| Background:Adipose-derived stem cells extracted from adipose tissue, are a kind of highly self-renewal and pluripotent stem cells. As an important source of stem cells, ADSCs participate in regeneration of various tissues and organs. ADSCs have sufficient source and can be easily obtained, thus having good prospects for clinical application. Currently, autologous ADSCs have been used for treatment of soft tissue defects, and its applications in treatment of myocardial ischemia and neurodegenerative diseases have entered clinical trials. Biological characteristics of ADSCs are one of the key factors affecting the success of stem cell therapy. With the increase of donor age, cellular senescence takes place. It has been confirmed that the biological functions of various stem cells change with age. Further research is necessary on whether the biological characteristics of ADSCs (proliferation, differentiation, migration, etc.) change with the increase of donor age.Objectives:To define SVF yield and senescent state of hADSCs among different age groups, and its expression in biological characteristics such as proliferation, differentiation, migration, etc. Provide more abundant information for in-depth knowledge of the effect of age factors on stem cell characteristics and its mechanism. Provide effective reference for the clinical application of autologous hADSCs in different age groups.Methods:24 clinical samples of human excised subcutaneous adipose tissue in right costal margin were obtained. According to the distribution of donor ages,24 cases of specimens were divided into three groups:(A) child group, age range:6 to 12 years old; (B) youth group, age range:22-27; (C) elder group, age range:60-73 years old. The primary (P0) hADSCs were abtained by enzymatic digestion method and the hADSCs were expanded to the third passage (P3) for assays. One-way ANOVA and Tukey multiple comparison tests were used to establish statistical significance between experimental groups.The test content includes:1. Dection of stromal vascular fraction(SVF)yield and the purity of hADSCs:Using Collagenase digestion method, nucleated cell in the fat tissue are obtained as SVF. Compare the difference of SVF content per unit volume of adipose in 3 groups; Obtain hADSCs by SVF adherent culture. Expression of mesenchymal stem cell surface markers in P3 hADSCs was analysed by flow cytometry. Compare the ratio of hADSCs which meet the requirements of the expression of mesenchymal stem cell surface markers in the three groups.2. Investigation of hADSCs senescent state and its related mechanism: Senescenct state of P5 hADSCs was preliminarily estimated by SA-β-Gal staining. Then the related mechanisms of cellular senescence were detected as followed:mitochondrial superoxide content was measured using MitoSOX TM staining of living hADSCs; hADSCs peroxynitrite content was analysed by flow cytometry; the expression levels of telomerase reverse transcriptase (hTERT) and histone deacetylase (SIRT1) mRNA in hADSCs of all age groups were detected by Real-time PCR.3.Detection of cell proliferation and apoptosis:The effect of age on hADSCs proliferation was detected by MTS and its mechanism was explored through cell cycle analysis and detection of cycle regulation gene. Cell apoptosis was investigated using Muse cell analyzer.4. Detection of hADSCs adipogenic and osteogenic differention ability:After dipogenic and osteogenic induction, the expression of key phenotypic marker in P3 hADSCs at different time points was detected by histochemical staining and Real-time PCR. The difference of multi-directional differentiation ability of hADSCs was detected among different age groups.5. Detection of cell migration ability and analysis of its related mechanism:hADSCs migration ability without chemokine interference was detected by wound healing assay, whereas their migration ability under the action of chemokine was tested by Transwell migration assay. Compare the migration abilities of various age groups. The underlying mechanism was explored by detecting the expression of chemokine receptors CXCR4 and CXCR7 of each age group.Results:1. The SVF yield of each age group was:Group A 7.28 × 105±5.52×104/ml; Group B 6.32×105±4.51×104/ml; Group C 5.53×105±9.88×104/ml. The cell viability was about 80%, and there was no significant difference among the three groups. Expression levels of positive surface markers of mesenchymal stem cells CD44, CD73, CD90 and CD 105 were high, whereas the expressions of negative markers CD34, CD11b, CD 19, CD45 and HLA-DR were lower than 5% in all age groups. The ratio of hADSCs which met the expression requirements of mesenchymal stem cell surface markers had no significant difference among all groups, which suggest that only the age factor could not influence the SVF yield and the purity of hADSCs in expansion.2. Results of SA-P-Gal staining showed that the staining ratio of P5 hADSCs in elder group