Objective To investigate the myogenic differentiation of mesenchymal stem cells (MSCs) after being transplanted into the local muscle tissues. Methods The serious muscleinjured model was established by the way of radiation injury, incising, and freezing injury in 36 mouses. Purified MSCs derived from bone marrow of male mouse and MSCs induced by5-azacytidine(5-Aza-CR) were transplanted into the local of normal muscle tissues and injured muscle tissues of femal mouse. The quantity of MSCs and the myogenic differentiation of implanted MSCs were detected by the method of double labeling, which included fluorescence in situ DNA hybridization (FISH) and immuno-histochemistry on the 1st, 3rd, 6th, 9th, 12th, and 15th day after transplantation. Results The quantity of implanted MSCs decreased as timepassed. MSCs’ differentiation into myoblasts and positive expression of desmin were observed on the 15th day in purified MSCs group and on the 6th day in induced MSCs groups. Conclusion MSCs could differentiate into myoblasts after being implanted into the local of muscle tissues. The differentiationoccurs earlier in the induced MSCs group than that in purified MSCs group.
Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
Objective To monitor the stem cell migration into the bone defect following an injection of the labeled mesenchymal stem cells (MSCs) by the enha nced green fluorescent protein (EGFP)technology and to provide insights into an application of MSCs for the fracture healing. Methods Isolated MSCs from the rabbit femur marrow were culture-expanded and were labeled by the transfection with the recombinant retrovirus containing the EGFP gene. Then, some labeled MSCs were cultured under the osteogenic differentiation condition and the phenotype was examined. After the fracture of their bilateral ulna, 18 rabbits were divide d into two groups. The labeled MSCs were injected into the aural vein at 1×107 cells／kg in the experimental group and the unmarked MSCs were injected in the control group 24 hours before surgery, and 1 and 24 hours after surgery, res pectively. Necropsies were performed 2 days after surgery in the two groups. The sections from the left defects were observed under the fluorescence microscope and the others were analyzed by the bright-field microscopy after the HE staining. Results The EGFP did not affect the MSCs viability. After the labeled cells were incubated in the osteogenic medium alkaline phosphatase, the calcium nodule s were observed. All the rabbits survived. The tissue of haematoma was observed in the bone defects and the fluorescent cells were found in the experimental gr oup, but no fluorescent cells existed in the control group. Conclusion The EG FP labeled MSCs can undergo osteogenic differentiation in vitro and can mig rate into bone defects after their being injected into the peripheral vein.
Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
ObjectiveTo investigate the feasibility of adipose-derived mesenchymal stem cells (ADMSCs) differentiating into corneal epithelium-like cells after transfection with Pax6 gene. MethodsThe adipose tissue from bilateral inguinal of healthy C57BL/6 mice (5-6 weeks old) was used to isolate and culture ADMSCs.The 3rd passage ADMSCs were subjected to treatments of non-transfection (group A),pcDNA3.1 empty vector transfection (group B),and recombinant plasmid of pcDNA3.1-Pax6 transfection (group C),respectively.At 48 hours after transfection,the cells in groups B and C were selected with G418.The cell morphology changes were observed under the inverted microscope.Pax6 protein and level of corneal epithelial cells specific molecular-cytokeratin 12 (CK-12) were measured by Western blot.Real-time fluorescence quantitative PCR was applied to measure the mRNA expression of CK-12. ResultsNo morphology change was observed in groups A and B.Two different cell clones were found in group C.No.1 selected clone showed a flagstone-like appearance that was similar to that of corneal epithelial cells;No.2 selected clone showed a net-like appearance,with 3-7 cell processes.The Western blot results showed the Pax6 protein expression in 2 clones of group C,but no expression in groups A and B; and CK-12 protein expression was only observed in No.1 selected clone of group C,and no expression in the others.The real-time fluorescence quantitative PCR results showed that the CK-12 mRNA expression level of No.1 selected clone of group C was 8.64±0.73,which was significantly higher than that of No.2 selected clone of group C (0.55±0.42),group B (1.36±0.40),and group A (1.00±0.00) (P<0.05),and there was no significant difference among groups A,B and No.2 selected clone of group C (P>0.05). ConclusionPax6 gene transfection could induce differentiation of ADMSCs into corneal epithelium-like cells which express CK-12 at both the mRNA and protein levels.This result provides a promising strategy of generating corneal epithelilcm-like cells for construction of tissue engineered cornea.
Objective To review the advances in repair of spinal cord injury by transplantation of marrow mesenchymal stem cells(MSCs). Methods The related articles in recent years were extensively reviewed,the biological characteristic of MSCs,the experimental and clinical studies on repair of spinal cord injury by transplantation of MSCs,the machanisms of immigration and therapy and the problems were discussed and analysed. Results The experimental and clinical studies demonstrated that the great advances was made in repair of spinal cord injury by transplantation of MSCs. After transplantation, MSCs could immigrate to the position of spinal cord injury, and differentiate into nervelike cells and secrete neurotrophic factors.So it could promote repair of injuryed spinal cord and recovery of neurologicalfunction. Conclusion Transplantation of MSCs was one of effective ways in repair of spinal cord injury, but many problems remain to be resolved.
