Objective To explore the effect of the platelet-rich plasma (PRP) on proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) in China goat in vitro. Methods MSCs from the bone marrow of China goat were cultured. The third passage of MSCs were treated with PRP in the PRP group (the experimental group), but the cells were cultured with only the fetal calf serum (FCS) in the FCS group (the control group). The morphology and proliferation of the cells were observed by an inverted phase contrast microscope. The effect of PRP on proliferation of MSCs was examined by the MTT assay at 2,4,6 and 8 days. Furthermore, MSCs were cultured withdexamethasone（DEX）or PRP; alkaline phosphatase (ALP) and the calcium stainingwere used to evaluate the effect of DEX or PRP on osteogenic differatiation of MSCs at 18 days. The results from the PRP group were compared with those from the FCS group. Results The time for the MSCs confluence in the PRP group was earlier than that in the FCS group when observed under the inverted phase contrast microscope. The MTT assay showed that at 2, 4, 6 and 8 days the mean absorbance values were 0.252±0.026, 0.747±0.042, 1.173±0.067, and 1.242±0.056 in the PRP group, but 0.137±0.019, 0.436±0.052, 0.939±0.036, and 1.105±0.070 in the FCS group. The mean absorbance value was significantly higher in the PRP group than in the FCS group at each observation time (P＜0.01). Compared with the FCS group, the positive-ALP cells and the calcium deposition were decreased in the PRP group; however, DEX could increase boththe number of the positiveALP cells and the calcium deposition. Conclusion The PRP can promote proliferation of the MSCs of China goats in vitro but inhibit osteogenic differentiation.
Objective To review the progress, methods and obstacles in the differentiation of embryonic stem cells(ESCs) into osteoblasts in vitro. Methods The recent literature concerned with the differentiation of ESCs into the osteoblasts was extensively reviewed and briefly summarized. Results ESCs was a good tool for derivation of obsteoblasts.Conclusion The study on the induction of ESCsinto the osteogenic lineage provides a model for analyzing the molecular processes of osteoblasts development in vivo and establishes the foundation for the use of ESCs in skeletal tissue repair.
Objective To investigate the effects of sodium hyaluronate solution on the proliferation and differentiation of myoblasts. Methods The 3rd subculture myoblasts from muscle of infant SD rat were cultured in four growth media, in which the concentrations of sodium hyaluronate were 0.05% (group A) , 0.1%( group B), 0.2% (group C)and 0 (group D, control group), respectively. The proliferation rate of myoblasts in each medium was observed through growth curves by means of count and MTT. At the same time, the subculture myoblasts were cultured in differentiated media in which the concentrations of sodium hyaluronate were 0 and 0.1%. The capacity of fusion of myoblasts was compared between two kinds of differentiated media. Results There were the nearly same proliferation curse in Groups A, B and C: increasing by logarithm at 2 days and reaching peak value at 4 days. The myoblasts in Group D increased slowly: increasing by logarithm at 3 days, doubling at 5 days and reaching peak value at 6 days. MTT has the similar curse to counting. The myoblast proliferation of Group B was more than that of the other groups. The peak value of myoblast fusion was 35% at 6 days in common differentiated media; slowly reached 11.7% at 7 days in the differentiated media in which the concentrations of sodiumhyaluronate was 0.1%.Conclusion Sodium hyaluronate at certain concentration can be a decent media for myoblasts, it can accelerate proliferation and differentiation of myoblasts.
Objective To review research progress of adipose tissuederived stromal cells (ADSCs).Methods The recent articles on ADSCs were extensively reviewed, and the culture and differentiation ability of ADSCs were investigated.Results A population of stem cells could be isolated from adult adipose tissue, they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling. The majority of the isolated cells were mesenchymal origin, with a few pericytes,endothelial cells and smooth muscle cells. ADSCs could be induced to differentiate intomultiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic and adipogenic cells, they could also differentiate into nerve cells.Conclusion ADSCs can substitute mesenchymal stem cells and become an alternative stem cells source for tissue engineering.
Objective To investigate the feasibil ity and effect of inducing adi pose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P lt; 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P gt; 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col IIimmunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.
