Wnt Signaling Mediates The Aging-Induced Differentiation Impairment Of Intestinal Stem Cells

Background/Aims:Stem cell aging is the basis for the development of aging-related diseases.With the occurrence of aging,intestinal homeostasis is difficult to maintain in the elderly.The incidence of intestinal diseases such as intestinal tumors increases sharply,and the ability to repair and regenerate after intestinal damage is weakened,which indicates that the core function of aging intestinal stem cells(ISCs)is changed.However,it is unclear what is the abnormality and its regulation mechanism of the core functions such as self-renewal and differentiation of intestinal stem cells during natural aging.This study focuses on the function of aging intestinal stem cell self-renewal and differentiation,in order to explore the mechanism of intestinal stem cell aging and the ways to delay aging.Methods:Freshly isolated crypts from 24-month-old(old)and 2-month-old(young)mice whole small intestine and the crypts were cultured in a standard culture system.On the 7th and 10 th day,the crypt culture phenotype was observed,and the total growth of the organoids was counted.The expression levels of ISCs differentiated genes(including Alpi,Atoh1,Defa24,Chga)and Wnt signal target genes(Axin2,Ascl2)were detected by qRT-PCR on the 7th day after plating.Subculture was performed on the 10 th day,the phenotype of crypt culture was observed and the total growth of organoids was counted after 14 days of primary culture.The aging of ISCs in aging mice was analyzed by observing the culture phenotype of crypts,the statistics of total growth of organoids and the results of qRT-PCR.The relationship between senescence ISCs and Wnt signaling levels was observed by down-regulation of Wnt signaling(reducing the concentration of Wnt activator R-spondin-1 in culture system to 1/3 of standard culture concentration)or up-regulating Wnt signal(adding Wnt3 a to standard culture system).Results:Under normal culture conditions,almost all of the crypts of young mice differentiated into normal crypt-villi structures,while most of the crypts of aging mice could not differentiate into crypt-villi structures,mainly forming large undifferentiated cysts.The cyst structure and phenotype remained after passage.The Wnt signaling levels is increased in aging.When the Wnt signal is down-regulated,the proportion of intestinal organoids that can differentiate into crypt-villi structure is significantly increased,and the large undifferentiated round cyst structure is significantly reduced,which is closer to the intestinal stem cell culture phenotype of young mice.This phenotype remained after passage.Conclusion:The ISCs from aging mice have a ditinct differentiation phenotype compared to the ISCs from young mice,and down-regulation of Wnt signaling pathway activity can partially reverse this phenotype.It provides new insights into the mechanism of intestinal aging and provides new ideas for the prevention and treatment of aging related intestinal diseases.