Objective To investigate the possibility of ectomesenchymal stem cell of human embryo facial process in differentiating into osteoblasts.Methods Ectomesenchymal stem cells of human embryo facial process were isolated and cultured in mineralized promoting solution containing 10 mmol/L β-glycerophosphate, 100 μg/ml ascorbic acid and 10 nmol/L dexamethasone supplemented with 15% FBS. The morphological change was observed by phase contrast microscopy. The characteristics of cells was identified by immunohistochemistry assay. Alkaline phosphatase activity was tested and the form of mineralized nodules was tested with Von Kossa staining. The expression of osteocalcin was identified by RT-PCR.Results There were significant changes in the shape of the cells after 3 days cultured in mineralized promoting solution. The cells became larger and the shape changed from fibroblast-like to multilateral. The result for anticollogen typeⅠstaining was positive. The alkaline phosphatase activity increased. Mineralized nodules were formed aftercultured 25 days by Von Kossa staining. RT-PCR assay showed induced cells expressed osteocalcin.Conclusion Ectomesenchymal stem cells of humanembryo facial process can be induced to differentiate into osteoblasts by mineralized promoting solution.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
ObjectiveTo investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats.MethodsThree lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining.ResultsAfter lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced (P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups (P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant (P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased.ConclusionSilencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.
ObjectiveTo investigate the possibility and effect of chitosan porous scaffolds combined with bone marrow mesenchymal stem cells (BMSCs) in repair of neurological deficit after traumatic brain injury (TBI) in rats.MethodsBMSCs were isolated, cultured, and passaged by the method of bone marrow adherent culture. The 3rd generation BMSCs were identified by the CD29 and CD45 surface antigens and marked by 5-bromo-2-deoxyuridine (BrdU). The chitosan porous scaffolds were produced by the method of freeze-drying. The BrdU-labelled BMSCs were co-cultured in vitro with chitosan porous scaffolds, and were observed by scanning electron microscopy. MTT assay was used to observe the cell growth within the scaffold. Fifty adult Sprague Dawley rats were randomly divided into 5 groups with 10 rats in each group. The rat TBI model was made in groups A, B, C, and D according to the principle of Feeney’s free fall combat injury. Orthotopic transplantation was carried out at 72 hours after TBI. Group A was the BMSCs and chitosan porous scaffolds transplantation group; group B was the BMSCs transplantation group; group C was the chitosan porous scaffolds transplantation group; group D was the complete medium transplantation group; and group E was only treated with scalp incision and skull window as sham-operation group. Before TBI and at 1, 7, 14, and 35 days after TBI, the modified neurological severity scores (mNSS) was used to measure the rats’ neurological function. The Morris water maze tests were used after TBI, including the positioning voyage test (the incubation period was detected at 31-35 days after TBI, once a day) and the space exploration test (the number of crossing detection platform was detected at 35 days after TBI). At 36 days after TBI, HE staining and immunohistochemistry double staining [BrdU and neurofilament triplet H (NF-H) immunohistochemistry double staining, and BrdU and glial fibrillary acidic protein (GFAP) immunohistochemistry double staining] were carried out to observe the transplanted BMSCs’ migration and differentiation in the damaged brain areas.ResultsFlow cytometry test showed that the positive rate of CD29 of the 3rd generation BMSCs was 98.49%, and the positive rate of CD45 was only 0.85%. After co-cultured with chitosan porous scaffolds in vitrofor 48 hours, BMSCs were spindle-shaped and secreted extracellular matrix to adhere in the scaffolds. MTT assay testing showed that chitosan porous scaffolds had no adverse effects on the BMSCs’ proliferation. At 35 days after TBI, the mNSS scores and the incubation period of positioning voyage test in group A were lower than those in groups B, C, and D, and the number of crossing detection platform of space exploration test in group A was higher than those in groups B, C, and D, all showing significant differences (P<0.05); but no significant difference was found between groups A and E in above indexes (P>0.05). HE staining showed that the chitosan porous scaffolds had partially degraded, and they integrated with brain tissue well in group A; the degree of repair in groups B, C, and D were worse than that of group A. Immunohistochemical double staining showed that the transplanted BMSCs could survive and differentiate into neurons and glial cells, some differentiated neural cells had relocated at the normal brain tissue; the degree of repair in groups B, C, and D were worse than that of group A.ConclusionThe transplantation of chitosan porous scaffolds combined with BMSCs can improve the neurological deficit of rats following TBI obviously, and also inhabit the glial scar’s formation in the brain damage zone, and can make BMSCs survive, proliferate, and differentiate into nerve cells in the brain damage zone.