The BMMSCs Derived From Juvenile Macaques Have the Ability to Promote Thymus Regeneration in Aged Macaques

The thymus gland is an important central immune organ in the human body and plays an indispensable role in the immune system. Aging thymic atrophy is critical factors leading to immune function decline and senile debilitation in aged people. Immune function decline is one of the important mechanisms of aging. Some research reports indicated that Stem cell therapy have the ability to promote tissue regeneration(cid:0)but reverse thymus atrophy or promote atrophy thymus regenerate by stem cells need to be con�rmed further.


Introduction
The world is undergoing a rapid transition to aging [1] .Aging is a ubiquitous phenomenon in the biological world, which leads to the degeneration or loss of a series of functions at the level of tissues and organs [2] .An important cause of aging is a series of functional and structural changes of the immune system, and the thymus is an important central immune organ of the human body.The thymus is the site of T cell differentiation, development and maturation, and determines the effectiveness of T cell immune response [3] .With the growth of age, the thymus gland will atrophy and degenerate, leading to the decline of the body's ability to ght infection, increasing the incidence of tumors and the decline of autoimmunity.Therefore, it is very necessary to study how to improve and delay the aging -related thymic degeneration changes [4] .
In recent years, many studies have shown that the treatment options for delaying thymus senescence include cell adoptive therapy, gene recombination therapy [5] , activated signaling pathway and bioengineering therapy [6] .However, these methods have certain risks and challenges.
Mesenchymal stem cells (MSCs) are a kind of cells with multidirectional differentiation potential and selfrenewal ability.Based on cell therapy, bone marrow mesenchymal stem cells have become a popular trend in cell regeneration therapy, due to their biological characteristics such as easy access, easy ampli cation, low immunogenicity, anti-in ammatory and immunomodulatory function [7] .Many clinical and basic studies have shown that bone marrow mesenchymal stem cells are effective in the treatment of thymus senescence and may be a new method to delay or reconstruct thymus senescence [8,9] .Rhesus macaques are non-human primates, and their genome sequencing is as high as 93% similar to human genomes [10] .The research results have more reference value than other animals, and have a high degree of homology with human genetic material.
At present, most of the researches are based on rodent models, and few researches on non-human primates.At the same time, the mechanism of bone marrow mesenchymal stem cell transplantation on the improvement of thymus function in aging macaque monkeys is still unclear, and its therapeutic effect is worthy of further exploration.

Experimental animal
Kunming Institute of Zoology, Chinese Academy of Sciences [2019-032 (Section)-01] provided 5 healthy female juvenile macaques, with an average age of 3 years and body weight of 2.0-3.0 kg;10 healthy elderly female macaques with an average age of 25 years weighing 4.0-5.0kg.All the macaques are stored in the animal room of the 920th Hospital of the PLA Joint Logistics Support Force.All the aged macaques showed senescence phenotype, dark hair color, poor mobility, obvious brain atrophy, pulmonary brosis, thymus and ovary atrophy.All animal studies were approved by the Laboratory Animal Ethics Committee of the supporting unit, animal certi cate number :SYXK (Military) 2012-0039.

Experimental protocols
Preparation and identi cation of BMMSCs from young macaques Bone marrow of healthy young female macaques with an average age of 3 years was collected by bone marrow puncture under aseptic conditions.BMMSCs were isolated and puri ed by differential attachment method and passage culture method.BMMSCs were obtained by four passage ampli cation times.The morphology and growth status of BMSCs were observed under inverted phase contrast microscope.The positive expression rates of CD29, CD34, CD90 and CD105 cells were detected by ow cytometry.BMMSCs were induced to differentiate into adipoblast, osteoblastic and chondroblast cells in vitro, and the differentiation ability was detected.

