C57 mice aged 10–12 months were used as natural ageing model mice (old group, n = 12), and C57 mice aged 4–5 months were used as controls (young group, n = 6). Compared with that of the young group, the reproductive function of the mice in the old group was significantly reduced, as evidenced by the following: (i) the number of follicles was significantly reduced (62.3 follicles per ovary vs. 32.7 follicles per ovary, p < 0.05, Fig. 2A), as shown and quantified by H&E staining of the ovaries; (ii) the proportion of proliferating cells in the ovaries was significantly reduced, as shown by immunohistochemistry (from 24.4 to 12.0%, Fig. 2B); and (iii) the levels of oestradiol (E2) and follicle-stimulating hormone (FSH) in serum were measured by ELISAs, which showed a significant decrease in E2 (from 655.0 pg/ml to 410.3 pg/ml, p < 0.05) and elevation in FSH (from 8.6 pg/ml to 28.4 pg/ml, p < 0.05) secretion (Fig. 2C).

Establishment of a natural ageing mouse model and evaluation of the therapeutic effects of MSCs on ovarian function. A The total number of follicles in the ovaries of the old and young groups. B The proportion of proliferating cells in the ovaries of the old and young groups. C Serum levels of oestradiol (E2) and follicle-stimulating hormone (FSH) in the old and young groups. D Representative images of vaginal smear staining. E Representative line charts of oestrous cycles in the old UC-MSC, old AD-MSC, old, old-saline and young groups. F The percent of time spent in the proestrus, estrus and metestrus/diestrus phases among the five groups. G The percent of proliferative cells based on Ki67 immunohistochemical staining of ovarian sections over 1 and 3 weeks after MSC transplantation. H Representative pathological images of Ki67 immunohistochemical staining of ovarian sections in each group over 1 week after transplantation. The error bars indicate SD. *p < 0.05

We further included the old-saline group (n = 17), old-AD-MSC group (n = 18) and old-UC-MSC group (n = 18) to observe the beneficial effects of orthotopic MSC transplantation on naturally aged mice and to compare the therapeutic effects of MSCs from different sources. After injection of 10 µl of MSC concentrate containing 3.5*105 MSCs or an equal volume of saline into each ovary, the mice were monitored for 8 consecutive days of the daily oestrous cycle. The mice from all groups were sacrificed over 1 and 3 weeks after transplantation, and their bilateral ovaries were removed for ovarian weight determination, H&E staining and immunohistochemical staining.

(1) Post-transplant oestrous cycle changes

The results revealed that UC-MSC ovarian transplantation improved the oestrous cycle and significantly shortened the metestrus/diestrus phase compared with those of the old group (from 59.4 to 41.7%, p < 0.05, Fig. 2D–F). AD-MSC ovarian transplantation also improved the oestrous cycle by decreasing the duration of the metestrus/diestrus phase (from 59.4 to 43.8%) and enhancing the duration of the estrus phase (from 28.1 to 40.3%), although the differences were not statistically significant.

(2) Post-transplant granulosa cell proliferation

The results of Ki67 immunohistochemical staining of ovarian sections suggested that over 1 week after transplantation, the proportion of proliferating cells was significantly increased in the old-AD-MSC group compared with the old and old-saline groups (23.8% vs. 12.0% and 12.9%, p < 0.05, Fig. 2G, H, Additional file 1: Figure S2), and the proportion of proliferating cells in the ovaries of the mice in the old-UC-MSC group was slightly increased (20.6%), but there was no significant difference compared with the old control group.

(3) Post-transplant ovarian weight changes

Over 3 weeks after AD-MSC transplantation, the ovarian weight (normalized by total body weight) of mice increased significantly (0.42 vs. 0.27, p < 0.05, Fig. 3A), and the ovarian weight of the mice in the UC-MSC group increased slightly (0.37), but there was no significant difference compared with that of the control group.

