Abstract: BACKGROUND: As the central organ of the immune system, the thymus serves as the primary site for T lymphocyte differentiation, development and maturation. Thymic involution initiates during adolescence, making it the first organ to undergo age-related degeneration in humans. There is still few current research on therapeutic interventions for thymic aging, and there are no studies in China addressing the potential of highly active umbilical cord mesenchymal stem cells to modulate thymic aging in the tree shrew. 
OBJECTIVE: To investigate the effects of highly active umbilical cord mesenchymal stem cells on the structure and function of thymus of aging tree shrews.
METHODS: The umbilical cord tissues from newborn tree shrews were obtained via cesarean section, and highly active umbilical cord mesenchymal stem cells were isolated and cultured using the tissue adherence method. Fourth-passage highly active umbilical cord mesenchymal stem cells were transfected using green fluorescent protein from GeneChip under conditions of a multiplicity of infection of 140 for 72 hours. Twenty female tree shrews with an average age of 7 years were randomly divided into elderly model group and elderly treatment group (10 per group). Ten 3-year-old female tree shrews were designated as the young control group. The aged treatment group received tail vein infusions of fourth-passage highly active umbilical cord mesenchymal stem cells at a dose of 1×10⁷ cells/kg, once daily for three consecutive days. The young control group and aged model group received no special treatment. After 4 months of routine feeding, thymus tissues were collected. Hematoxylin-eosin staining was used to examine thymic structure. Masson staining was utilized to assess thymic fibrosis. Immunohistochemical staining was applied to detect the expression of senescence markers p21 and p53. Immunofluorescence staining was employed to evaluate the expression of proliferative proteins Ki67 and proliferating cell nuclear antigen. Reactive oxygen species fluorescence staining was used to measure reactive oxygen species levels in thymic tissues. Immunohistochemical staining was utilized to quantify CD3+ total T lymphocyte counts in the thymus. Enzyme-linked immunosorbent assay was employed to determine serum thymosin β4 levels in tree shrews. DAPI counterstaining of thymic nuclei was performed to observe the distribution of highly active umbilical cord mesenchymal stem cells in thymic tissues. 
RESULTS AND CONCLUSION: (1) Green fluorescent protein from GeneChip transfected tree shrew highly active umbilical cord mesenchymal stem cells were observed in the tree shrew thymus of the elderly treatment group. (2) Compared with the elderly model group, the thymus of the elderly treatment group exhibited significantly improved histoarchitecture, including increased thymic parenchyma (P < 0.05), reduced adipose infiltration, distinct corticomedullary demarcation, decreased collagen fiber proportion, and a trend toward rejuvenation. (3) Senescence markers p21 and p53 expression levels were downregulated in the elderly treatment group compared with the elderly model group (P < 0.01 and P < 0.05). (4) Compared with the elderly model group, the elderly treatment group showed a non-significant increasing trend in Ki67 expression (P > 0.05), elevated proliferating cell nuclear antigen expression (P < 0.05), and significantly reduced reactive oxygen species levels (P < 0.01). (5) Compared with the elderly model group, the proportion of CD3+ T lymphocytes displayed an increasing trend in the elderly treatment group, though the difference was not statistically significant (P > 0.05). (6) Serum thymosin β4 levels were significantly increased in the elderly treatment group compared with the elderly model group (P < 0.01). (7) These findings demonstrate that highly active umbilical cord mesenchymal stem cells ameliorate age-related thymic structural degeneration, suppress senescence marker expression, enhance thymocyte proliferative activity and thymic function in aged tree shrews.

Key words: stem cells, umbilical cord mesenchymal stem cells, tree shrew, thymus, aging, aging marker molecule, proliferating gene, immunity