Bone Marrow Mesenchymal Stem Cells Derived From Juvenile Macaques Reversed Ovarian Aging in Elderly Macaques
Authors: Tian Chuan , He Jie , An Yuanyuan , Yang Zailing , Yan Donghai , Pan Hang , Lv Guanke , Li Ye , Wang Yanying , Yang Yukun , Zhu Gaohong , He Zhixu , Zhu Xiangqing , Pan xinghua 
Published: April 2021

Abstract
Background
Female sex hormone secretion and reproductive ability decrease with ageing. Bone marrow mesenchymal stem cells (BMMSCs) have been postulated to play a key role in treating ovarian senescence; however, the curative effect and mechanism are not clear.
Methods
We used the macaque ovarian senescence model and observed the structural and functional effects of juvenile BMMSCs in the treatment of ageing macaque ovaries. Moreover, to elucidate the molecular regulatory mechanism by which BMMSCs reverse ovarian senescence, RNA sequencing (RNA-seq) of the ovaries was used to identify key genes and signalling pathways associated with transcriptome profile changes.
Results
(1) The Rhesus monkey ovarian aging models were an average of 24 years old and had the following sex hormone levels: 0.28 ± 0.11 mIU/mL hFSH, 0.017 ± 0.009 mIU/mL hLH, 0.24 ± 0.042 ng/mL Testo, 51.86 ± 18.37 pg/mL ESTRDL, 0.13 ± 0.012 ng/mL Prog, 0.013 ± 0.012 hCG, and 11.96 ± 2.96 pmol/l AMH. The ovarian organ index was 0.057 ± 0.021, and the HE staining results showed almost no follicular structure, with only local atresia follicles observed. For young rhesus monkeys, the average age was 7 years old, and sex hormone levels were as follows: 0.043 ± 0.03 mIU/mL hFSH, 0.007 ± 0.009 mIU/mL hLH, 0.57 ± 0.15 ng/mL Testo, 123.2 ± 26.26 pg/mL ESTRDL, 0.28 ± 0.014 ng/mL Prog, 0.05 ± 0.012 hCG and 11.96 ± 2.96 pmol/l AMH. The ovarian organ index was 0.011 ± 0.005, and the HE staining results showed all levels of follicles, suggesting that senile ovaries occur in old macaques. (2) P4 generation BMMSCs presented a typical cell morphology, staining positive for Oil Red O, Alizarin Red, and Alcian Blue. The positive rates of CD29, CD34, CD90, and CD105 on the cell surface were 98, 0.98, 98.8, and 99.8%, respectively, in line with mesenchymal stem cell standards. (3) The PET-CT results showed that the ovarian volume in the elderly treatment group increased, the lesions decreased, and the metabolism was vigorous. (4) The level of sex hormone secretion generally recovered to the level of the follicular phase for the 3rd, 6th and 8th months after the treatment of BMMSCs. (5) The HE results showed that all levels of follicles were observed in the young control group, and the medulla and stroma were neatly arranged, whereas primitive, primary, secondary, and atretic follicles were observed in the elderly treatment group. In addition, the medulla and stroma had obvious boundaries with a small amount of calcium nodules in the young control group. while those in the elderly control group had essentially no follicular structure, with only atretic follicles were seen locally that were filled with connective tissue. Masson staining results showed that the proportion of collagen fibres was 10.61 ± 1.83% in the young control group, 56.79 ± 3.58% in the elderly control group, and 23.71 ± 2.4% in the elderly treatment group. TUNEL staining results showed that cell apoptosis was 1.07 ± 0.04%, in the young control group was, 25.93 + 2.49% in the old control group, and 6.98 + 1.35% in the old treatment group. The immunohistochemistry results showed 114 ± 17, 73 ± 6, and 118 ± 18 blood vessels in the young control group, the elderly control group, and the elderly treatment group, respectively. Immunofluorescence staining showed a lack of expression of enhanced green fluorescence protein (E-GFP) in BMMSCs without from the old control group, while green fluorescence was observed in the old treatment group. (6) After the treatment of BMMSCs, 1258 genes were identified as being differentially expressed. The 3D-PCA trace showed that the ovaries of the macaques in the elderly treatment group shifted to that observed in the young group. The genes that were upregulated with age were downregulated after the stem cell treatment. Genes that are downregulated with age were upregulated after stem cell therapy, and the top 20 PPI network genes were enriched for the progesterone-mediated maturation of follicles, oocytes, and cell cycle categories.
Conclusions
BMMSCs derived from juvenile macaques can reverse ovarian aging in elderly macaques