Animal grouping and BMMSCs transplantation treatment of macaque
The 15 rhesus macaques had no abnormal changes after 1 week of normal feeding and were randomly divided into groups, namely 5 in the juvenile group (n=5), 4 in the elderly group (n=4) and 6 in the elderly treatment group (n=6).In the elderly treatment group, BMMSCs were transplanted via femoral vein at a dose of 1×10 7 cells/kg, once every other day, for 3 consecutive infusions; the juvenile group and the elderly group were infused with equal volume of normal saline at the same time.Routine feeding was carried out, and the macaques of each group were sacri ced 6 months after the last transplantation, and the materials were collected.
Imaging observation of thymus PET-CT scans were performed on macaques in the juvenile group, macaques in the elderly treatment group before treatment, 3 months after treatment in the elderly treatment group, and 6 months after treatment in the elderly treatment group.Rhesus macaques were fasted for at least 6 hours before PET-CT scan, blood glucose measured after anesthesia <8 mmol/L, intravenous injection of 18F-FDG at a dose of 0.3 mCi/kg (by body weight), and rest for 60 to 90 minutes, PET-CT scan.CT uses conventional wholebody spiral scanning, tube voltage 120kV, tube current 260 mA, pitch 0.561, rotation speed 0.5 s/cycle, layer thickness 3.75 mm, interval 3.75 mm, matrix 512 × 512, FOV 50 cm × 50 cm; subsequent PET scan , Each bed is collected for 2.5 min.

Evaluation of thymus function in macaques
Periphereal blood was collected from femoral vein of macaques before treatment and 30, 60 and 90 days after treatment.Flow cytometry was used to detect the output levels of T cells and regulatory T cells in peripheral blood of rhesus monkeys, and ELISA was used to analyze the levels of simian thymosin α and simian thymosin in peripheral blood of rhesus macaques.
Evaluation of thymus tissue structure in macaques 3% pentobarbital sodium (1kg/ml) was used to anesthetize and kill the macaque monkeys in each group, and the thymus tissue was obtained.After the thymus tissue was removed, the thymus tissue was xed with 4% PFA solution for 24h.The thymus tissue was dehydrated and para n-embedded in sections with a thickness of 4μm.He staining was used to observe the changes of thymus tissue structure, immuno uorescence staining was used to observe the changes of thymus cortex and medulla structure, Masson staining was used to observe the changes of collagen ber deposition , TUNEL staining was used to observe the apoptosis of thymus cells and gene expression was observed by immunohistochemistry staining .

Transcriptomics sequencing analysis of thymus tissue gene
For sample collection and preparation, rst perform RNA extraction and detection, and secondly perform library construction and quality inspection.After the library is constructed, use Qubit2.0Fluorometer for preliminary quanti cation, dilute the library to 1.5ng/ul, and then use agilent 2100 bioanalyzer the insert size of the library is tested.After the insert size meets the expectation, qRT-PCR will accurately quantify the effective concentration of the library (the effective concentration of the library is higher than 2nM) to ensure the quality of the library and perform online sequencing.The main links of data analysis include data accusation, sequence alignment to reference genome, new transcript prediction, gene expression level quanti cation and differential expression analysis.Use DESeq2R software (1.16.1) to perform differential expression analysis between the two comparison sets (Two biological replicates per group).Through DESeq2, genes with adjusted P values <0.05 were assigned as differentially expressed genes.The corrected P value and |log2foldchange| are used as a threshold for signi cant differential expression, and a linear model is used to screen out aging-related genes.

Statistical analysis
All data are analyzed by SPSS 21.0 statistical analysis software.The statistical results are expressed as `x±s.The statistical comparison of differences between groups is performed by one-way ANOVA, P <0.05 is considered signi cant.

