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BACKGROUND: Retinal degeneration causes blindness, and cell replacement is a potential therapy. The purpose of this study is to formation of pigmented neurospheres in a simple medium, low-cost, high-performance manner over a short period of time while expressing markers of RPE cells and the activation of specific genes of the pigment cells. Also, these neurospheres have the ability to produce a monolayer of retinal pigment epithelium-like cells (RPELC) with the ability of photoreceptor outer segment phagocytosis. METHODS: BMSC were isolated from pigmented hooded male rats and were immunoreactive to BMSC markers, then converted into neurospheres, differentiated into pigmented spheres (PS), and characterized using Retinal pigment epithelium-specific 65 kDa protein (RPE65), Retinaldehyde-binding protein 1 (CRALBP) and orthodenticle homeobox 2 (OTX2) markers by immunocytochemistry, RT-PCR and RT-qPCR. The PS were harvested into RPELC. The functionality of RPELC was evaluated by phagocytosis of fluorescein-labeled photoreceptor outer segment. RESULTS: The BMSC immunophenotype was confirmed by immunostained for fibronectin, CD90, CD166 and CD44. These cells differentiated into osteogenic and lipogenic cells. The generated neurospheres were immunoreactive to nestin and stemness genes. The PS after 7–14 days were positive for RPE65 (92.76–100%), CRALBP (95.21–100%) and OTX2 (94.88–100%), and after 30 days RT-PCR, qPCR revealed increasing in gene expression. The PS formed a single layer of RPELC after cultivation and phagocyte photoreceptor outer segments. CONCLUSION: Bone marrow stromal stem cells can differentiate into functional retinal pigmented epithelium cells in a simple, low-cost, high-performancemanner over a short period of time. These cells due to expressing theRPELCgenes andmarkers can be used in cell replacement therapy for degenerative diseases including age-relatedmacular degeneration as well as retinitis pigmentosa.
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Animals , Humans , Male , Rats , Blindness , Bone Marrow , Epithelium , Fibronectins , Gene Expression , Genes, Homeobox , Immunohistochemistry , Nestin , Phagocytes , Phagocytosis , Retinal Degeneration , Retinal Pigment Epithelium , Retinaldehyde , Retinitis Pigmentosa , Stem CellsABSTRACT
Objective To study the effect of OK on the differentiation of bone marrow stromal stem cells (BMSCs) through the TGFβ/Smad signaling pathway in rats. Methods We removed the bilateral ovaries of rats to replicate OP model, and isolated and cultured BMSCs. BMSCs isolated from nomal rats were cultured as control group, BMSCs isolated from OP rats were divided into 5 groups, OP model group was regularly cultured, positive control group was treated with alendronate sodium (1 μmol/L), low, medium and high OK treatment group were treated with 50 mL/L, 100 mL/L and 200 ml/L OK respectively.After 24 h incubation, all cells were collected. The proliferation rate of BMSCs was determined by MTT method, and ELISA method was employed to detect the contents of bone formation markers, RT-qPCR was used to determine the m RNA expression level of TGFβ and the protein expression levels of TGFβ, p-Smad2/3 and of Smad2/3 were detected by Western blot. Results Compared with control group, the proliferation rate of the BMSCs in OP model group was reduced, concentrations of bone formation markers (OC, PINP and BALP) were reduced, m RNA and protein expression levels of TGFβ, as well as the phosphorylated level of Smad2/3 were downregulated.The difference was statistically significant (P＜0.05). After treatment with OK, compared with model group, all the above effects were ameliorated in different degree, a dose dependent manner was observed in OK treatment group, and the treatment effects of alendronate sodium (1 μmol/L), 100 mL/L and 200 mL/L OK group were statistically significant (P ＜0.05).Conclusion OK can promote the proliferation of osteoblasts by activating TGFβ/Smad signaling pathway to achieve the effect of treating postmenopausal OP.