was higher than that of child group. Expression results of mitochondria superoxide in MitoSOX TM staining assay showed that hADSCs of child and youth groups were negative, while the elder group samples were positive. Flow cytometry assay results showed an age-depedent increase of peroxynitrite expression.There was statistical difference between elder group and child group. Moreover, hTERT and SIRT1 expression levels of hADSCs in elder group had a downward trend compared with that of child and youth groups. The above results suggest that cellular senescence level of hADSCs in elder group is higher.3. Cell proliferation assays showed that,with age, hADSCs growth rate trended to decline, but there was no significant difference in growth rate of hADSCs among different age groups. Cell cycle analysis indicated that for the elder group, the ratio of hADSCs in S phase was significantly lower than those of the other two groups. The expression level of protooncogenes c-Jun, c-Fos also tended to decline with increasing age. The expression of cyclin dependent kinase inhibitors P21 was significantly higher than other groups, suggesting cell cycle arrest may occur in elder group, and the progress from G1 to S phase slowed down. Meanwhile,the distribution of hADSCs apoptosis and expressions of apoptosis related genes Bcl-2、NFκB、caspase8 were similar among all age groups. The above results indicate that along with increasing age,the cell cycle of hADSCs had changed, thus influenced their proliferation potential.4. In the early stage of adipogenic induction(D4), the expression of adipogenic marker genes CEBPa, PPARy, adiponectin, LPL and FABP4 in child group were significantly higher than those from youth and elder groups, but these differences gradually disappeared in the late phase (D8, D12). Oil red O staining and semi-quantitative analysis showed that the production of triglycerides at different time points after induction had no significant difference among three age groups.,and the expressions of lipid synthase genes and energy metabolism genes had no significant difference between groups, indicating that age had no significant effect on the adipogenic differentiation potential of hADSCs.In the early stage of osteogenic differentiation (D7), expressions of osteogenic related genes RUNX2, OCN, OPN, BMP2 in elder group declined significantly compared with those of child group. In the late stage (D21), semi-quantitative detection of alizarin red staining showed that the mineralization levels of the elder group was significantly lower than that of child group. Moreover, the expression of mineralization related gene OPN was also lower. It indicates that the osteogenic differentiation of hADSCs in elder donors is significantly lower than that of child group.5. The results of cell migration assay showed there was a significant decline of cell migration ability in elder groups compared with child group. Further research found that the expression levels of chemokine receptors CXCR4 and CXCR7 of hADSCs in elder group tended to be lower than those of child group. It indicates that the decrease of chemokine receptor expression may be one of the reasons for the decline of its migration ability.Conclusions:1. Donor age has no significant effect on SVF yield of thorax subcutaneous fat, and purity of hADSCs expanded in vitro.2. The effect of age on senescent state of hADSCs:hADSCs of older individuals have higher senescence level. The oxidative stress caused by elevated levels of intracellular ROS content may be one of the reasons.3. The effect of age on proliferation of hADSCs:With the increase of donor age, hADSCs trend to proliferate slowly, which may result from age-related cell cycle arrest.4. The effect of age on differentiation potential of hADSCs:For elder individuals, adipogenic differentiation potential has no significant change, whereas osteogenic differentiation ability of hADSCs decreases, indicating that the application feasibility of autologous hADSCs for osteogenesis reduces in elderly patients.5. The effect of age on migration ability of hADSCs:hADSCs migration ability of elderly donors decreases, that may partly be caused by decreased expression of chemokine receptors CXCR4 and CXCR7 on cell surface.In conclusion, age has no significant influence on SVF yield of excised adipose in the chest; but along with the age growth, cellular senescence level of hADSCs increase, the proliferation, differentiation and migration abilities decrease accordingly. This study not only has detected changes of biological expressions of stem cells, but also has carried on the preliminary exploration to the corresponding mechanism. It provides more abundant information for insight of age influences on characteristics of stem cell and its mechanisms. It provides also effective reference for the clinical application of hADSCs for different age groups. |
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