Preparation and identi cation of juvenile BMMSCs
Primary BMMSCs showed colony growth, and the cells were evenly dispersed on the bottom of the bottle.When the cell fusion rate reaches 80%~90%, it will be subcultured and expanded.With the subculture, the P4 generation cells began to become uniform in morphology, with less impurities, and grew in a long spindle shape, broblast-like growth, high cell density, growth in a whirlpool shape, and good growth status (Fig. 1A).
BMMSCS obtained by differential adherence method and subculture method are long spindle-shaped and broblast-like; after in vitro lipogenic induction and staining with Oil Red O, the cell morphology becomes round and red-stained lipid droplets can be seen in the cell.Rhesus monkey BMMSCs were induced in vitro to form osteoblasts.After staining with alizarin red, a large number of obvious dark red calcareous nodules were seen; Rhesus monkey BMMSCs were induced to form cartilage in vitro and stained with acian blue, the morphological changes were obvious, and the proteoglycan synthesis in cartilage tissue , visible blue (Fig. 1C).

Thymus formal and functional changes after MSC treated by PET-CT imaging observation
The PET-CT of the thymus in the juvenile group and the elderly treatment group showed that the thymus position was normal.The density of the thymus in the juvenile group was as high as 63HU, and SUVmax>1; in the elderly group, most of the thymus was replaced by adipose tissue before treatment, only a few ne ber cord-like structures were seen, the density was the same as the adipose tissue, PET SUVmax<1; March and June after treatment The density of thymus tissue gradually increased, the CT value gradually increased, reaching 68HU, PET SUVmax>1 Fig. 2;Table1 .
Changes in peripheral blood T cell and regulatory T cell output levels By ow cytometry, the percentage of CD3 + T cells in the peripheral blood of macaques in the elderly treatment group increased at rst and then decreased, and increased signi cantly at 30 d and 60 d compared with before treatment (P 0.05), but there was no signi cant difference at 90 d (P 0.05)(Fig.3A);The percentage of CD3 + CD4 + T cells in peripheral blood increased slowly, and there was no signi cant difference between before treatment and 30,60,90 days after treatment (P 0.05);The percentage of CD3 + CD8 + T cells in peripheral blood increased rst, then decreased and then increased (Fig. 3B).
The percentage of regulatory T cells in peripheral blood showed a decreasing trend, and there was signi cant difference between before and after 30, 60 and 90 days of treatment (P 0.05) (Fig. 3C).
Changes of thymosin α and simian thymosin in peripheral blood of macaques The levels of thymosin α were rstly increased and then decreased after BMMSCs infusion, and were signi cantly increased after 30 days of treatment compared with before treatment (P 0.01);After 60 d and 90 d of treatment, there was no signi cant difference (P 0.05) (Fig. 3d).
The levels of thymopoietin in macaques increased at rst and then decreased, and increased at 30 and 60 days after treatment compared with before treatment, and decreased at 90 days after treatment, but there was no signi cant difference (P 0.05) (Fig. 3e).

Thymus tissue structural regeneration
The juvenile group (Fig. 2-A) showed that the thymus tissue envelope was relatively intact, with a clear junction between the cortex and the medulla, the parenchymal area of the thymus is large and full.In the elderly group of macaques, the thymus gland was signi cantly atrophied and degenerated, the skin and medulla junction were blurred and the cortical area was lled with adipose tissue(Fig.5A).In the treatment of BMMSCs, the elderly treatment group(Fig.5A) increased the parenchymal area of the thymus compared with the elderly group, the structure of the cortex and medulla area improved and clear boundaries began to appear, the thymocytes increased, and the fat tissue lling decreased.
By immuno uorescence staining (Fig. 5B), the cortex and medulla junction structure gradually appeared in the elderly treatment group, and the thymus structure was dense.The expression of CK5 was highest in the juvenile group and lowest in the elderly group.The expression in the elderly treatment group was signi cantly higher than that in the elderly group (P 0.05); the expression of CK8 was the highest in the juvenile group and the lowest in the elderly group.The expression in the elderly treatment group was signi cantly higher than that in the elderly group (P<0.01)The expression of CK5+CK8 was highest in the juvenile group, and the lowest in the elderly group.The expression of CK5 in the elderly treatment group was signi cantly higher than that in the elderly group (P<0.05), and the expression of CK8 showed an upward trend compared with the elderly group.
Masson staining of tissue sections, the juvenile group are arranged neatly and densely, the collagen bers are blue, the collagen deposition area is small, and the degree of brosis is low(Fig.5C); with the aging, the elderly group A large number of collagen bers appeared in the thymus tissue, the arrangement was disordered, the collagen deposition area increased, and the brosis was obvious(Fig.5C); the collagen ber deposition area of the elderly treatment group was signi cantly lower than that of the elderly group, and the degree of thymic brosis was signi cantly reduced(Fig.5C).
Tunel staining of rhesus macaque thymus tissue(Fig.5D)showed that the juvenile group had the least thymocyte apoptosis, the elderly group had the most thymocyte apoptosis, and the elderly treatment group had signi cantly fewer apoptotic cells than the elderly group.

