Abstract: Objective To evaluate if cardiac function and myocardial perfusion in acute ischemia myocardial transplanted by autologous bone mesenchymal stem cells (MSC) can be improved. Methods Sixteen New Zealand rabbits were studied.The left anterior descending coronary artery under the first diagonally branch was ligated to result in acute myocardial ischemia models,the sixteen models were divided into two groups with randomed number table. Control group(n=8): 0.6ml αminimum essential medium was injected into myocardium; transplanted group (n=8): 0.6ml medium of autologous MSC marked with 5-bromium,2-deoxy-uridine (BrdU) was injected into myocardium. Echocardiography were erformed to measure left ventricular ejection fraction(LVEF),as well as the displacement and strain of apex segment of left ventricle pre-ichemia,beforeand 4 weeks after treatment; the target myocardial tissues were harvested 4 weeks after treatment,double immunohistochemistry staining of anti-BrdU and anti-troponin T(TnT) were used to evaluate the survival and differentiation of implanted MSC; immunohistochemistry staining of anti-CD146 endothelium factor were used to evaluate the density of capillary vessels in treated myocardium. Results Double immunohistochemistry staining showed that positive cells were found in transplanted group and not found in control group. Anti-CD146 immunohistochemistry staining showed density of capillary vessels of transplanted group was significantly more than that of control group(Plt;0.05) ; LVEF,displacement and strain of cardiac apex of transplanted group improved significantly more than those of control group(Plt;0.05). Conclusion Transplanted to acute myocardium ischemia models of rabbits, MSC can differentiate into myocardium-like cells in myocardial microenvironment,and improve global and part cardiac systolic function and then improving perfusion of ischemia myocardium.
Objective To summarize the advancement of breast cancer stem cells and genotyping and analyze the correlation between the two. Methods Relevant literatures about breast cancer stem cells and genotyping, which were published recently were collected and reviewed. Results Cancer stem cell origin theory was supported by researches of correlation between breast cancer stem cells and genotyping, which also explained the complexity of intrinsic subtypes and heterogeneity of breast cancer. Conclusions A new way can be detected to study the formation mechanism and biological characteristics of breast cancer at the cellular and molecular level by researches of correlation between breast cancer stem cells and genotyping, which are expected to provide new strategies and tools for diagnosis and treatment of breast cancer.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
RCBTB1 gene associated hereditary retinopathy is an extremely rare inherited retinal disease (IRD) discovered recently. The mutation of RCBTB1 gene can lead to a variety of IRD clinical phenotypes, such as early retinitis pigmentosa and delayed chorioretinal atrophy. The hereditary mode of RCBTB1 gene associated retinopathy is autosomal recessive. RCBTB1 gene plays an important role in maintaining mitochondrial function and anti-oxidative stress defense mechanism of retinal pigment epithelium cells. In the future, it is necessary to further determine whether there is a genotypic and phenotypic correlation in the age of onset of RCBTB1 gene associated retinopathy or multi-organ involvement, and evaluate the safety and efficacy of adeno-associated virus-mediated RCBTB1 gene replacement therapy in animal models, to explore the feasibility of gene replacement therapy and stem cell therapy.
ObjectiveTo study the external biocompatibility bewteen the mouse induced pluripotent stem cells (miPSCs) and poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx). MethodsAfter we recovered and subcultured miPSCs, we divided them into two groups. There was one group cultured with material of PHBHHx films outside the body. We observed the adhesive pattern of miPSCs on film by fluorescence of 4, 6-diamidino-2-phenylindole (DAPI) staining. The cell vitality was detected by cell counting kit-8 (CCK-8). The morphology of miPSCs attached on the film was visualized under scanning electron microscope (SEM). We used the traditional petri dish to culture miPSCs and detect the cell activity by CCK-8. ResultsMiPSCs can adhere and proliferate on PHBHHx films. The result of cell vitality which detected by CCK-8 showed that there was a statistical difference in OD value between culturing on PHBHHx films and traditional cultivation (0.617±0.019 vs. 0.312±0.004, P < 0.05). ConclusionThere are adhesion and proliferation on the surface of cells patch made by miPSCs co-culturing with PHBHHx film. Compared with traditional culturing in the cell culture dish, culturing in PHBHHx films have great advantages in the process of adhesion and proliferation. PHBHHx can be used as one of the scaffold for stem cells treating various disease.