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Objective To explore the effect of telomerase reverse transcriptase(TERT) gene transfected bone marrow stem cell(BMSC)on the memory function and hippocampal CA1 region synaptic plasticity in vascular dementia rat.Methods A total of 60 rats were randomly divided into the negative control group(group A),model group(group B),conventional BMSC group(group C) and transfected BMSC group(group D).The related indicators in each group were detected by using the Morris maze test,RTPCR and Western blot respectively.Results The escape latency period in the group C and group D was significantly longer than that in the group B,which in the group D was significantly longer than that in the group C.Compared with the group A,the expressions of brain-derived neurotrophic factor(BDNF)mRNA,TERT mRNA,SYP mRNA and protein in the group B,group C and group D were significantly decreased.The synaptic cleft arrange in group A was clear with more SYN positive ceils.The synaptic cleft in the group D was clearer,and the number of SYN positive cells was close to that in group A.Conclusion TERT transfected BMSC has obvious therapeutic effect on vascular dementia rats and its mechanism may be related to the promotion of BDNF,TrkB expression and the improvement of synaptic plasticity.
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Objective To investigate the biological features of the mouse bone marrow stromal stem cells (BMSCs) transfected by humanβnerve growth factor(β-NGF) .Methods BMSCs of GFP transgenic mouse were isolated and cultured .Theβ-NGF re-combinant plasmid vectors were transferred into the cultured BMSCs by LipofectamineTM 2000 .The expression of β-NGF was detec-ted with ELISA .Results The β-NGF recombinant plasmid vectors were successfully transferred into BMSCs of GFP transgenic mouse ,the expression of β-NGF in transfected cells appeared .In addition ,the expression ofβ-NGF could effectively protect the BM-SCs which were injured in transfection process .Conclusion BMSCs of GFP transgenic mouse after transfecton with human β-NGF have the proliferation and differentiation capacity ,and possess the expression ability of β-NGF protein .
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BACKGROUND:Fol owing physicochemical treatment and high-temperature calcinations, heterogeneous biological bone becomes a ceramic-like heterologous bone forming a similar structure to the human bone that is a natural network pore structure, which is conducive to seed cel adhesion and proliferation. OBJECTIVE:To observe the feasibility of constructing tissue-engineered bone through combination of sintered bone and bone marrow mesenchymal stem cel s to repair alveolar defects. METHODS:Sheep bone marrow mesenchymal stem cel s as seed cel s were combined with the high temperature sintered bone as scaffold materials to construct tissue-engineered bone. Under general anesthesia, sheep bilateral mandibular first premolars were removed in batches, the alveolar ridge space between the distal root and mesial root of the second premolar to form a bone defect area of 5 mm×5 mm×5 mm. Twelve experimental sheep were equal y randomized into tissue-engineered bone group and sintered bone group, which were implanted with tissue-engineered bone and sintered bone, respectively, at the left surgical area of the mandible. The right surgical area was considered as blank control group. RESULTS AND CONCLUSION:After high-temperature calcinations, the sintered bone was chalk in color, exhibiting a porous structure as the natural cancel ous bone. The porosity was (66.10±1.32)%, and the pore size was between 137.44μm and 538.72μm. After 24 hours of bone marrow mesenchymal stem cel s inoculated to the sintered bone, a large number of cel s are visible adherent to the scaffold;up to day 7, extracel ular matrix was secreted and there was no clear boundary between the cel s and the matrix. X-ray films showed that the tissue-engineered bone and pure sintered bone implants were embedded in the surgical area, and there was a low-density shadow at the edge of the sintered bone. Hematoxylin-eosin staining showed bone trabecular formation at the experimental side, but no obvious bone formation at the control ed side. Tissue-engineered bone prepared by bone marrow mesenchymal stem cel s and sintered bone can better repair sheep alveolar bone defects, which is an ideal seed cel and scaffold material for smal range bone defects.
