Transplantation of menstrual blood-derived mesenchymal stem cells (MbMSCs) promotes the regeneration of mechanical injuried endometrium

Abstract

Purpose: The ultimate cause of intrauterine adhesions (IUAs) is the substantial destruction of the endometrium, which makes the regeneration of endometrium difficult. The purpose of this study was to observe menstrual blood-derived mesenchymal stem cells (MbMSCs)'s effect on the endometrial regeneration with different methods of transplantation. We also studied whether MbMSCs transfected with the FGF2 gene can improve the regenerative effect.

Methods: 75 female SD rats with endometrium removed were used as IUA models. These IUA models were divided into 5 groups: group A was the IUA control, group B received a scaffold transplant, group C received a scaffold+MbMSC transplant, group D received a scaffold+FGF2-MbMSC transplant, and group E received FGF2-MbMSCs injected via the tail vein. After the intervention, 5 rats from each group were sacrificed on the 7^th day and the 28^th day respectively. The distribution of MbMSCs in endometrium was traced using enhanced green fluorescent protein. The endometrial morphology, number of endometrial glands, area of endometrial fibrosis and immunohistochemistry (IHC) of Ki67, VEGF, and CD31 were evaluated. In addition, the fertility of all groups was tested.

Results: On the 7^th day after transplantation, enhanced green fluorescent protein showed that there were more MbMSCs in the uterine cavity of group D than that of group E. The endometrial morphology of groups A and B was atrophic and thinned with a high proportion of fibrosis in the endometrium. The endometrium of groups C, D and E was thickened, contained more glands, exhibited reduced fibrosis, and had increased expression of Ki67, VEGF and CD31. The endometrial regenerative effect from high to low was D > C > E with significant differences between each two groups. The fertility test verified the regenerative effect.

Conclusions: These results suggest that the injection of MbMSCs into the tail vein was an effective way to stimulate endometrial regeneration, but the effect was not so well as the intrauterine transplant of MbMSCs with scaffold. The FGF2-transfected MbMSCs exhibited enhanced regenerative effect.

Keywords: FGF2 transfect; Intrauterine adhesions (IUAs); endometrial regeneration; menstrual blood-derived mesenchymal stem cells (MbMSCs); scaffold.

Figure 1

The micro view of IUA rat model. A. HE staining of rat uterine after 7 days; the IUA uterine cavity disappeared and gland quantity decreased sharply. Neutrophil cells and red blood cells also filled in IUA endometrium. Scale bar = 200 µm. B. HE staining of rat uterine after 7 days at high magnification. Scale bar = 100 µm. C. Massion staining of rat uterine after 7 days. Scale bar = 200 µm. D. Massion staining of rat uterine after 7 days at high magnification. Scale bar = 100 µm. E. Gland quantity decreased significantly in the 7 d and 28 d IUA group compared with that of normal groups, P < 0.01. F. Endometrial thickness decreased significantly in the 7 d and 28 d IUA group compared with that of normal groups, P < 0.01. G. Area of endometrial fibrosis increased significantly in the 7 d and 28 d IUA group compared with that of normal groups, P < 0.01.

Figure 2

A. Shows the moment after transplantation of MbMSCs+scaffold to the IUA model rat and suturing of uterine. B. Shows the 7^th day after MbMSCs+scaffold transplantation: the scaffold can be seen after the incision of uterus. C. Shows 28^th days after MbMSCs+scaffold transplantation: the scaffold was mostly been absorbed and can’t be easily seen.

Figure 3

(A and B) show the expression of enhanced green fluorescent protein of FGF2 gene transfecten to P3-MbMSCs under the fluorescence microscope: (A) shows the fluorescence expression of MbMSCs was weak on the 7^th day after transfection. (B) shows that P4 FGF2 MbMSCs which were passaged from transfected P3-MbMSCs. P4 FGF2 MbMSCs had returned to spindle shape, well grown and have high fluorescence expression. (C and D were P5 FGF2-MbMSCs, which were no different from that of non transfected MbMSCs (C was 40 times and (D) was 100 times magnification respectively).

Figure 4

The electrophoretic map of PCR TM4-TOPO-FGF2 recombinant plasmid was as above: 1: negative control (blank vector- control group); 2: negative control (ddH2O); 3: positive control (GAPDH); 4: pseudopositive particle enzymolysis marker; 5-10: FGF2 enzymolysis positive clone plasmid 1-6. The figure right shows the expression of FGF2 mRNA in FGF2-MbMSCs infected with different MOI was significantly higher than that of the control group.

Figure 5

The expression of FGF2 in the culture medium of the MbMSCs was significantly increased 6 d and 11 d after transfection (*P < 0.001) when compared with the blank transfection group and the MbMSC control group. This suggests that the FGF2 gene integrated into the MbMSCs can be transferred to the progeny and stably expressed. There was no significant difference between the blank transfection group and the MbMSC control group. The expression of FGF2 in the culture medium at 11th d after transfection was higher than the expression level 6th d (all P < 0.05).