Objective To investigate the possibility of enhancing the inducing rate of adipose-derived stem cells (ASCs) into epidermal cells in the medium containing all-trans retinoic acid (ATRA) by supplementing with HaCaT condition medium. Methods ASCs were isolated and identified by detecting the expression of CD34, CD45, CD73, CD90, and CD105 with flow cytometry and differentiating into adipose and osteoblast lineage in the induction medium. The air-liquid interface cell culture model was established with the Transwell Room. The induction medium A contained ATRA, epidermal growth factor (EGF), and keratinocyte growth factor (KGF), while the induction medium B contained ATRA, EGF, KGF, and HaCaT condition medium. Experiment was divided into three groups cultured for 12 days: induction medium A (group A), induction medium B (group B), basic medium (group C). The epidermal cell surface markers: cytokeratin (CK) 14, 15, 16, 19 (Pan-CK) were detected by flow cytometry and CK14 were identified by immunofluorescence stain. Results After induction for 12 days, flow cytometry showed that the positive rate of Pan-CK in group B [(22.0±3.5)%] was higher than that in group A [(11.9±2.7)%], which were both higher than that in group C [(1.1±0.3)%], and the differences were statistical significantly (P<0.01). Immunofluorescence stain showed that the positive rate of CK14 in group B was higher than that in group A [(19.5±7.0)%vs. (10.8±5.7)%, P<0.01], and the expression of CK14 was negative in group C. Conclusion HaCaT condition medium can enhance the ability of ASCs differentiation into epidermal cells in the culture medium containing ATRA.
Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
Objective To establish a safe, effective, and economic feeder-free culture system which is suitable for the culture of human parthenogenetic embryonic stem cells (hPESCs) in vitro. Methods hPESCs were cultured with mTeSRTMl medium (control group) and human foreskin fibroblasts-conditional medium (hFFs-CM) (experimental group). The growth status of hPESCs in both feeder-free culture systems were observed with inverted microscope. Alkaline phosphatase (ALP) analysis and karyotype analysis were used to study the biological characteristics of hPESCs. The expression of hPESCs pluripotent marker Oct-4 was analyzed by RT-PCR. Differentiation experiment in vivo and in vitro was applied to observe the differentiation potential of hPESCs into three germ layers. Results hPESCs had regular morphology with difficulty in differentiation in both culture systems. No obvious difference was observed in morphology and expansion speed of hPESCs between 2 groups. After subcultured for 15 passages in vitro, hPESCs in 2 groups could maintain normal female diploid karyotype 46, XX and pluripotency. The expression of Oct-4 mRNA was positive in 2 groups. hPESCs in 2 groups could form embryonic body in differentiation experiment in vitro and could develop into teratomas containing three germ layers in nude mice. Conclusion Feeder-free culture system of hFFs-CM can sustain the growth of hPESCs and keep hPESCs undifferentiated state for long. A feeder-free culture system of hPESCs is successfully established, which can support the growth of hPESCs, reduce the contamination from animals, decrease the cost of culture, and satisfy the clinical large-scale application.
ObjectiveTo observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis, and the therapeutic effects of glial cell derived neurotrophic factor (GDNF) and/or ciliary neurotrophic factor (CNTF) based on neural stem cells (NSC) on mouse photoreceptor cells. MethodsA total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group, with 80 and 20 mice respectively. Twenty C57BL/6J mice aged 14 days were assigned as wild-type group (WT group). Mice in control group and WT group did not receive any interventions. At 2, 4, and 6 months of age, immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones, rod-bipolar cells, and cone-bipolar cells within the retinal of mice while electroretinography (ERG) examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes. At 14 days of age, the mice in the experimental group were intravitreally injected with 2 μl of CNTF-NSC, GDNF-NSC, and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC (GDNF/CNTF-NSC). Those mice were then subdivided into the CNTF-NSC group, the GDNF-NSC group, and the GDNF/CNTF-NSC group accordingly. The contralateral eyes of the mice were injected with 2 μl of control NSC without neurotrophic factor (NTF) as their own control group. At 2 and 4 months of age, the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes. At 4 months of age, the differentiation of grafted NSC and the expression of NTF were observed. Statistical comparisons between the groups were performed using a two-way ANOVA. ResultsCompared with WT group, the density of cones in the peripheral region of the control group at 2, 4 and 6 months of age (F=285.10), rod-bipolar cell density in central and peripheral retina (F=823.20, 346.20), cone-bipolar cell density (F=356.30, 210.60) and the scotopic amplitude of a and b waves (F=1 911.00, 387.10) in central and peripheral retina were significantly decreased, with statistical significance (P＜0.05). At the age of 4 and 6 months, the density of retinal cone cells (F=127.30) and b-wave photopic amplitude (F=51.13) in the control group were significantly decreased, and the difference was statistically significant (P＜0.05). Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes, and stably expressed CNTF and GDNF at high levels. Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages: CNTF-NSC group, at 2 months of age: the whole, central and peripheral regions were significantly different (F=31.73, 75.06, 75.06; P＜0.05); 4 months of age: The difference between the whole area and the peripheral region was statistically significant (F=12.27, 12.27; P＜0.05). GDNF/CNTF-NSC group, 2 and 4 months of age: the whole (F=27.26, 27.26) and the peripheral area (F=16.01, 13.55) were significantly different (P＜0.05). In GDNF-NSC group, there was no statistical significance at all in the whole, central and peripheral areas at different months of age (F=0.00, 0.01, 0.02; P＞0.05). ConclusionsCLN7 neuronal ceroid-lipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing, aligning with both morphological and functional observations. Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells. Nevertheless, the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect. GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.
OBJECTIVE To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.