Objective To investigate the feasibility of imaging of bone marrow mesenchymal stem cells (BMMSCs) labeled with superparamagnetic iron oxide(SPIO) transplanted into coronary artery in vivo using magnetic resonance imaging (MRI), and the redistribution of the cells into other organs. Methods BMMSCs were isolated, cultured from bone marrow of Chinese mini swine, and double labeled with SPIO and CMDiI(Cell TrackerTM C-7001). The labeled cells were injected into left anterior descending coronary artery through a catheter. The injected cells were detected by using MRI at 1 week,3weeks after transplantation. And different organs were harvested and evaluated the redistribution of transplanted cells through pathology. Results The SPIO labeled BMMSCs injected into coronary artery could be detected through MRI and confirmed by pathology and maintained more than 3 weeks. The SPIO labeled cells could be clearly imaged as signal void lesions in the related artery. The pathology showed that the injected cells could be distributed into the area of related artery, and the cells injected into coronary artery could be found in the lung, spleen, kidney, but scarcely in the liver, the structures of these organs remained normal. Conclusion The SPIO labeled BMMSCs injected into coronary artery can be detected by using MRI, the transplanted cells can be redistributed into the non-targeted organs.
Objective To study the growth characteristics of umbil ical cord MSCs (UCMSCs) in vitro and its effect on the nerve regeneration after spinal cord injury (SCI). Methods UCMSCs isolated from pregnant rats umbil ical cord were cultured and purified in vitro. Sixty female Wistar rats weighing (300 ± 10) g were randomized into three groups (n=20per group). UCMSCs group (group A) in which UCMSCs suspension injection was conducted; DMEM control group (groupB) in which 10% DMEM injection was conducted; sham group (group C) in which the animal received laminectomy only.Establ ish acute SCI model (T10) by Impactor model-II device in group A and group B. The recovery of the lower extremity was observed using BBB locomotor scoring system, neurofilament 200 (NF-200) immunofluorescence staining was performed to detect the neural regeneration, and then the corticospinal tract (CST) was observed using the biotinylated dextran amine (BDA) tracing. Results Cultured UCMSCs were spindle-shaped fibrocyte-l ike adherent growth, swirl ing or parallelly. The USMSCs expressed CD29, but not CD31, CD45, and HLA-DR. The BBB score was higher in group A than group B 4, 5, and 6 weeks after operation, and there was a significant difference between two groups (P lt; 0.05). The BBB scores at different time points were significantly lower in groups A and B than that in group C (P lt; 0.05). UCMSCs was proved to survive and assemble around the injured place by frozen section of the cords 6 weeks after injury. NF-200 positive response area in groups A, B, and C was (11 943 ± 856), (7 986 ± 627), and (13 117 ± 945) pixels, respectively, suggesting there was a significant difference between groups A, C and group B (P lt; 0.05), and no significant difference was evident between group A and group C (P gt; 0.05). BDA anterograde tracing 10 weeks after operation demonstrated that more regenerated nerve fibers went through injured area in group A, but just quite few nerve fibers in group B went through the injuried cavity. The ratios of regenerative axons amount to T5 axons in group A and group B were smaller than that of group C (P lt; 0.05). Conclusion UCMSCs can prol iferate rapidly in vitro, survive and differentiate to neurons after being grafted into injured spinal cord. The transplantation of UCMSCs is effective in promoting functional recovery and axonal regeneration after SCI.
Purpose To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. Methods The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. Results Two to three days after transplantation,yellowish-white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. Conclusion The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina-like structure. (Chin J Ocul Fundus Dis，2000，16：213-284)
ObjectiveTo determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration. MethodsHair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups. ResultsThe isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences (P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant (P<0.05). ConclusionLentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
Objective To introduce the basic research and cl inical appl ication of stem cells transplantation for treating diabetic foot. Methods The recent original articles about the stem cells transplantation for treating diabetic foot were extensively reviewed. Results Transplanted different stem cells in diabetic foot could enhanced ulceration heal ing in certain conditions, increase neovascularization and avoid amputation. Conclusion Stem cells transplantation for treating diabeticfoot may be a future approach．
Objective To introduce the cells and cell-transplantation methods for periodontal tissue engineering. Methods Recent l iterature about appl ication of cell-based therapy in periodontal tissue engineering was extensively reviewed, the cells and cell-transplantation methods were investigated. Results Mesenchymal stem cells were important cell resourcesfor periodontal tissue engineering, among which peridontal l igament stem cells were preferred. Bone marrow mesenchymal stem cells had several disadvantages in cl inical appl ication, and adipose-derived stem cells might be a promising alternative; different transplantation methods could all promote periodontal regeneration to some extent. Single-cell suspension injection could only promote a l ittle gingival regeneration, and tissue engineered scaffolds still needed some improvement to be used in periodontal regeneration, while cell sheet technique, with great cell loading abil ity and no need of scaffolds, could promote regeneration of cementum, periodontal l igament, and alveolar bone under different conditions. Conclusion Multipotent stem cells are fit to be used in periodontal tissue engineering; improvement of cell-transplantation methods will further promote periodontal regeneration.