Figure 6

As shown in the figure, enhanced green fluorescent protein traced the distribution of MbMSCs in endometrial tissue (7 days after treatment). (A) shows FGF2-MbMSCs directly injected into uterine cavity through scaffold and (B) shows FGF2-MbMSCs injected into tail vein. It can be seen that FGF2-MbMSCs directly injected into the uterine cavity with scaffold are more and brighter compared with the cells injected into the tail vein. (C) shows the number of GFP cells of scaffold+FGF2-MbMSCs group is more than scaffold+MbMSCs group, which is more thanFGF2-MbMSCs-V group both on 7^th day and 28^th day.

Figure 7

The Uterine contour picture of 5 groups at 7 and 28 days after MbMSCs transplantation. The upper row were pictures at 7^th day and the lower row were pictures at 28^th day. On 7^th days after transplantation, group A which is control group had hydrous dilatation in part of the uterine cavity and atrophy in some other part of the uterine cavity. There was no significant difference in groups B, C, and D with the inspection of the naked eye, while Group E had edema. On the 28^th day after transplantation, group A showed that part of the uterine cavity was more dilated by hydronephrosis, and some part atrophied more obviously. Hydronephrosis was the most serious. In group B. Hydronephrosis seemed to be alleviated in group C. The appearance of the uterus was nearly recovered in group D which is the treated by FGF2 transfected MbMSCs+scaffold. In Group E which is treated by injecting of FGF2 transfected MbMSCs into the tail vein, the appearance of the uterus was kind of recovered, and there was hydronephrosis in part of the uterine cavity.

Figure 8

The endometrial morphology after MbMSCs treatment: The upper row including (A) from IUA control group and (B/C) from the scaffold control group shows endometrial atrophy, thinning, high proportion of fibrosis. The lower row including (D-F) show endometrial thickening, gland regeneration, and reduced proportion of fibrosis in the FGF2 transfected MbMSCs group. (G) Shows the fibrotic area of endometrium decreased in the scaffold+MbMSCs and scaffold+FGF2-MbMSCs group. (H) Shows the endometrial thickness increased in the scaffold+MbMSCs and scaffold+FGF2-MbMSCs group with P < 0.01.

Figure 9

Representative immunostaining of CD31, VEGF and Ki67 positive cells of the endometrium on the 7^th day and 28^th day after treatment. Scale bar = 20 µm. A: There was a significant difference expression of CD31 in endometrium among the five groups after treatment (P < 0.001). The expression of MVD in the endometrium of all groups on the 28^th day after treatment was higher than that on the 7^th day with was statistically significant difference (P < 0.001), which means the regeneration could be maintained after 7 days. On the 7^th day after treatment, MVD was highest expressed in scaffold+MbMSCs group, then scaffold+FGF2-MbMSCs group and then FGF2-MbMSCs-V group. There was no significant statistical difference between control group and scaffold group (P = 0.136), There was significant difference between the other groups (all P < 0.001). MVD was IUA control group < Scaffold group < FGF2-MbMSCs-V < scaffold+MbMSCs < scaffold+FGF2-MbMSCs from low to high 28 days after treatment, and there was significant statistical difference between each two groups (all P < 0.001). B: There was no significant difference in the expression of VEGF between 7^th day and 28^th day in control group and scaffold group. In the other 3 groups, the expression of VEGF in the endometrium on the 28^th day after treatment was lower than that on the 7^th day with statistically significant difference (all P < 0.001). There were significant differences among the five groups (P < 0.001). The results showed that the expression of group ABECD increased in turn, and the difference between each two was statistically significant (all P < 0.001). On the 28^th day, the highest expression was found in scaffold+FGF2-MbMSCs group, which was statistically more than other groups (P < 0.001). C: On the 7^th day after MbMSCs treatment, there was significant difference among the 5 groups (P < 0.001). Control group was the lowest in expression but there was no significant difference between control group and scaffold group (P = 0.180). The other 3 groups were all significantly higher than group scaffold group (all P < 0.001). The expression from low to high was FGF2-MbMSCs-V < scaffold+MbMSCs < scaffold+FGF2-MbMSCs with significant difference between each two groups (all P < 0.001). On the 28^th after MbMSCs treatment, there was significant difference among the 5 groups (P < 0.001). The expression from low to high was control group < Scaffold group < FGF2-MbMSCs-V < scaffold+MbMSCs < scaffold+FGF2-MbMSCs with significant difference between each two groups.

Figure 10

This figure shows the fertility rate and the number of gestational sac of 5 groups. There is no difference between control group and scaffold group. The other 3 groups had a higher number of gestational sac and fertility rate than control group and scaffold group. The number of gestational sac from high to low was scaffold+FGF2-MbMSCs > scaffold+MbMSCs > FGF2-MbMSCs-V. (*P < 0.05, **P < 0.01).