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Objective To label adipose-derived stromal cells (ADSCs) with different concentrations of ultrasmall superparamagnetic particles of iron oxide (USPIO) to investigate the biological characteristics of ADSCs treated with these USPIO,and determine the optimal concentration of USPIO in labeling ADSCs in vitro. Methods USPIO with different concentrations were prepared,and the particles were endocytosed by ADSCs generated from rat adipose tissue. Eight groups (negative control group,blank control group,and 11.25,22.5,45,90,135 and 180 μg/mL treatment groups) were chosen. Labeling efficiency and cellular uptake were analyzed by Prussian blue staining. Meanwhile,proliferation capacity and viability of ADSCs were evaluated by Alamar blue assay and Cell Counting kit-8. Results ADSCs could be effectively labeled with USPIO: approximately 95％ ADSCs were labeled when they were incubated with USPIO for 24 h under the concentration of USPIO was 45 μg/mL;and approximately 100％ ADSCs were labeled when the concentration of USPIO was 90 μg/mL and above. The CCK-8 and Alamar blue tests showed that USPIO of different concentrations (11.25-90 μg/mL) had little influence on cell growth viability,and no significant difference was noted between each 2 concentration groups (P＞0.05). In a word, 45-90 μg/mL USPIO were the optimal choice for transplantion of ADSCs in vivo. Conclusion ADSCs from the adipose tissue can be effectively labeled with USPIO with minimal effect on cell proliferation and viability.
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<p><b>OBJECTIVE</b>To investigate the biological features of human decidua basalis-derived mesenchymal stem cells (PDB-MSCs) in vitro and identify their capacity of multilineage differentiation.</p><p><b>METHODS</b>PDB-MSCs were harvested from the decidua basalis of term placental by enzymatic digestion and density gradient centrifugation, and the growth characteristics and morphological changes of the MSCs were observed by inverted microscope. The proliferative ability of the cells was assessed by Cell Counting Kit-8. The cell cycle and expressions of the surface markers (CD29, CD44, CD73, CD90, CD34, CD45, and CD14) of the MSCs were identified by flow cytometry. Multilineage differentiation capacity of the cells was tested by inducing their differentiation toward osteoblasts, adipocytes and chondroblasts in vitro.</p><p><b>RESULTS</b>MSCs isolated from human decidua basalis of term placental exhibited a morphology similar to that of bone marrow-derived MSCs, and grew into colonies in in vitro culture, where the cells proliferated rapidly after passage with a cell doubling time of 2.21∓0.21 days. More than 70% of the cells stayed in the resting stage (G(0)/G(1)) and showed positivity for CD29, CD44, CD73 and CD90, but not for CD14, CD34 or CD45. After induction, the cells showed positive results of alizarin red staining, oil red O staining and Alcian blue staining.</p><p><b>CONCLUSION</b>Human decidua basalis contains a rich source of MSCs, which can be easily isolated and cultured without affecting their capacity of multilineage differentiation. The PDB-MSCs may have the potential as a novel source of stem cells.</p>
Subject(s)
Female , Humans , Pregnancy , Cell Differentiation , Physiology , Cell Separation , Cells, Cultured , Decidua , Cell Biology , Mesenchymal Stem Cells , Cell Biology , Multipotent Stem Cells , Cell Biology , Placenta , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector pDsRed2-N1-SDF-1alpha and observe its expression in the mouse bone marrow mesenchymal stem cells.</p><p><b>METHOD</b>SDF-1alpha gene sequence with XhoI, EcoRI restriction enzyme cutting site was amplified from the total RNA of mouse smooth muscle cells by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the eukaryotic expression vector pDsRed2-N1 encoding red fluorescent protein gene, and the insertion was verified by endonuclease digestion and DNA sequencing. Mouse bone marrow mesenchymal stem cells identified with immunofluorescence assay for vimentin expression were transfected with the constructed plasmid pDsRed2-N1-SDF-1alpha, and the expression of sdf-1alpha was detected using immunofluorescence assay.</p><p><b>RESULTS</b>The DNA fragment amplified by PCR from the total RNA was identical to SDF-1alpha from the gene library, and an identical DNA fragment was also amplified from the recombinants. Sequence analysis confirmed the successful insertion of SDF-1alpha into the pDsRed2-N1 vector and the eukaryotic expression vector pDsRed2-N1-SDF-1alpha was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-1alpha expression 24 h after transfection with the recombinant vector.</p><p><b>CONCLUSION</b>The pDsRed2-N1-SDF-1alpha eukaryotic expression vector constructed is capable of expression of SDF-1alpha fusion protein in the mouse bone marrow mesenchymal stem cells.</p>
Subject(s)
Animals , Female , Mice , Bone Marrow Cells , Cell Biology , Metabolism , Chemokine CXCL12 , Genetics , Genetic Vectors , Mesenchymal Stem Cells , Metabolism , Mice, Inbred C57BL , Recombinant Fusion Proteins , Genetics , TransfectionABSTRACT