Objective To investigate the feasibility of differentiation of the marrow mesenchymal stem cells (MSCs) into the cells of the skin appendages andthe mechanism of their involvement in the wound healing. Methods The bone marrow was collected from Wistar rats by the flushing of the femurs, MSCs were isolated and purified by the density gradient centrifugation. Then, the MSCs were amplified and labelled with 5-bromo-2′-deoxyuridine (BrdU). The full-thickness skin wounds with an area of 1 cm×1 cm were made on the midback of the homogeneous male Wistar rats. At the same time, 1×106/ml BrdU-labelled MSCs were infused from thepenile vein. The specimens were harvested from the wound tissues on the 3rd dayand the 7th day after operation and were immunohistochemically stained by either BrdU or BrdU and pan-keratin. Results The BrdU positive cells appeared in thehypodermia, the sebaceous glands, and the hair follicles of the wounds, as wellas the medullary canal of the femurs. The double-staining showed the BrdU positive cells in the sebaceous glands and the hair follicles of the wounds expressedpan-keratin simultaneously. Conclusion During the course of the wound healing, MSCs are involved in the wound repair and can differentiate into the cells ofthe skin appendages under the microenvironment of the wound.
Objective To investigate the division, prol iferation and differentiation abil ities of nestin+/GFAP+cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs). Methods Twelvemale SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group inwhich the spinal cord injury model was establ ished by aneurysm cl ip compression method, and control group in which no processing was conducted. At 5 days after model ing, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cellsuspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was appl ied to induce differentiation. Immunohistochemistry detection and flow cytometry were appl ied to observe and analyze the type of cells and their capabil ity of division, prol iferation and differentiation. Results At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 ± 0.71 and 1.12 ± 0.38, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Concerning cell cycle, the proportion of S-phase cell and prol iferation index of the model group (15.49% ± 3.04%, 15.88% ± 2.56%) were obviously higher than those of the control group (5.84% ± 0.28%, 6.47% ± 0.61%), indicating there were significant differences between two groups (P lt; 0.01). In the model group, primary cells gradually formed threedimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multi ple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of β-tubul in III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ ol igodendrocyte, β-tubul in II+ neuron and GalC+ cell body and protruding were observed. Conclusion Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the abil ity of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
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 induce embryonic stem cell (ESC) to differentiate into endothel ioid cells using a simple adhesive culture method, and to provide a new cells seed source for vascular tissue engineering or cell therapy. Methods SV129-derived ESC were seeded at 2 × 104/cm2 and maintained undifferentiated on ESC culture medium in the presence of 1 000 U/mL leukaemia inhibitory factor (LIF). Embryoid body (EB) formatted when ESC cultured in suspension in the lack of LIF. At 4 days, EB was transferred to 0.1% gelatin coated dish and cultured with medium supplementary of VEGFto be induced differentiation. The characteristics of differentiated cells were determined by immunohistochemistry staining, flow cytometry (FCM), 1, 1-dioctadecyl-3, 3, 3, 3-tetramethyl indocarbocyanine-labeled acetylated low density l ipoprotein (DiIAc- LDL) takeup test, and TEM detection. Results Differentiated cells were morphologically characterized as endothel ial cells. They could takeup DiI-Ac-LDL, be stained positive by Flk-1 and CD31. The CD31 positive cells reached above 90% when measured by FCM. Furthermore, Weibel-Palade bodies were detected and tight junctions were found when differentiated cells were examined by TEM. Conclusion Using a simple adhesive culture method and by suppl ied with VEGF alone, ESCs can be induced to differentiate into endothel ioid cells. The differentiation method is simple and economic, and can provide seed cells for vascular tissue engineering or cell-therapy.
OBJECTIVE: To investigate the biological characteristics of continuously subcultured human embryonic skeletal myoblasts, and choose the optimal seeding cells for muscle tissue engineering. METHODS: Human embryonic skeletal myoblasts were subcultured in vitro. The growth curve, rate of myotube formation(RMF) were used to evaluate the proliferative and differentiation ability of myoblasts, and to investigate the influence of fibroblasts contamination on myoblasts. RESULTS: The beginning 6 passages of myoblasts showed b proliferative and differentiation ability. From the 8th to 20th passage, the rate of fibroblasts contamination was increased, it mainly showed the growth characteristics of fibroblasts with increased proliferation and low differentiation. After subcultured to the 20th passage, the degeneration of myoblasts was obvious. CONCLUSION: The myoblasts within 6 passages should be used as the seeding cells of muscle tissue engineering because of b proliferative ability and high rate of myotube formation.