Changes in protein expression of aging and apoptosis related genes
The expression of P21 gene was the lowest the young group and the highest in the elderly group.The elderly treatment group was signi cantly lower than the elderly group (P<0.05).The expression of P53 gene was the lowest in the young group and the highest in the elderly group.The elderly treatment group was signi cantly lower than that of the elderly group (P>0.05).Sirt1 gene expression was highest in the juvenile group and lowest in the elderly group.The elderly treatment group had a higher trend than the elderly group (P>0.05).Sirt3 gene expression in the elderly group was lower than that in the young group (P<0.05), and the elderly treatment group had a higher trend than the elderly group (P>0.05).Caspase3 and Bax gene expression was the lowest in the young group and highest in the elderly group.The elderly treatment group was signi cantly lower than the elderly group (P<0.01); the Bcl-2 gene expression was the lowest in the elderly group, and the elderly treatment group had higher expression than the elderly group (P<0.05).And slightly higher than the juvenile group(Fig.6A).

Changes of gene Transcription felies after MSC treated
Using linear model analysis, a of 312 differentially expressed genes related to thymic tissue aging were detected based on P<0.05, among which 305 genes were up-regulated with age.7 genes were downregulated with age.Based on thymic aging-related genes, 3D PCA analysis was performed on the expression matrix results of thymus tissues of various ages, and it was found that the overall transcriptome characteristics of the elderly treatment group (dark blue) had a very obvious tendency compared with the elderly group (purple).The trend of the direction(Fig.7A) .At the same time, cluster analysis was used to discover the changes in thymic senescence-related gene expression before and after BMSC transplantation (Fig. 7B).And through trajectory analysis, we found the trajectory of thymic agingrelated genes(Fig.7C and D).The results showed that, compared with the elderly group, the thymus expression characteristics after BMSC transplantation tended to be the expression characteristics of the young rhesus monkey thymus.The GO enrichment analysis of genes that were up-regulated with aging and decreased after treatment, according to the -log P value corresponding to each item, sorted from large to small to get a total of 17 items (Fig. 7C).Through GO enrichment analysis, we found that genes that are up-regulated with aging and decreased after treatment are mainly enriched in cytokine-cytokine receptor interaction (Cytokine-cytokine receptor interaction), nephron tubule formation (Nephron tubule formation), and synaptic signaling (Synaptic signaling), positive regulation of ion transport, regulation of osteoclast differentiation (Regulation of Osteoclast Differentiation) and other pathways.