Therapeutic effects of MSCs on follicles and ovarian blood vessels. A Bar graph of the ovarian weight of mice in each group, showing a significant increase in ovarian weight over 3 weeks after AD-MSC transplantation. B Representative pathological images of primordial, primary, secondary and antral follicles. C The total number of follicles in each group indicated that the total follicle number did not increase significantly after MSC transplantation. D Number of primary follicles in the ovaries of mice showing a significant increase after UC-MSC and AD-MSC transplantation. E Proportion of follicles in each group 1 week after transplantation. F Proportion of follicles in each group 3 weeks after transplantation. G Representative histochemical staining images of Cd31 in each group and the corresponding bar graph in mouse ovaries over 3 weeks after transplantation. H Representative histochemical staining images of Vegf in each group and the corresponding bar graph in mouse ovaries over 3 weeks after transplantation. The error bars indicate SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

(4) Post-transplant follicle count

The results of H&E staining of ovarian sections showed that the total number of follicles in the mouse ovaries did not increase significantly after MSC ovarian transplantation (Fig. 3B, C, Additional file 1: Figure S3). The follicles at each stage were observed and counted, and the results showed that the number of primary follicles increased significantly after UC-MSC and AD-MSC transplantation (Fig. 3D). Over 1 week and 3 weeks after transplantation, the number of primary follicles was significantly increased in the old-AD-MSC group compared with the old group (from 7.8 to 14.1 on W1, from 7.5 to 13.7 on W3, p < 0.05), and over 3 weeks after transplantation, the number of primary follicles was significantly increased from 7.3 to 13.4 in the old-UC-MSC group compared with the old-saline group (p < 0.05). Subsequent calculation of the proportion of follicles at each stage showed a slight increase in the proportion of primary follicles in the old-AD-MSC group over 1 week after transplantation (Fig. 3E), and over 3 weeks after transplantation, the proportion of primary follicles in mouse ovaries was significantly increased in both the old-UC-MSC (35.4% vs. 22.4% and 20.2%, p < 0.05) and old-AD-MSC (31.1% vs. 20.2%, p < 0.05) groups (Fig. 3F).

(5) Post-transplant angiogenesis

Histochemical staining of Cd31 and Vegf in the mouse ovaries of both the old-AD-MSC and old-UC-MSC groups indicated a significant increase in angiogenesis in the ovaries of mice 3 weeks after MSC transplantation (p < 0.05, Fig. 3G, H). Among them, the increased levels of Cd31 (p < 0.001) and Vegf (p < 0.0001) in the ovaries of the old-AD-MSC group were significantly greater than those of the old-UC-MSC group.

To further evaluate the clinical utility of UC-MSCs and AD-MSCs, we conducted safety assessments on UC-MSCs and AD-MSCs, including tumorigenicity tests, acute toxicity tests, immunogenicity studies and biodistribution studies (tracking MSC migration in vivo).

(1) Tumorigenicity test

As shown in Fig. 4A, B, we conducted a comparative experiment using BALB/c nude mice aged 4–6 weeks, which were divided into three groups: a positive control group (injected with ES-2 cells, n = 8), an AD-MSC group (injected with AD-MSCs at passage 10, n = 20), and a UC-MSC group (injected with UC-MSCs at passage 10, n = 20). The positive control group was sacrificed on Days 18–20 after transplantation based on tumour size, while the MSC groups were sacrificed after 16 weeks. Tumorigenicity was evaluated using two methods: tumour nodules at the injection site and H&E staining of pathological sections after sampling.

Tumorigenicity test of orthotopic transplantation of MSCs. A Flowchart overview of the establishment of an orthotopic xenograft tumour model in mice. B Flowchart overview of the evaluation of the tumorigenic potential of MSC transplantation. C Photographs and pathological section images of xenograft tumours established in situ. D Representative image of mice in the ES-2 injection group. E Photographs of ovaries in the AD-MSC injection group. F Photographs of ovaries in the UC-MSC injection group. G Representative pathological images of ovaries and other organs in the MSC transplantation group

The results showed that all samples from the 8 mice in the positive control group became tumorigenic after bilateral ovarian transplantation of ES-2 cells (tumorigenic rate: 100%, Fig. 4C, D), of which one died on Day 16 after transplantation (postmortem diagnosis showed that it was caused by malignant ascites). No tumorigenesis was observed either in the AD-MSC group or in the UC-MSC group after 16 weeks (tumorigenic rate: 0%, Fig. 4E, F). H&E staining of ovary sections confirmed the above results (Additional file 1: Figure S4). Additionally, H&E staining of mouse organs (intestine, uterus, spleen, kidney, liver, and lung) showed that mice in both the AD-MSC and UC-MSC groups were healthy with no tumorigenesis (Fig. 4G). During the follow-up process, no obvious abnormalities in the body weight, appearance, behaviour, or health status of mice were found in the AD-MSC group or the UC-MSC group.