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Objective To study the effects of serum of the Zhuartgguqiang jin tablets on the proliferation and osteogenic differentiation of bone marrow stromal stem cells (BMSCs) of aged rats.Methods The BMSCs were obtained from male SD rats of 18 months.The cell surface markers CD34,CD31,CD29 were detected by flow cytometry.The proliferation of BMSCs treated by different concentration of medicated serum (2.5％,5％,10％) at 24,48,72hours was detected by CCK-8 method.The BMSCs were divided into 4 groups,which were osteogenic induction group,the Chinese medicated osteogenic induction group,Chinese medicine group,the blank group.After the BMSCs were cultured for 7 days,alkaline phosphatase (ALP) vitality was detected.After the BMSCs were cultured for 14 days,the BMSCs were stained by alizarin red,the mRNA of ALP and osteocalcin(OC) was detected.Results The expressionof CD29 was positive,and the expression of CD31 and CD34 were negative.2.5％,5％ of the medicated serum could promote cell proliferation.The ALP vitality of osteogenic induction group[202.76 ± 15.44(U/gprot)],the Chinese medicated osteogenic induction group [240.48 ± 18.55 (U/gprot)],Chinese medicine group [178.87 ± 17.29 (U/gprot)]were significantly higher than that of blank group [111.24 ± 20.71 (U/gprot)] (t =22.50,7.985,3.535,all P ＜0.01).The ALP activity of Chinese medicated osteogenic induction group was higher than that of osteogenic induction group (t =3.103,P ＜ 0.05).The ALP gene relative OD value of osteogenic induction group (0.40 ± 0.20) and Chinese medicated osteogenic induction group (0.60 ± 0.06) were higher than the blank group (0.09 ± 0.03) (t =2.372,9.547,all P ＜0.01).The ALP gene expression of Chinese medicated osteogenic induction group was obviously higher than that of the osteogenic induction group(P ＜0.05).The OC gene relative OD value of osteogenic induction group(0.58 ± 0.09) and Chinese medicated osteogenic induction group(0.76 ± 0.11) were higher than the blank group(0.41 ± 0.02) (P ＜ 0.05,P ＜ 0.01).The OC gene expression of Chinese medicated osteogenic inductiongroup was obviously higher than that of the osteogenic induction group(t =2.673,P ＜ 0.05).Conclusion Medicated serum of Zhuangguqiangjin tablets can promote the proliferation and osteogenic differentiation of BMSCs of aged rats,which may be the mechanism of prevention senile osteoporosis.
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Objective: To observe the effects of He-Ne laser therapy instrument on bone marrow mesenchymal stem cell (BMSC) growth and neural differentiation potential. Methods: The primary BMSCs from Sprague-Dawley rats were taken using whole bone marrow culture. The passage 3 cells were randomly divided into an experimental group (He-Ne laser therapy instrument treated for 10 min) and a control group (exposing in the air for the same time) according to whether they were treated with the He-Ne laser therapy instrument or not. The cell proliferation activity was measured with cell counting kit-8 (CCK-8) assay. Apoptosis detection was performed using flow cytometry. The cell vitality was detected by Trypan blue staining. Human neural stem cell differentiation potential was identified by immunofluorescence technique. Results: Circled digit oneCells showed a fibroblast-like adherent growth. The phenotypic identification results revealed that the expressions of CD29 and CD90 were positive, but the expression of CD45 was negative, and it is consistent with the characteristics of the BMSC phenotype. Circled digit twoThere was no significant difference in the proliferative activity between the experiment group and the control group, P > 0.05. Circled digit threeThere were no significant differences between the early apoptosis rate (1.10 ± 0.26%) in the experiment group and that (1.43 ± 0.06%) in the control group, as well as the cell viability (96.2 ± 0.6%) in experiment group and that (96.2 ± 0.7%) in the control group. Circled digit fourSimilar to the control group, BMSCs in the experiment group showed the suspended neurospheres after being cultured in neural stem cell culture medium. Immunofluorescence staining showed that the nestin staining of neurosphere cells was positive. Conclusion: He-Ne laser therapy instrument has no significant effects on the proliferation and viability of BMSCs. It does not cause apoptosis and change the potential of BMSCs to neural stem cell differentiation.
