viability, proliferation, cell cycle and apoptosis of mESCs. In addition, the labeled cells show persistent low MR signals on T2*WI. After differentiation, the mESDECs express endothelial cell markers similar to MAECs, incorporate Dil-Ac-LDL, and form vascular-like channels. After all, CLIO-Tat-labeling does not affect angiogenesis property of mESDECs either.Conclusion From these data taken together, we concluded that mESCs could be efficiently and safely labeled with CLIO-Tat (50μg/ml) and the labeled cells could be reliably detected by MRI in vitro. T2*-weighted gradient echo acquisitions provide the greatest sensitivity to the presence of intracellular CLIO nanoparticles. We also demonstrated that mESCs labeled with CLIO-Tat or CLIO compared to their unlabeled counterparts had similar functional property and endothelial differentiation capacity. The mESDECs display characteristics similar to murine aortic endothelial cells (MAECs).Part ThreeCLIO-Tat Labeling and in vivo Magnetic Resonance Tracking of Transplanted Embryonic Stem Cell-Derived Endothelial CellsObjective The objectives of this study were to determine the feasibility and effect of using CLIO-Tat to label mESDECs for monitoring their retention and migration in vivo by magnetic resonance imaging (MRI), and to evaluate the treatment efficiency of the implantation of CLIO-Tat labeled mESDECs in the model of murine hind limb ischemia. Materials and Methods Before transplantation into murine hind limbs, mESDECs and mESCs were incubated with CLIO-Tat-FITC or CLIO for 24 h. After 7 days of ligation of the murine femoral artery, the CLIO-Tat-FITC labeled mESDECs, CLIO-Tat-FITC labeled mESCs, CLIO labeled mESDECs or PBS were transplanted into the ischemic hind limbs. The magnetic resonanceimaging (MRI) was carried out at day 0 and 1-4 weeks, respectively. At the 4th week, the blood supply of the ischemic hindlimb was measured with laser Doppler flowmetry and the impaired score of limb function was assessed. After MRI, the hind limbs were excised, and the segment in which injections were performed were thin cut and stained with hematoxylin-eosin and Prussian blue staining. Additionally, the angiogenesis of the ischemic muscular tissue was measured by microvessel counting (MVC) using immunostaining with anti-factorⅧ-related antigen monoclonal antibody. Results After labeling of mESDECs with CLIO-Tat(50μg/ml) complexes, There were intracytoplasmatic particles in nearly every cell in the Prussian blue staining and immunfluorescence staining. After transplantation, the injected sites containing labeled with mESDECs and mESCs could all be detected through MRI and were confirmed on pathology. T2*WI show persistent low MR signals that lasted up to 4 weeks. Laser Doppler flowmetry, impaired score of limb function and MVC show improvement of the blood supply in those labeled cell populations. Postmortem histology and immunohistochemistry confirm teratoma formation after injection of undifferentiated mESCs but not mESDECs. Conclusion We concluded that mESDECs could be efficiently and safely labeled with CLIO-Tat and the labeled cells could be reliably detected by MRI in vivo. CLIO-Tat labeled mESDECs could stimulate vascular regeneration and promote the blood flow of ischemic tissues. Furthermore, transplantation of predifferentiated rather than undifferentiated mESCs would be more suited for avoiding teratoma formation.