ObjectiveTo comprehensively analyze and compare the biological difference between bone marrow mesenchymal stem cells (BMSCs) and placenta-derived MSCs (PMSCs) in hypoxia and to extend the knowledge for seed cells selection.
MethodsThe domestic and foreign related literature about the effects of hypoxia microenvironment on proliferation, apoptosis, differentiation, paracrine secretion, migration, and homing ability of BMSCs and PMSCs were summarized and analysed.
ResultsPMSCs proliferated much faster and more sensitive to the hypoxia than BMSCs; in addition, PMSCs showed stronger survivability. Similar to BMSCs, PMSCs can home to hypoxic-ischemic tissues efficiently, secrete a lot of growth factors and differentiate into tissue-specific cells to stimulate tissue regeneration.
ConclusionPMSCs as the seed cells will have broad application prospects in the regenerative medicine.
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.
To investigate the effect of BMSCs on the repair of digestive tract injury and its mechanisms.Methods Recent l iterature on the effect of BMSCs on the repair of digestive tract injury was reviewed. Results BMSCs had the potency of self-repl ication, prol iferation and multipotential differentiation, which played an important role in the repair of digestive tract injury. The probable mechanisms included: BMSCs’ abil ity of migrating to the injured tissue and inhibiting the host immune response; BMSCs’ dedifferentiation and redifferentiation; BMSCs’ direct differentiation into the epithel ial cellsor the stem cells of digestive tract; BMSCs’ fusion with the stem cells or the mature epithel ial cells of digestive tract; BMSCs’ participation in the reconstruction of injured microenvironment. Conclusion BMSCs participates in the repair of digestive tract injury and has a bright future in the treatment of digestive system disease.
【Abstract】 Objective To investigate the possibil ity of BMSCs seeded into collagen Ⅰ -glycosaminoglycan (CG)matrices to form the tissue engineered cartilage through chondrocyte inducing culture. Methods Bone marrow aspirate of dogs was cultured and expanded to the 3rd passage. BMSCs were harvested and seeded into the dehydrothemal treatment (DHT)cross-l inked CG matrices at 1×106 cells per 9 mm diameter sample. The samples were divided into experimental group and control group. In the experimental group, chondrogenic differentiation was achieved by the induction media for 2 weeks. Medium was changed every other day in both experimental group and control group. The formation of cartilage was assessed by HE staining and collagen Ⅱ immunohistochemical staining. Results The examinations under the inverted phase contrast microscopeindicated the 2nd and 3nd passage BMSCs had the similar morphology. HE staining showed the BMSCs in the experimental group appeared polygon or irregular morphology in the CG matrices, while BMSCs in the control group appeared fibroblast-l ike spindle or round morphology in the CG matrices. Extracellular matrix could be found around cells in the experimental group. Two weeks after seeded, the cells grew in the CG matrices, and positive collagen Ⅱ staining appeared around the cells in the experimentalgroup. There was no positive collagen Ⅱ staining appeared in the control group. Conclusion It is demonstrated that BMSCs seeded CG matrices can be induced toward cartilage by induction media.
Objective To establ ish a two-dimensional biological printing technique of hBMSCs so as to control the cell transfer process and keep cell viabil ity after printing. Methods Bone marrow (5 mL) was obtained from healthy volunteer. The hBMSCs were regularly subcultured to harvest cells at passage 2, which were adjusted to the single cell suspensionat a density of 1 × 106/mL. The experiment was divided into 3 groups: printing group 1 in which cells underwent propidium iodide (PI) fluorescent label ing, then were transferred by rapid prototype biological printer (interval in x-axis 300 μm, interval in y-axis 1 500 μm), and laser scanning confocal microscope was appl ied to observe cell fluorescence; printing group 2 in which cells received no PI label ing and were cultured for 2 hours after transfer, Live/Dead viabil ity Kit was adopted to detect cell viabil ity and laser scanning confocal microscope was appl ied to observe cell fluorescence; half of the cells in printing group receiving no Live/Dead viabil ity Kit detection were cultured for 7 days, then inverted microscope was used to observe cell morphology, routine culture was conducted after the adherence of cells, the growth condition of cells was observed dynamically; control group in which steps were the same as the printing group 2 except that cell suspension received no printing. Results Laser scanning confocal microscope observation on the cells in printing group 1 revealed the “cell ink droplets” were distributed regularly and evenly in the two-dimensional layer and each contained 15-35 cells, meeting the requirement of designing two-dimensional cell printing. The cells in printing group 2 went through cell viabil ity test, laser scanning confocal microscope observation showed the fluorescence of cells 30 minutes after cell incubation. There was no significant difference between the control group and the printing groups in terms of cell viabil ity. The printed cells presented normal adherence, good morphology and good growth state 7 days after routine culture. Conclusion Biological printing technique can real ize the oriented, quantificational and regulardistribution of hBMSCs in the two-dimensional plane and lays the foundation for the construction of three-dimensional cellprinting or even organ printing system.
Objective To review researches of BMSCs in tumor therapy. Methods The recent relevant l iterature was extensively reviewed. The tropism of BMSCs to cancer, the effect of BMSCs on tumor growth and the appl ication of BMSCs in tumor therapy were summarized. Results BMSCs has the tropism to tumor and may inhibit or enhance growth of tumor. BMSCs as gene-del ivery vehicle for gene therapy had obtained certain therapeutic efficacy. However, BMSCs can become tumorigenic. Conclusion BMSCs is a good gene-del ivery vehicle for gene therapy. The relationship of BMSCs and tumorcells should be studied deeply for enhance the safety of BMSCs in gene therapy of tumor.
