Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
ObjectiveTo study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry.
ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups.
ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.
【Abstract】 Objective To explore the interventional effect of platelet lysate (PL) on osteogenic differentiation ofBMSCs by induction in rats in vitro. Methods Twenty-four clean-grade adult Wistar rats, weighing from 250 g to 300 g, maleor female, were included in this study. PL was obtained through three times of centrifugation and repeated freeze-thaw for the blood aspirated from cardiac cavities in 16 Wistar rats. ELISA assay was conducted to detect the concentration of growth factors PDGF, TGF-β1, IGF-1 and VEGF in PL. The BMSCs harvested by flushing femurs of 8 adult Wistar rats were isolated, cultivated and expanded in vitro. The cells at the 4 passage were performed for osteogenic differentiation by induction in three groups of A (5% PL of final concentration in basic induction medium), B (1% PL of final concentration in basic induction medium), and C (no presence of PL in basic induction medium as a control). The morphological changes of the cells were dynamically observed with inverted phase contrast microscope during the whole period. At different time-points, ALP staining (7 days) and ALP/TP (2, 8, 12 days) of the cells were detected to evaluate ALP activity, and the mineral formation in extracellular martrix was examined with Al izarin red staining which provided quantitative analysis of mineral deposits. Results ELISA assay showed that the content of PDGF, TGF-β1, IGF-1 and VEGF in PL reached (300 ± 30), (140 ± 25), (80 ± 35), (70 ± 20) pg/mL, respectively. Morphological observation displayed BMSCs in group A or B gradually turned from spindle-shape to square- or polygon-shape as the morphorlogical type of osteoblast-l ike cells at 7 days. The cells in group A showed slower shape changesbut higher prol iferation than that in group B or C. Moreover, at the 20 days, the cells in group A still displayed dense gro wth and produced obviously decreased amount of mineral deposits in ECM when compared with group B or C. At the 7 days, the cells ofgroup A showed smaller amount of granules positive for ALP staining in cytoplasm when compared with groups B and C, and displayed marked reduction in ALP activity assay at the 2, 8, and 10 days compared with that of groups B and C (P lt; 0.05). At the 20 days, Al izarin red staining showed the number of mineral deposits in groups A, B and C were 7.67 ± 1.10, 12.87 ± 0.81 and 15.59 ± 0.25, respectively, while the area of mineral deposits were (161 778.70 ± 44 550.80), (337 349.70 ± 56 083.24), and (415 921.70 ± 71 725.39) pixels, respectively. The number of mineral deposits and the area of mineral deposits in group A were smaller than those in groups B and C (P lt;0.05). But there was no statistically significant difference between groups B and C (P gt; 0.05). Conclusion PL is a kind of system carrying various growth factors. Exposure of PL inhibits both ALP activity and mineral formation of BMCs in a dose-dependent way under the osteogenic induction environment.
Objective To investigate the feasibility of Y27632 to induce transdifferentiation from human retinal pigment epithelial (hRPE) cells into neuron-like cells in vitro. Methods The third to sixth generation of primary hRPE cells were cultured with 2% fetal bovine serum + Dulbecco's modified eagle medium/F12 culture solution, with (experimental group) or without (control group) 10 mu;mol/L Y27632. At 3, 6 hours and 1, 3, 5, 7 days after induction, the morphologic changes of RPE cells were observed by inverted microscope. The expression rate of CK18, Map2, NF200 and Pax6 at 3 days after induction in the experimental and control group were detected by immunofluorescent staining. chi;2 test was employed for comparison between the two groups. Results 50.1% cells of the experimental group formed axon-like processes and interconnected each other with typical neuron-like appearance. The expression rates of CK18, Map2, NF200 and Pax6 in the experimental group were 43.88%, 31.90%, 57.45% and 65.79%, while the above indexes in the control group were 93.97%, 4.49%, 22.37% and 8.33% respectively. Compared the expression rate of CK18 (chi;2=64.763), Map2 (chi;2=23.634), NF200 (chi;2=21.261) and Pax6 (chi;2=25.946) between the two groups, the differences were significant (P＜0.01). Conclusion The hRPE cells can be trans-differentiated into neuron-like cells in vitro by Y27632.
Objective To investigate the feasibility of differentiation of invitro induced rat bone marrowderived mesenchymal stem cells（rMSCs） into retinal pigment epithelial （RPE） cells.Methods The rMSCs from BrwonNorway (BN) rats were isolated and cultured by adherent screening method. RPE cells lysate made by repeated freezethawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin（CK）and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells. The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously. The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate, rMSCs can differentiate into RPE cells.
Objective
To investigate the regulating effect of Notch-1 on retinal progenitor cells (RPC) differentiating into retinal ganglion cells (RGC).
