ObjectiveTo establish the system of isolation, cultivation, and identification of the neural stem cells (NSCs) from subventricular zone (SVZ) of neonatal mice so as to seek for the appropriate seed cells for potential therapeutic interventions of neurological disorders. MethodsNSCs were isolated enzymatically and mechanically from SVZ of neonatal mice and cultured. The cellular morphology was observed by inverted microscopy. Immunocytochemical stainings of anti-Nestin and anti-SOX-2 were used to identify NSCs of passage 3. To study the differentiation of NSCs, NSCs were plated into 24-wells in the medium supplemented without epidermal growth factor (EGF) and basic fibroblastic growth factor (bFGF) for 3 or 7 days. To compare the differentiation and proliferation potential of NSCs with different cultivation time, the BrdU pulse-labeling method and MTT test were used. To identify neurons and astrocytes, the anti-β-tubulin Ⅲ (Tuj-1) and anti-glial fibrillary acidic protein (GFAP) staining were used. ResultsThe cells of the SVZ can be isolated and cultured in vitro, and these cells began to form neurospheres after cultured for 3 days at primary passage. While cultured for 7 days, these cells formed more neurospheres, and the volume of the neurospheres became bigger than neurospheres cultured for 3 days. In addition, after cultured for 7 days, the phenomena of fusion of neurospheres and adherent differentiation of neurospheres were observed under inverted microscope. These cells were provided with the typical phenotype of NSCs. The immunofluorescence staining results revealed that these cells showed positive immunoreactivity to Nestin and SOX-2. During the 4 hours BrdU pulse, the number of proliferated NSCs cultured for 3 days (75.817±2.961) was significantly higher than that of NSCs cultured for 7 days (56.600±4.881) (t=3.366, P=0.028). The results of MTT assay revealed that the absorbance (A) value of NSCs cultured for 3 days (0.478±0.025) was significantly higher than that of NSCs which were cultured for 7 days (0.366±0.032)(t=2.752, P=0.011). After cultivated without EGF and bFGF, the percentage of Tuj-1 and GFAP positive cells in NSCs was 23.1%±3.7% and 23.7%±3.8% for 3 days and was 40.1%±3.6% and 37.1%±4.5% for 7 days, respectively, all showing significant differences (t=3.285, P=0.030; t=3.930, P=0.017). ConclusionThe NSCs from SVZ of neonatal mice have potentials of self-renewal and multipotential differentiation in vitro. With different cultivation time, the potentials of proliferation and differentiation of NSCs are different.
OBJECTIVE: To explore the pluripotential and possible clinical application of adult stem cells. METHODS: The original articles on adult stem cells were extensively reviewed and the recent advances were summed up. RESULTS: Adult stem cells were located at different tissues of human beings and had the pluripotentiality of self-renewal and differentiation. Some adult stem cells, such as in marrow, nerve, muscle, fat, skin, liver, tissues, had the ability to differentiate into the unrelated cell type. CONCLUSION: The pluripotential, ubiquitous distribution and plasticity of adult stem cells offered a new way in regeneration medicine, such as cell therapy and tissue engineering.
Objective To explore the effects of mechanical stretch with variant frequencies on the alignment and differentiation of the multilayer myotubes cultured in vitro, and to select the optimized cultural condition of regenerative skeletal muscle tissue with stress loading cultured in vitro. Methods C2C12 myoblasts cultured in vitro in the groove casts of Sylgard 184 were induced into the multilayer myotubes. Meanwhile the myoblasts were treated with various mechanical stretch withcells tensile instrument, at the amplitude of 10% and the frequency of 0 (group A), 0.25 (group B), 0.50 (group C), and 1.00 Hz (group D) for 1 hour, 3 times a day. The myotubes morphology was observed by inverted phase contrast microscope at 5, 7, and 10 days after continuous mechanical stretch. And the expressions of mRNA for myogenic differentiation antigen (MyoD), Myogenin, Desmin, and myosin heavy chain (MyHC) were detected by RT-PCR and real-time fluorescent quantitative PCR (QRT-PCR), respectively. Results The mechanical stretch could promote the al igned fusion and increase the number of myotubes. Indeed the multilayer myotubes arranged more closely in group B at 7 days. At the same group, as the time went on, the mRNA expressions of MyoD gradually decl ined in each group. There were significant differences in mRNA expressions of MyoD between 5 days and 7, 10 days (P lt; 0.05). The mRNA expressions of Myogenin, Desmin, and MyHC were highest at 7 days. There were significant differences between different time points (P lt; 0.05), except the mRNA expression of Desmin of group B between 7 and 10 days (P gt; 0.05). At the same time, with the increase of frequency, the highest mRNA expressions of MyoD, Myogenin, Desmin, and MyHC were in group B. There were significant differences at the same time between group B and the other groups (P lt; 0.05), except mRNA expression of Desmin at 5 days between groups B and C, and mRNA expression of MyHC at 10 days between groups A and B (P gt; 0.05). Conclusion Low frequency (0.25 Hz) and suitable time (7 days) periodic mechanical stretch is beneficial to the differentiation of the multilayer myotubes cultured in the groove casts of Sylgard 184, but as the stretch time goes on the aging of myotubes will be accelerated.