Objective To supply references to tissue-engineered skin cl inical appl ications with autogenic BMSCs composited collagen membrane to repair swine full-thickness cutaneous deficiency. Methods Twenty mL bone marrow were obtained respectively from 4 swine, autogenic BMSCs were cultured and passed to the 3rd passage. The fresh bovine tendontreated by means of chemically cross-l inked was made 5 cm diameter collagen I (Col I) membrane. The 2 × 107/mL P3 swine autogenic BMSCs labeled DAPI were planted to sterile Col I membrane for 24 hours incubation, then the tissue-engineered skin was constructed. The five full-thickness skin defect of 5 cm diameter was excised to the muscle from forward to backward on the back midl ine two sides of swine. The tissue-engineered skin were implanted in the experimental group, while Col I membrane was implanted in control group. After 3 and 8 weeks of implantation, the two swine wound surface heal ing circumstance was observed and further evaluated with histology analysis and TEM. After 3 weeks of implantation, the experimental group were observed with fluorescence microscopy and staining for glycogen. Results After 3 weeks of implantation, the wound surface of control group were observed nigrescence, scab and putrescence, and after 8 weeks of implantation, also evident putrescence and scar. The wound surface of experiment group was al ive after 3 weeks implantation, appearance was leveled off and flexible without evident scar. The wound surface recovered well after 8 weeks of implantation, wound surface heal ing rate was significantly difference between the two groups (P lt; 0.01). After 3 weeks of implantation, control group were observed acestoma hyperplasia and no epidermal coverage by histology analysis. The experimental group was showed integrity epidermis and dermis structure. The basal layer was crimson and continuously positive with glycogen staining. After 8 weeks of implantation, the experimental group and control group were emerged normal skin structure. After 3 weeks of implantation in control group, a lot of neutrophil ic granulocytes and fibroblasts were noticed, but no epidermal structure was observed under TEM. In the experimental group, a lot of epidermal cells were observed, dermatome connection among epidermal cells and hemidermosome connection between basilar membrane cells and basal membrane were observed in epidermis. In the dermis experimental group, blood capillary endothel ial cells were noticed. Furthermore, considerable collagen fiber deposit was found in the surrounding tissue of fibroblasts. After 3 weeks of implantation, BMSCs labeled with DAPI were located reconstructed epidermal basement membrane and dermis by fluorescence microscopy. Conclusion Tissue-engineered skin which is composited with autogenic BMSCs as seed cells and collagen membrane were potential prospects in appl ication of repairing swine full-thickness cutaneous deficiency.
Objective To investigate the effects of the recombinant plasmid pIRES-hBMP-2-hVEGF165 on differentiation and maturation of hBMSCs in vitro. Methods The co-expressing vector of hBMP-2 and hVEGF165 was constructed. The BMSCs were isolated and cultured from health adult human denoted marrow. By the l ipofection method, the reconstructed plasmids pIRES-hBMP-2-hVEGF165, pIRES-hBMP-2, pIRES-hVEGF165 and pIRES neo empty vector, weretransfected to hBMSCs (groups A, B, C and D). The untransfected cells were harvested as control group (group E). After4 weeks of culture, RT-PCR was employed to assay the hBMP-2, hVEGF165 and osteocalcin mRNA expression in hBMSCs. The expressions of hBMP-2 and hVEGF165 of BMSCs were assayed by Western blot. The level of ALP activities of BMSCs was determined. Col I was also determined by immunohistochemical staining. Results Compared to group E, the hBMSCs in group A secreted high level of hBMP-2, hVEGF165, Col I and osteocalcin; osteocalcin and Col I expressed at high level in group B, and hVEGF165 expressed at high level in group C. Otherwise, the expression of hVEGF165 in group B and the expressions of hBMP-2 and Col I in group C resemble to that of groups D and E, no expression or few expression was observed. The activities of ALP in groups A, B, C, D and E were 0.91 ± 0.03, 0.90 ± 0.02, 0.64 ± 0.03, 0.67 ± 0.01 and 0.66 ± 0.02, respectively. The activity of ALP of groups A and B were significantly increased compared with that of group E (P lt; 0.05); there was no significant difference among groups C, D and E (P gt; 0.05). Conclusion The recombinant plasmid pIRES-hBMP-2-hVEGF165 can be successfully transfected into BMSCs with cation l iposome-mediated transfection method, the exogenous hBMP-2 and hVEGF165 genes can be expressed constitutively in the transfected BMSCs, and it can enhance the differentiation abil ities of BMSCs.
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide (SPIO) in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The label ing efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblau stain and MTT were performed to calculate the cell’ s activity. Rabbits were randomly divided into experimental group (n=8) and control group (n=4), the labeled BMSCs and non-labeled BMSCs (5 × 105/mL) were injected into their own intervertebral discs (L1,2, L2,3, L3,4 and L4,5), respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl’s fluid to observe cell survival and distribution. Results The label ing efficiency of BMSCs with SPIO was 95.65% ± 1.06%, the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell prol iferation within 7 days between non-labeled and labeled cells (P gt; 0.05). After 8 weeks of operation, the injected cells was al ive. ConclusionLabeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 μmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 μmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