Methods
RPC of 14-day embryonic SD rats were induced and differentiated in the culture medium with Notch-1 antisense oligonucleotides (experimental group) or missense oligonucleotides (control group) for 14 days. The condition of growth and differentiation of the cells were observed daily under the phase-contrast microscope. RGC were identified by Thy1.1 immunocytochemistry methods and the cellular number was counted.
Results
RPC in both of the two groups differentiated into various retinal cells, including Thy1.1-positive RGC. The percentage of RGC derived from RPC was 31.19%plusmn;6.90% in experimental group and 16.57%plusmn;4.31% in control group, and the difference was statistically significant (t=9.84，Plt;0.001).
Conclusion
Notch-1 may down-regulate the differentiation of RPC, and the inhibition of Notch-1 may promote RPC differentiating into RGC.
(Chin J Ocul Fundus Dis, 2007, 23: 101-103)
Objective
To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes and its mechanisms.
Methods
Eighteen male adult Sprague Dawley rats [weighing, (200±20) g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor (EGF), hepatocyte growth factor (HGF), oncostatin M (OSM), and basic fibroblastic growth factor (bFGF). Passage 3 HUCMSCs were divided into 3 groups: cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serum and 5?mL/ L serum from rats with hepatic fibrosis (group A), in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from normal rats (group B), and in DMEM/F12 containing 10% fetal bovine serum (group C). The morphological changes of the cells were observed. The expressions of α-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunofluorescence. The protein levels of albumin (ALB), tryptophan 2, 3-dioxygenase (TPH2), and CYP3A4 and MAPK/ERK signal pathway protein (P-ERK) were detected using Western blot. The content of blood urea nitrogen (BUN) was measured by diacetyl m onoxime method.
Results
HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were (21.42±0.32) pg/mL and (17.57±0.31) pg/mL respectively, showing significant difference (t=14.989, P=0.000); the concentrations of OSM were (129.96±0.65) pg/mL and (98.44±1.32) pg/mL respectively, showing significant difference (t=37.172, P=0.000); the concentrations of HGF were below the detection limit and (1.03±0.12)?ng/ mL respectively; and the concentrations of bFGF were lower than the detection limit in both groups. No morphological changes of cells were observed in both groups at 7 days, and there was no significant difference between groups. At 7 days after culture, the cells in group A could express human hepatocyte biomarkers of AFP, CK18 and hepatocyte-specific-function proteins of ALB, TPH2, and CYP3 A4 while cells in groups B and C did not. Western blot showed that cells in each group could express P-ERK protein. The relative level of P-ERK protein in group A was significantly higher than that in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The BUN concentration of group A [(0.74±0.07)?mmol/ L] was significantly higher than that of groups B [(0.40±0.04)?mmol/ L] and C [(0.38±0.04) mmol/L] (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05).
Conclusion
Under the condition of hepatic fibrosis, the level of HGF will increase while EGF and OSM will decrease. The formed blood microenvironment?will activate MAPK/ERK signal pathway in HUCMSCs, induce them differentiate into hepatocytes.
Objective
To investigate the possibility of commitment differentiation of embryonic stem cells induced by the medium of cultured retinal neurons of SD rats.
Methods
The medium from cultured retinal neurons of SD rats were collected, sterilized and mixed with DMEM medium according to 2∶3 proportion, ES cells were cultured with these mixed medium and were observed under the phase contrast microscope daily, the induced cells were identified by NF immunohistochemistry methods.
Results
The ES cells cultured with these mixed medium can differentiate into neuron-like structure, and the induced cells were positive in NF immunofluorescence staining.
Conclusion
The medium from cultured retinal neurons of SD rats can induce ES cells commitment differentiation into neuron-like structure.
(Chin J Ocul Fundus Dis, 2002, 18: 134-136)
Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.
Objective To observe the differentiation effect of rabbit amnion-derived stem cells (ADSC) induced into neural cells.Methods ADSC of New Zealand female rabbits were isolated and cultured. Its mRNA level of Fibronectin, Nestin and Vimentin were detected by real-time quantitative polymerase chain reaction. The selfreplication ability of ADSC was confirmed by monoclonal formation experiments. These ADSC were further induced into neural cells in vitro. Five days after induced differentiation, the expression of -tubulin and glial fibrillary acidic protein (GFAP) were detected by immunofluorescent staining. Results ADSC were separated from amnion tissue gradually after 24 hours. There were polygonal cells gathered around the amnion tissue at 72 hours, and were distributed compactly around the amnion at 120 hours. The morphology of cleavage daughter cells was basically the same as parent cells. ADSC has the ability of self-replication. The Nestin, Vimentin, Fibronectin mRNA expressions in ADSC were 15.79, 1.91, 7.65 times those in spleen cells. The differences were statistically significant(Z=-5.243, -3.972, -2.524; P＜0.05). The beta;-tubulin expression was found in cytoplasm of most cells. The GFAP expression was found in cytoplasm in some cells. Conclusions ADSC has self-replication ability. It can be induced into neurons and neuroglial cells under the right conditions.