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Objective To study the effects of 1,25-(OH)_2-vitamin D_3[1,25-(OH)_2D_3] on proliferation,differentiation, and secretion of osteoprotegerin and RANK ligand (RANKL)in cultured marrow mesenchymal cells of rhesus monkey (RhBMSCs). Methods Three different concentrations of 1,25-(OH)_2D_3 (10~(-12) ,10~(-10), and 10~(-8)mol/L)were added to the cultured RhBMSCs in vitro. MTT was used to observe cell proliferation and ELISA technique was used to measure the concentration of alkaline phosphatase (ALP), osteocalcin, osteoprotegerin, and RANKL. Mineralization nodus was identified via alizarin red staining. Results Under different concentration of 1,25-(OH)_2D_3, RhBMSCs proliferation were promoted within 7 days but were suppressed beyond 7 days. No significant dose-dependent manner was found. Differentiation of RhBMSCs into osteoblast was promoted by 1,25-(OH)_2D_3. The levels of ALP and osteocalcin in groups with various concentrations of 1,25-(OH)_2D_3 were higher than those of control group [ALP(ng/ml) ,7 d:31.40±1.25,26.50±0.50,28.47± 0.25 vs 13.48±0.26;10 d:33.37±0.68,35.30±1.57,33.27±0.67 vs 17.14±0.55;13 d:35.37±0.12,30.47± 0. 25 , 30. 27±1.25 vs 16.55 ± 1.13 ; osteocalcin (ng/ml), 7 d:4.47±0. 29,4.00 ±0. 60,3.73±0.78 vs 1.63± 0.55;10 d:5.63±0.57,5.17±0.15,4.30±0. 10 vs 2.17±0. 15;13 d:7.03±0.15,5.53±0.25,5.27±0.31 vs 2.23±0. 55 ; all P < 0. 05], but no typical mineralization nodus was found in either group. Secretions of osteoprotegerin and RANKL from RhBMSCs were stimulated by 1,25-(OH)_2D_3 [osteoprotegerin (pg/ml), 7 d: 72.57±0.67,68.00±1.75,64.23±0.87 vs 30. 13±1.72; 10 d:62.03±1.62,51.80±1.30,28.93±0.95 vs 18.13±1.40;13 d:65.13±0.71,62.43±2.11,44.93+1.63 vs 36.70±0.95 ;RANKL(pg/ml) ,7 d:74.33+0.61, 82.37±2.15,85.23±0.45 vs 70.83±1.71 ;10 d:83.30±0.46,86.70±0.56,88.23±0.91 vs 74.20±1.83;13 d: 81.70±1.81,81.07±0.95,84.70±1.41 vs 72.73±0.97 ;all P<0.05]. The secretion of RANKL was increased at first and then decreased, whereas the secretion of osteoprotegerin had the opposite tendency. The secretions of RANKL and osteoprotegerin were both in a dose-dependent manner. Conclusions 1,25-(OH)_2D_3 promotes differentiation of RhBMSCs into osteoblasts,resulting in increased secretions of osteoprotegerin and RANKL
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E were all found in the above three aspects (P
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Objective To study the model of separation and culture and the growth characteristics and osteogenic capability of bone marrow stromal stem cells (BMSCs). Methods BMSCs were isolated from adult rats using gradient centrifugation and anchoring culture. The passage cells were induced to differentiate into osteoblasts by means of an differentiation induction medium containing dexamethasone,?-glycerophophate and Vitamin C. Alkaline phosphatase (ALP) activity and the ability to form mineralized nodules were examined. Results Colonies of stromal stem cells were formed in the primary culture and contacted one another at the 14th day. In the passage culture, cells became bigger than those in the primary culture and could be subcultured one generation in 5～7 days. After the culture in the differentiation induction medium, the ALP activity was enhanced significantly and the mineralized nodules were formed. Conclusion BMSCs can be obtained easily and have strong proliferation ability and osteogenic capacity. This study suggests that BMSCs can become seeding-cells in bone tissue engineering.