Objective To investigate effect of intravitreal injection of FK506 on the survival of human retinal pigment epithelial (RPE) cells heter oplastically transplanted into the subretinal space of rabbits.Methods The immortalized human RPE cells were genetically labeled by retrovirus vector carrying a green fluorescent protein (GFP). A total of 50 μl RPE cells suspension with 4×103 cells/μl which expressed GFP were injected into the subretinal space of both eyes of 18 white rabbits and 10 gray rabbits. The left eyes of all of the rabbits were injected of 5 μl FK506 (5 μg/μl) intravitreally once a week during the first 5 weeks, then once every other week until the 20th week and the right eyes were as the control. The histological sections of heteroplastic RPE cells were observed by epifluorescent microscope.Results GFP-expressing cells could be seen after 1 week, 2, 3, 4, 6, 10, 11, 14, 18, 20, 23, 24, 25, 26, 33, and 54 weeks in white rabbits and after 4 , 5, 6, 7, 14, 18, 20, and 26 weeks in gray rabbits. The configuration and integrality of the RPE-GFP cells in the left eyes which had been intravitreally injected of FK506 1-14 weeks after transplantation were better than those in the right eyes without injection. After 18 weeks, the condition of heteroplastic cells with few difference in both eyes in 7 white and 3 gray rabbits were found. After 1-6 weeks, focal and disseminated lymphocytes around the choroidal small vessles of right eyes in 6 white and 3 gray rabbits could be seen while the infiltration of the lymphocytes in the left eyes was much reduced.Conclusion Intravitreal injection of a small amount of FK506 at the first 3 months after transplantation may significantly improve the survival of heteroplastic RPE cells in the subretinal space of rabbits. (Chin J Ocul Fundus Dis，2003，19：333-404)
Objective To investigate the synergetic effect and possibil ity of repairing spinal cord injury (SCI) by transplantation of olfactory ensheathing cells (OECs) and chondroitinase ABC (ChABC) in adult rats. Methods Three adult male SD rats were used to isolated olfactory bulb and primarily cultured OECs. In the 8th or 9th day, OECs were transplanted, the concentration of cells was modulated to 1 × 105/μL. Fifty-four SD rats were made the models of T8 spinal cord crush injury and divided into 4 groups. In group A (control, n=36), injured site was not treated; in groups B, C and D (n=6), OECs, ChABC and OECs+ChABC were injected into injured site, respectively. At 1, 2, 3, 7 and 14 days after injury, the BBB score system was used to evaluate the motion function. At 0, 1, 2, 3, 7, 14 days in group A and at 14 days in groups B, C, D after injury, the maximal transverse diameter and gross area of necrosis were evaluated on HE stained sections. The immunofluorescence double label ing staining for gl ial-fibrillary acidic protein (GFAP)/CS56, GFAP/growth associated protein 43(GAP-43) and GFAP/neurofilament 160(NF160) was carried out to evaluate the regeneration of nerve fiber. Results At 14 days after injury, there were significant difference in the BBB scores between group A and groups B, C, D (P lt; 0.05), and between groups B, C and group D (P lt; 0.05), HE staining showed that the formation of cavity was observed in each group at 14 days after injury. There were significant difference in the maximal transverse diameter and gross area of necrosis between groups B, C, D and group A (P lt; 0.01), and between groups B, C and group D (P lt; 0.01). The immunofluorescence staining indicated that expression of GFAP were more intense in group A than in other groups, and the cavity of the lesion site was apparent, but it was moderate in groups B and C. The expression of GAP-43 was more intense in group D than in groups B and C. The expression of NF160 was more intense in group D. Conclusion Transplantation strategy of OECs combined with ChABC was effective in the repair of SCI in some extent.
Purpose To investigate the development of embryonic stem cells (ESC)in the subretinal space． Methods ESC were cultivated in suspension for 4 days till they developed into cell aggregates,i.e.embryonic body(EB).ESC as well as EB combined with or without RA were respectively transplanted into vitreous cavity and subretina1 space in SD rats,and the subretinal transplanted eyes,transient ischemia-reperfusion injuries were made by ligating the ophthalmic artery for 40 seconds before the transplantation .The experimental eyes were enucleated for histological and immunohistochemical assays after 14～28 d. Results The EB was found to develope into photoreceptors induced by RA in the subretinal space under an ischemia-reperfusion condition，and EB transplantation without RA induction induced multiple differentiations in the subretinal space.The single injection of RA without EB induced hyperplasia of the neural retinal cells.ESC transplanted into vitreous cavity rapidly proliferated and developed into atypical hyperplastic mass. Conclusion EB derived from ESC can differentiate into photoreceptors induced by RA in the host subretinal space under an ischemia-reperfusion condition. (Chin J Ocul Fundus Dis，2000，16：213-284)