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.
Objective To investigate the influence of Nogo extracellular peptide residues 1-40 (NEP1-40) gene modification on the survival and differentiation of the neural stem cells (NSCs) after transplantation. Methods NSCs were isolated from the cortex tissue of rat embryo at the age of 18 days and identified by Nestin immunofluorescence. The lentiviruses were transduced to NSCs to construct NEP1-40 gene modified NSCs. The spinal cords of 30 Sprague Dawley rats were hemisected at T9 level. The rats were randomly assigned to 3 groups: group B (spinal cord injury, SCI), group C (NSCs), and group D (NEP1-40 gene modified NSCs). Cell culture medium, NSCs, and NEP1-40 gene modified NSCs were transplanted into the lesion site in groups B, C, and D, respectively at 7 days after injury. An additional 10 rats served as sham-operation group (group A), which only received laminectomy. At 8 weeks of transplantation, the survival and differentiation of transplanted cells were detected with counting neurofilament 200 (NF-200), glial fibrillary acidic portein (GFAP), and myelin basic protein (MBP) positive cells via immunohistochemical method; the quantity of horseradish peroxidase (HRP) positive nerve fiber was detected via HRP neural tracer technology. Results At 8 weeks after transplantation, HRP nerve trace showed the number of HRP-positive nerve fibers of group A (85.17 ± 6.97) was significantly more than that of group D (59.25 ± 7.75), group C (33.58 ± 5.47), and group B (12.17 ± 2.79) (P lt; 0.01); the number of groups C and D were significantly higher than that of group B, and the number of group D was significantly higher than that of group C (P lt; 0.01). Immunofluorescent staining for Nestin showed no obvious fluorescence signal in group A, a few scattered fluorescent signal in group B, and b fluorescence signal in groups C and D. The number of NF-200-positive cells and MBP integral absorbance value from high to low can be arranged as an order of group A, group D, group C, and group B (P lt; 0.05); the order of GFAP-positive cells from high to low was group B, group D, group C, and group A (P lt; 0.05); no significant difference was found in the percentage of NF-200, MBP, and GFAP-positive cells between group C and group D (P gt; 0.05). Conclusion NEP1-40 gene modification can significantly improve the survival and differentiation of NSCs after transplantation, but has no induction on cell differentiation. It can provide a new idea and reliable experimental base for the study of NSCs transplantation for SCI.
Objective To introduce growth and differentiation factor 5 (GDF-5) gene into hBMSCs using recombinant adenovirus vector and to investigate the effect of GDF-5 gene expression on hBMSCs osteogenic differentiation. Methods Recombinant adenovirus GDF-5 (Ad-GDF-5) containing green fluorescent protein (GFP) and Ad-GFP were amplifiedand tittered. hBMSCs at passage 3 were infected with two viruses at different titers. At 2 days after intervention, GFP expression was observed using fluorescence microscope, and GDF-5 expression in hBMSCs was detected by RT-PCR. Adherent hBMSCs at passage 3 were randomly divided into 4 groups: experimental group (GDF-5 gene transfection), osteogenic induction group, Ad- GFP infection group, and control group. Cell differentiation was detected by inverted phase contrast microscope observation, fluorescence microscope observation, reverse transcription fluorescence quantitative PCR, immunofluorescence staining, and von Kossa staining at different time points after intervention. Results The titer of Ad-GDF-5 and Ad-GFP was 1.0 × 109 pfu/mL and 1.2 × 109 pfu/mL, respectively. hBMSCs was efficiently infected by Ad-GDF-5 and Ad-GFP, and expressed target gene and GFP gene. At 1-7 days after intervention, morphology and growth pattern of the hBMSCs in the experimental group and the osteogenic induction group were transformed into osteoblast-l ike cells, whereas the cells in the other two groups were still maintained their original morphology and growth pattern. Reverse transcription fluorescence quantitative PCR detection: at 4 days after intervention, GDF-5 expression in the experimental group was obviously higher than that of other groups (P lt; 0.05); ALP, Col I, and OC gene expression in the experimental and the osteogenic induction group were superior to those of theAd-GFP infection and the control group (P lt; 0.05); Col I gene expression in the osteogenic induction group was greater than that of the experimental group (P lt; 0.05). Immunofluorescence staining: at 4 days after intervention, the cells in the osteogenic induction group and the experimental group expressed and secreted Col I, and no expression of Col I was evident in the other two groups. At 10 days after intervention, the cells in the osteogenic induction and the experimental group were positive for von Kossa staining, and the results of the other two groups were negative. Conclusion GDF-5 gene can be transferred into hBMSCs via adenovirus vector and be expressed stably. It can facil itate the osteogenic differentiation of the hBMSCs and lay a foundation for the further study of this kind of gene transferred hBMSCs effect on bone tissue repair.