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AIM:To evaluate the biocompatibility between copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) and bone marrow stem cells (BMSCs). METHODS: Canine BMSCs were isolated and cultured. The cells in passage 3-4 were seeded onto the PHBV films and three-dimensional foams. The seeded cells were observed under inverted microscope for morphology and cell attachment onto the PHBV films at 1, 2 or 3 weeks after seeding. With 4% paraformaldehyde formalin and staining, the protein content in seeded cells was determined by bicinchoninic acid assay (BCA). The content of DNA was quantified using the Hoechst 33258 assay. RESULTS: Observation under inverted microscope showed that the PHBV fabric was fairly thickness, lucency is weak. Unser contrast phase microscope, PHBV fabric was uneasy to be observed. Most cells attached onto the PHBV films 2 h after seeding, and extended well and acquired a spindle fibrecyte-like morphology 3 d later. Moreover, on the three-dimensional foams, the seeded cells lay in micropores and grew tri-dimensionally. The conjunction of cells appeared about 1 week, and extended at 3 weeks, with a large amount of extracellular matrix around cells. The content of DNA and protein has no significant difference with control group. CONCLUSION: As a kind of tissue engineering material for BMSCs seeding, PHBV has an excellent biocompatibility.
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AIM:To explore the survivorship and the mechanism of the intravenous administration of bone marrow stromal stem cells(BMSCs) for treating permanent focal cerebral ischemia in rats.METHODS:After purified,proliferated,and marked with BrdU,the BMSCs were injected intravenously into rats 1 d after focal cerebral ischemia.Modified neurological severity score(mNSS) was evaluated before and following 1,7,14 and 28 d after middle cerebral artery occlusion(MCAO).Rats were executed at 1,7,14 and 28 d after MCAO.Brain sections were stained with hematoxylin and eosin(HE) for determining the infarct volume.Slides were stained by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL) and immunostaining for cleaved caspase-3 method for apoptosis detection and mechanism exploration in situ.RESULTS:mNSS in BMSCs-transplanted group at 14th day and 28th day of MCAO was significantly lower than that in control group(P
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AIM: To investigate the inducing action of retinal cells of rat neonates on the adult rat bone marrow stromal cells (BMSCs).METHODS: The full marrow from adult Wistar rat were cultured and passaged repeatedly to harvest the pure BMSCs. In vitro, the BMSCs were induced by retinal cells of rat neonates in transwell.The morphological changes of the BMSCs were observed under phase contrast microscope, the specific markers of the induced cells were ~identified immunocytochemically with nestin, Map-2, neuron-specific enolase(NSE),neurofilament(NF), Thy 1.1 and glial fibrillary acidic protein(GFAP) antibodies. The mRNA of nestin, NF, NSE, Thy 1.1 and Ran were detected in the induced cells by RT-PCR.RESULTS: The BMSCs were induced by reinal cells of rat neonates into the retina-like neurons, which showed the typical neural morphology and expression of the specific retinal neural antigens.CONCLUSION: The BMSCs from the adult rat can be induced into retina-like cells by retinal cells of rat neonates. [
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To observe the possibility of differentiation of bone marrow stromal stem cells of adult dog into myogenic cells. In a hope of providing a new cell line for treatment of myocardial infarction and heart failure. Bone marrow stromal stem cells were isolated by their adherence to plastic in vitro . 5 azacytidine was used to induce stem cells to differentiate into cardiomyotes in the experimental group, and same volume of culture media was used in the control group. The cultured cells were exarnined with inverted contrast phase microscope and immunochemistry. 4 weeks after induction, the shape of most of cultured cells changed from fusiform into rod like contiguration in experimental group, immunohistochemistry showed that the induced cells were positive for Desmin, ? smooth muscle actin, and Troponin I. The shape of cultured cells remained fusiform in control group, immunohistochemistry showed that they were mildly positive for Desmin and ? smooth muscle actin and negative for Troponin I. Adult canine bone marrow stromal stem cells were able to differentiate into myogenic cells after induction in vitro .