Discussion
Thymus glands gradually shrink and degenerate with age, and aging and immune function degradation are inseparable.In this study, the anatomical structure and function of thymus tissue were analyzed by PET-CT imaging.We found that the light transmittance of thymus tissue increased signi cantly, and SUVmax showed a gradual increase trend.It has been reported in the literature that PET-CT can be used to track the transplantation of differentiated BMMSCs and the model of myocardial infarction to restore cardiac function [11] .Therefore, PET-CT is an important tool for tracking the anatomical structure and function of thymus tissue.
T cells are a kind of extremely active cell population derived from hematopoietic stem cells and mature in the thymus, accounting for about 60% of peripheral blood lymphocytes.According to our results, CD3 + T cells, CD3 + CD4 + T cells and CD3 + CD8 + T cells were increased in naturally aging macaque monkeys after infusion of juvenile BMMSCs compared with before treatment, suggesting that BMSCs can improve thymus output function [12] and increase thymus T cell output.Observed 90 days after the treatment of CD3 + T cells proportion is on the decline, guess may be associated with the regulation of extracellular vesicles, and no statistical differences compared with before treatment, cell function has the timeliness, not once and for all, have reference to the future clinical cell therapy, cell therapy should have certain treatment, as well as the dynamic observation.
The results of this experiment showed that after BMSC infusion, the overall level of T cells of immune cells increased, and Tregs decreased.As can be seen from the results, Tregs showed a downward trend on the 30 and 60 days after treatment, reached the lowest point on the 60th day, and showed an in ection point and an upward trend on the 90 day.We speculate that there is a direct relationship between the number of cells and the time of infusion.It has been reported that in vitro co-culture of MSC and immune cells, when the number of MSC cells exceeds a certain amount, the number of Tregs is inhibited, and when the number of MSC cells is below a certain number, the proliferation of Tregs can be promoted.
Thymus epithelial cells and their secreted thymosin and thymosin are the main components of the thymic microenvironment, which allow thymocytes to migrate and interact with each other, helping thymocytes to develop and mature, and both of them can promote the differentiation of thymocytes [13] .However, cytokines secreted by thymus epithelial cells and hormone-mediated signal transduction showed a decreasing trend during aging.The experimental results showed that thymosin α and thymosin were increased in the thymus of aging macaques after the infusion of bone marrow mesenchymal stem cells, which proved that BMSCs can improve the secretion function of thymus [14] and enhance the immune function [15,16] , and play an important role in maintaining the homeostasis of the microenvironment of thymus.
Zhan et al. [17] used human adipose-derived mesenchymal stem cells to treat thymus damage during chemotherapy and found that human adipose-derived mesenchymal stem cells expanded the area of the thymus damaged by chemotherapy and promoted the recovery of the thymus.This experiment found that after treatment with BMMSCs, compared with the elderly group, the senile rhesus macaques in the elderly treatment group increased in volume, the skin and medulla structure improved and began to appear boundaries, thymocytes increased, and adipose tissue decreased; and it was conducive to the deposition of collagen bers in the thymus tissue The improvement of thymus tissue reduces the degree of brosis.
We infer that the effect of BMMSCs on the thymus is mainly through the differentiation of thymocytes and the paracrine and immunomodulatory effects of BMMSCs [18] , which may be related to the immunomodulatory cytokines secreted by BMMSCs, such as transformation Growth factor-β, hepatocyte growth factor, vascular endothelial growth factor, broblast growth factor.thereby exerting an immunomodulatory effect in the thymus and promoting the improvement of thymus tissue.
By immuno uorescence staining, it can be found that the cortex and medulla junction structure gradually appeared in the elderly treatment group, and the thymus structure was dense.The expression of CK5, CK8 and CK5+CK8 in the thymus of macaque was highest in the juvenile group, and the lowest in the elderly group.The expression in the elderly treatment group was higher than that in the elderly group.Upward trend.We can speculate that BMMSCs can improve the effect of aging thymus and gradually transform the structure of thymus to normal structure.Zhan et al. [19] analyzed CK8 and CK5 immuno uorescence and found that human adipose-derived mesenchymal stem cells can repair the damaged thymus during chemotherapy, improve the proliferation of thymic epithelial cells, and can restore normal in the chemically damaged thymus in mice structure.