Objective To study the effect of core-binding factor α1(Cbfa1)on the mesenchymal stem cells(MSCs) osteoblastic differentiation.Methods The MSCs were isolated from Japan white rabbits and cultured in vitro. The 3rd generation MSCs were infected with Cbfa1 recombinant adenovirus. The expression of Cbfa1 was detected by immunofluorescence after being infected for 3 days and the proliferation was estimated by MTT method from the 1st day to the 7th day. Then the MSCs were divided into four groups: the commonly cultured group, the simply induced group, the control adenovirus treatment group, and the Cbfa1 adenovirus treatment group. The expressions of mRNA for a various of osteoblast gene markers such as alkaline phosphatase, osteocalcin, osteopontin and type I collagen were analyzed based on reverse transcriptase polymerase chain reaction (RT-PCR). The change of adipose and myoblastic differentiation gene marker PPARγ2 and MyoD expression were detected by RT-PCR respectively.Results Positive staining of Cbfa1 was found in the MSCs infected with Cbfa1 adenovirus, and there was no significant difference in cell proliferation among the experimental groups（Pgt;0.05）. The RT-PCR indicated that all the osteoblast gene markers except type I collagen were up-regulated in the Cbfa1 adenovirus treatment group. In contrast, the expressions of PPARγ2 and MyoD were restrained. Conclusion Cbfa1 can directly promote the differentiation of MSCs into osteoblasts.
Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors’ il iac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were establ ished. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures of hBMSCs were divided into four groups: 10 ng/mL TGF-β3 group (group A), 200 ng/mL BMP-7 group (group B), combination group of TGF-β3 and BMP-7 (group C) and blank group as the control (group D). Histological observation, RT-PCR and RQ-PCR were appl ied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day (P lt; 0.05). The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day (P lt; 0.05). The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.
Stem cells have been regarded with promising application potential in tissue engineering and regenerative medicine due to their self-renewal and multidirectional differentiation abilities. However, their fate is relied on their local microenvironment, or niche. Recent studied have demonstrated that biophysical factors, defined as physical microenvironment in which stem cells located play a vital role in regulating stem cell committed differentiation. In vitro, synthetic physical microenvironments can be used to precisely control a variety of biophysical properties. On this basis, the effect of biophysical properties such as matrix stiffness, matrix topography and mechanical force on the committed differentiation of stem cells was further investigated. This paper summarizes the approach of mechanical models of artificial physical microenvironment and reviews the effects of different biophysical characteristics on stem cell differentiation, in order to provide reference for future research and development in related fields.
ObjectiveTo investigate the effect of transforming growth factor β3 (TGF-β3) at different concentrations on the differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. The TSCs were stimulated with TGF-β3 at the concentrations of 5.0, 2.5, 1.0, and 0 ng/mL. At 1, 3, and 5 days, the mRNA expressions of tendogenic differentiation related genes[collagen type Ⅰ,tenascin C (TNC), tenomodulin (TNMD), scleraxis (Scx)], osteogenic differentiation related genes[Runt related transcription factor 2 (Runx2) and alkaline phosphatase (ALP)], chondrogenic differentiation genes (Sox9 and collagen type Ⅱ),and adipogenic differentiation genes[AP2 and peroxisome proliferator-activated receptor γ (PPARγ)] were measured by real-time quantitative PCR (qRT-PCR). ResultsTSCs could differentiated in different directions after treated with TGF-β3 at different concentrations at different time points. TGF-β3 was able to induce TSCs differentiated into tenocytes, which was related to the concentration and time of duration, and the two factors have interaction. Stimulation of TGF-β3 at low concentration and for short time could inhibit non-tendogenic differentiation of TSCs, but at high concentration and for long time, TGF-β3 enhanced TSCs differented into osteocytes or chondrocytes. ConclusionEffects of TGF-β3 on TSCs differentiation are complicated and depend on the concentration and time of duration, which may be a key factor between tendogenic and non-tendogenic differentiations of TSCs.