In summary, the tumorigenicity was 100% (8/8) when ovarian cancer cells were injected into the ovaries of BALB/c nude mice, confirming that BALB/c nude mice are highly susceptible to ovarian cancer cells. When MSCs were injected into the ovaries of BALB/c nude mice, tumorigenicity was 0%. Therefore, AD-MSCs and UC-MSCs were not tumorigenic when orthotopically transplanted into the ovaries of BALB/c nude mice (p < 0.0001).

(2) Acute toxicity test

For the acute toxicity test, 4-week-old C57BL/6 mice were used and divided into a normal control group (n = 6) and a UC-MSC transplantation group (n = 12). The mice were weighed on the day after the initiation of the experiment, sequentially on Day 7 and Day 14, and each organ sampling was completed on Day 14. Whether the mice possessed acute toxic reactions to UC-MSC transplantation in vivo was determined by observing general indices (appearance, behaviour, mental status, etc.), mortality, and weight changes and anatomical and pathological examination of each organ during the experimental period. The results indicated that both groups of mice survived and were in good mental condition, with no significant abnormalities observed in their appearance or behaviour. In terms of weight changes, the mice in both groups showed a tendency to increase in weight, and the mean weight fluctuated from 16.9–18.7 g in the control group to 16.3–18.2 g in the treatment group, with no significant difference between the groups (p > 0.05, Additional file 1: Figure S5A). Histological anatomy (volume, colour, texture) and pathological findings of each organ did not show significant abnormalities in either group (Additional file 1: Figure S5B).

In summary, the results showed that after the transplantation of UC-MSCs, the mice were in good general condition without any accidental death or significant abnormalities, and there were no significant differences in their body weight growth compared with the control group. Therefore, it can be concluded that there was no significant acute toxic reaction after UC-MSC transplantation into mice. Similarly, the mice in the AD-MSC group were generally in good condition without any accidental deaths, and no significant abnormalities were found in the histological and pathological examinations, which confirmed that AD-MSCs had no acute toxic reactions.

(3) Immunogenicity study

For the immunogenicity study, 9-month-old C57BL/6 mice were used and divided into three groups: the sham group (injected with PBS, n = 6), AD-MSC transplantation group (injected with AD-MSCs, n = 6) and UC-MSC transplantation group (injected with UC-MSCs, n = 6). Tail tip blood was collected on Days 1, 3 and 6 after injection. Mice were sacrificed on Day 6, and the material was harvested. The immunogenicity of MSCs was determined by measuring the blood routine on Days 1, 3 and 6 and the concentration of immune molecules (IL-10, TNF-α, IFN-γ) in the serum of mice on Day 6. The results showed that there were no differences in the white blood cell (WBC), lymphocyte (Lym), eosinophil (Eos) and basal (basophil) counts among the groups. On Day 6, a difference in the content of monocytes (Mon) appeared between the UC-MSC group and sham group, with the UC-MSC group having higher levels than the sham group (0.35*109/L vs. 0.74*109/L, p < 0.05) (Fig. 5A). In terms of immune molecules, there were no significant differences in serum TNF-α, IL-10 and IFN-γ concentrations among the groups (Fig. 5B).

Immunogenicity study and biodistribution study of MSC transplantation. A The number of WBC (white blood cells), Neu (neutrophils), Lym (lymphocytes), Mon (monocytes), Eos (eosinophils) and Bas (basophils) in the mouse serum on Day 1, Day 3 and Day 6. B The concentration of immune molecules (IL-10, TNF-α, IFN-γ) in the serum of mice on Day 6. C Images of UC-MSCs under bright field (top) and green fluorescence (bottom) after PKH67 fluorescent dye labelling. D Representative images of ovary, uterus and spleen sections showing UC-MSC distribution on Day 1 and Day 7 after injection. E Images of AD-MSCs under bright field (top) and green fluorescence (bottom) after PKH67 fluorescent dye labelling. F Representative images of ovary, uterus and spleen sections showing AD-MSC distribution on Day 1 and Day 7 after injection. The error bars indicate SD. *p < 0.05

In summary, the data revealed that the number of immune cells did not significantly increase in the majority of mice after MSC transplantation, and there were no significant increases in the expression of immune molecules in mice after MSC transplantation, thus indicating the low immunogenicity of both AD-MSCs and UC-MSCs.