Whether it is caused by oxidative stress or telomere, it is related to the activation of transcription factors and the tumor suppressor gene P53 and its downstream gene P21 [20] .We observed that the expression of P21 and P53 gene protein was the lowest in the young group and the highest in the elderly group.The elderly treatment group was signi cantly lower than the elderly group (P<0.05).Therefore, we speculate that the reduction of P53 and P21 can promote stem cell division, accelerate cell growth, and improve thymic senescence [21] .
Sirt1 is related to multiple signal transduction pathways, and participates in neuroprotection, glucose and lipid metabolism, cell senescence and cell apoptosis and other reactions, thereby exerting gene regulation.Sirt3 is a mitochondrial deacetylase, which is abundantly expressed in the heart, brain and adipose tissue, and plays different roles in metabolic regulation, cell proliferation and gene stability [22] .Sirt1 and Sirt3 gene protein expression was highest in the juvenile group, and lowest in the elderly group.The elderly treatment group had a higher tendency than the elderly group.In this study, the expression of Sirt1 and Sirt3 increased after BMMSCs transplantation treatment.It is speculated that Sirt1 and Sirt3 promote the repair of thymic tissue in the elderly, and Sirt1 and Sirt3 play an important role in maintaining the youthful state of thymus tissue [23] .
In this study, the protein expression of apoptosis-related genes Caspase3 and Bax gene was the highest in the elderly group, signi cantly decreased after treatment in the elderly group, and the lowest in the young group.The protein that inhibits the expression of apoptosis gene Bcl-2 was the lowest in the elderly group, and the expression in the elderly treatment group increased after treatment, and was slightly higher than that in the young group.Secondly, the results of Tunel staining of thymus tissue showed that with age, thymocyte apoptosis increased, but after BMMSCs transplantation treatment, thymocyte apoptosis decreased.Therefore, it is speculated that BMMSCs may improve thymic tissue aging by reducing thymocyte apoptosis and aging [24,25] .
mRNA is a large class of RNA molecules, which transmit genetic information from DNA to ribosomes, serve as a template for protein synthesis and determine the amino acid sequence of the peptide chain of gene expression products.The detection of mRNA can indirectly re ect the expression of genes [26] .In this study, the expression matrix of thymus tissue was obtained through gene transcriptomics sequencing, and linear model analysis was used.Based on P<0.05, a total of 312 differentially expressed genes related to thymic tissue aging were detected, which increased with age 305 genes were up-regulated, and 7 genes were down-regulated with age.Based on thymic aging-related genes, 3D PCA analysis was performed on the expression matrix results of thymus tissues of various ages, and it was found that the overall transcriptome characteristics of the elderly treatment group had a very obvious trend toward the youth group compared with the elderly group.At the same time, cluster analysis was used to nd the changes in thymic senescence-related gene expression before and after BMMSCs transplantation, and the trajectory of changes in thymic senescence-related genes was found through trajectory analysis.The results showed that, compared with the elderly group, the thymus expression characteristics after BMMSCs transplantation tended to be the expression characteristics of the young rhesus monkey thymus.The GO enrichment analysis of genes that were up-regulated with aging and decreased after treatment, were sorted according to the -log P value corresponding to each item in descending order to get a total of 17 items.Through GO enrichment analysis, we found that genes that are up-regulated with aging and decreased after treatment are mainly enriched in Cytokine-cytokine receptor interaction, Nephron tubule formation, and synaptic signaling.(Synaptic signaling), Positive regulation of ion transport, Regulation of Osteoclast Differentiation, etc.In general, after BMMSC transplantation to treat senescent macaques, the transcriptome characteristics related to aging were signi cantly reversed.
In summary, juvenile BMMSCs can improve the structure and function of the thymus of senile macaques.However, in the process of improving the structure and function of the thymus, which related genes and signal pathways are involved in the regulation of BMMSCs, there are still shortcomings in this study.We will establish a more comprehensive set, systematic evaluation criteria and in-depth study of molecular mechanisms.

Conclusions
1. BMMSC transplantation can promote the reconstruction of thymus structure, improve the T cell output capacity and thymus hormone production function in aging macaques.

Figure 1 Preparation
Figure 1

Figure 2 PET
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Figure 3 Changes
Figure 3