Objective To investigate the method for inducing OABAergic neurons from adipose-derived mesenchymal stem cells (ADSCs) and observe the effect of transplantation of these neurons in the treatment of parkinsonian rats. Methods ADSCs isolated from rat inguinal fat pads were digested with collagenase, cultured and passaged in vitro, from which neural stem cells were induced using the neural stem cell culture medium prepared by our institute and identified for the stem cell markers. The neural stem cells obtained were further induced using the GABAergic neuron culture medium. After identification for the marker GAD65, the GABAergic neurons or the neural stem cells were stereotaxically transplanted into the subthalamic nucleus of the Parkinsonian rats, and the behavioral changes of the rats were observed at 2, 4 and 8 weeks after the cell transplantation. Results The neural stem cells differentiated from the ADSCs expressed the stem cell markers including nestin and neuron-specific enolase. After the second induction, the cells were positive for GAD65 as identified by immunofluorescence staining. Four weeks after transplantation of the neural stem cells and GABAergic neurons into the subthalamic nucleus, the parkinsonian rats exhibited significantly improved rotational behavior induced by apomorphine, and the improvement was especially obvious in rats with GABAergic cell transplantation. Conclusion GABAergic neurons can be induced from the rat ADSCs and transplantation of these neurons into the subthalamic nucleus can produce obvious behavioral improvement in rat models of Parkinson disease.
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Objective To describe a simple and efficient method to obtain large quantities of higllly purified Schwann cells(SCs).Methods SCs were isolated from the sciatic and brachial nerves of 3-to 5-day-old newborn SD rats by collagenase digestion.The isolated SCs were plated at the density of3x105/mL for primary culture,several rounds oftrypsin digestion were performed within 72 h to purify SCs. Results Compared with the purification using 1.25 g/L trypsin digestion in serial differential detachment procedures, Our protocol allowed easier and more efficient separation of the SCs from the fibroblasts.Immunocytochemical staining showed that the purity of the SCs exceeded 95%.Conclusion The purification protocol of the SCs we established can be easily carried out and yields well reproducible results to obtain large quantities ofhighly purified SCs for transplantation studies.
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Objective To establish a method for precise localization of the boundaries of the glial scars in chronic spinal cord injury for their complete resection to provide the experimental basis for neural stem cell transplantation studies. Methods Chronic spinal cord injury was induced in beagl edogs via partial transections of the thoracic segment of the spinal cord. Three months after the operation, with the posterior-medial midpoint of the inferior edge of the first vertebral plate above the bony window defined as the point-of-origin of a 3-dimensional space, the distances from the superior and inferior margins of the injured spinal segment to the point-of-origin were measured on anteroposterior magneti cresonance images (MRI). B-mode ultrasound was used to detect the signal variations in the spinal cord .Immunohistochemistry was performed on the longitudinal sections of the spinal cord for microscopi cmeasurement of the glial fiber lengths in comparison with the length determined by MRl. Results MRI defined a greater length (14.7±0.94 mm) of glial scars with abnormal signals than the actually injured length (10 mm). Ultrasound detected obvious signal changes in the injured spinal cord with distinct boundaries of the injuries. The glial scar length in the spinal cord defined pathologically (18.6± 1.19 mm) exceeded that defined by anteroposterior MRI scanning. Conclusions Anteroposterior MRI scanning combined with stereotactic localization allows approximate determination of the glial scar boundary in the injured spinal cord, and the discrepancies can be adjusted according to the result of pathological measurements. Ultrasonic inspection helps detect the residue scar tissues aRer surgica lresection to ensure complete glial scar resection.
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Objective To investigate the effects of the variables of superconducting knife diameter and freezing time of Endocare CryocareTM surgical system on the necrosis extent and micro-pathological changes of the normal frozen brain tissue of dogs. Methods Argon-helium refrigeration superconducting knives of 2 and 3mm in diameter were respectively inserted into the right frontal lobe of dog brains, and the brains were frozen for 3 and 5min, respectively. The procedure was repeated one time. 48h later, the brains were perfused and harvested for the pathological observation under light microscope and electron microscope and the determination of necrosis area. Results 48h after refrigeration, the frozen brains presented hemorrhagic necrosis and were obviously divided into the central necrosis, inflammatory reaction zone, bleeding and edema zone. The boundaries were clear. Survival of brain cell structure was not found in the edge of frozen area. Conclusion Two freeze-thaw cycles with the freezing time of 3 and 5min are enough to cause the death of frozen brain tissue.