(4) Biodistribution study

To assess MSC migration in mice, we selected 8-week-old C57BL/6 mice, which were orthotopically injected with PKH67 fluorescent dye-labelled UC-MSCs (n = 6) or AD-MSCs (n = 6). Three mice from each group were sacrificed on Day 1, and the remaining three were sacrificed on Day 7 post-transplantation. Tissue sections were prepared from each organ to observe the distribution of MSCs in mice after transplantation. Our results showed that on Day 1, PKH67-labelled MSCs were mainly detected in the ovaries, with a small amount found in the uterus and spleen (Fig. 5C–F). On Day 7, MSCs were still mainly concentrated in the ovaries, with a small amount found in the uterus but no signal detected in the spleen. PKH67-labelled MSCs were not detected in any other organs (brain, intestine, heart, kidney, liver, lung, stomach) on either Day 1 or Day 7 (Fig. 5D, F, Additional file 1: Figure S6).

Overall, the results of the biodistribution study revealed that fluorescence was predominantly enriched in the ovaries, with lower levels observed in the uterus and spleen, indicating a low degree of nondeterministic distribution of MSCs. Taken together, the results suggest that both AD-MSCs and UC-MSCs possess high safety profiles when transplanted into mouse ovaries.

For each sample, we sequenced an average of 44.5 million raw reads (ranging from 40.7 to 49.4 million reads), with mapping rates of approximately 95.2% (ranging from 94.9 to 95.5%, sequencing quality statistics in Additional file 2: Table 1). An average of 34,921 genes were detected. We compared the upregulated genes in each group, and 34 shared genes were discovered (Fig. 6A, Additional file 2: Table 2). Enrichment analysis of these genes revealed that the regulation of the MAPK cascade was enhanced after UC-MSC and AD-MSC transplantation, whether for a short-term (1 week) or long-term (3 weeks) period (Fig. 6B), with a significant increase in Map4k1 expression (Fig. 6C). Protein‒protein interaction network and MCODE component analyses were further performed, and a network mainly composed of Cd3e, Cd247, Map4k1, Zap70 and Itk was identified (Fig. 6D) and supported by immunohistochemical experiments (Additional file 1: Figure S7). These genes might play an indelible role in improving ovarian function after MSC transplantation. Next, unique upregulated genes in each group were analysed to identify different mechanisms of UC-MSC and AD-MSC treatment on ovarian ageing in mice (Fig. 6E, F). Over 3 weeks after AD-MSC transplantation, 473 unique upregulated genes were discovered, while only 135 unique upregulated genes were discovered for the UC-MSC transplantation group after 3 weeks. This finding suggested that after AD-MSC transplantation, changes in ovarian function were more intense, with enrichment in cell‒cell adhesion and positive regulation of the immune response. Moreover, short-term effects were mainly concentrated in kinase activity, GPCR signalling, chemokine signalling and the Wnt signalling pathway, while long-term effects were enriched in the activation of immune function. This result suggested differences in the mechanism of short-term and long-term effects.

Transcriptome changes in mouse ovaries after MSC transplantation. A The number of upregulated genes in each group after MSC transplantation. Among them, 34 genes were shared among the groups. B Bar graph of the top 12 enriched terms across the 34 shared genes, coloured by p values. Network of enriched terms (left) coloured by cluster ID, where nodes that share the same cluster ID are typically close to each other. C Relative expression levels of Map4k1, a key gene in the MAPK pathway, in each group. The error bars indicate SD. *p < 0.05. D Protein‒protein interaction network and MCODE components identified through analysis of the 34 shared genes. E Heatmap of DEGs in each group. F Bubble graphs of enriched terms across unique up-regulated genes in each group. Old-UC-MSC-W1 versus old-saline-W1 (top left, 337 genes), old-AD-MSC-W3 versus old-saline-W3 (bottom left, 473 genes), old-UC-MSC-W3 versus old-saline-W3 (top right, 135 genes), old-AD-MSC-W1 versus old-saline-W1 (bottom right, 145 genes)