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. 2020 Jul 13;11(1):280.
doi: 10.1186/s13287-020-01806-4.

Quantification of the CM-Dil-labeled human umbilical cord mesenchymal stem cells migrated to the dual injured uterus in SD rat

Jia-Hua Zheng  1 , Jing-Kun Zhang  1 , De-Sheng Kong  1 , Yan-Biao Song  2 , Shuang-Dan Zhao  1 , Wen-Bo Qi  1 , Ya-Nan Li  1 , Ming-le Zhang  1 , Xiang-Hua Huang  3
Affiliations

Quantification of the CM-Dil-labeled human umbilical cord mesenchymal stem cells migrated to the dual injured uterus in SD rat

Jia-Hua Zheng et al. Stem Cell Res Ther. .

Abstract

Background: Human umbilical cord mesenchymal stem cell (hUC-MSC) therapy is considered as a promising approach in the treatment of intrauterine adhesions (IUAs). Considerable researches have already detected hUC-MSCs by diverse methods. This paper aims at exploring the quantitative distribution of CM-Dil-labeled hUC-MSCs in different regions of the uterus tissue of the dual injury-induced IUAs in rats and the underlying mechanism of restoration of fertility after implantation of hUC-MSCs in the IUA model.

Methods: In this study, we investigated the quantification of the CM-Dil-labeled hUC-MSCs migrated to the dual injured uterus in Sprague Dawley rats. Additionally, we investigated the differentiation of CM-Dil-labeled hUC-MSCs. The differentiation potential of epithelial cells, vascular endothelial cells, and estrogen receptor (ER) cells were assessed by an immunofluorescence method using CK7, CD31, and ERα. The therapeutic impact of hUC-MSCs in the IUA model was assessed by hematoxylin and eosin, Masson, immunohistochemistry staining, and reproductive function test. Finally, the expression of TGF-β1/Smad3 pathway in uterine tissues was determined by qRT-PCR and Western blotting.

Results: The CM-Dil-labeled cells in the stroma region were significantly higher than those in the superficial myometrium (SM) (71.67 ± 7.98 vs. 60.92 ± 3.96, p = 0.005), in the seroma (71.67 ± 7.98 vs. 23.67 ± 8.08, p = 0.000) and in the epithelium (71.67 ± 7.98 vs. 4.17 ± 1.19, p = 0.000). From the 2nd week of treatment, hUC-MSCs began to differentiate into epithelial cells, vascular endothelial cells, and ER cells. The therapeutic group treated with hUC-MSCs exhibited a significant decrease in fibrosis (TGF-β1/Smad3) as well as a significant increase in vascularization (CD31) compared with the untreated rats.

Conclusion: Our findings suggested that the distribution of the migrated hUC-MSCs in different regions of the uterine tissue was unequal. Most cells were in the stroma and less were in the epithelium of endometrium and gland. Injected hUC-MSCs had a capacity to differentiate into epithelial cells, vascular endothelial cells, and ER cells; increase blood supply; inhibit fibration; and then restore the fertility of the IUA model.

Keywords: CM-Dil; Differentiate; Human umbilical cord mesenchymal stem cells; Intrauterine adhesions; Quantification.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Human umbilical cord mesenchymal stem cells (hUC-MSCs) characterization and CM-Dil-labeled hUC-MSCs (a–d). After 1–2 days of inoculation, the tissue masses began to adhere to the wall, and 6–7 days after inoculation, the cells could be seen climbing out of the tissue mass, mostly in fusiform shape; the cell fusion reached more than 80% in about 2 weeks, showing a long and fibrous mesenchymal-like morphology, and then following cell passage (a). The flow cytometry indicated that CD29, CD73, CD90, and CD105 were positive markers; CD34, CD45, and HLA-DR were the negative markers of hUC-MSCs at passage 3 (b). Ninety percent of CM-DiI-labeled cells were observed under a fluorescence microscope (c). The stained cells presented the same proliferation rate with hUC-MSCs at passage 3 (d)
Fig. 2
Fig. 2
In vivo CM-Dil-labeled hUC-MSC tracing. DAPI located in the nucleus (blue), hUC-MSCs located in the cell membrane and cytoplasm (red), the superposition color of red and blue was magenta representing the living cells, and dead cells were dyed red (arrow). The number of the cells was the most on the 1st week after treatment, and the survival number decreased gradually with the extension of treatment time
Fig. 3
Fig. 3
The red fluorescent cells were observed in the rat uterus regions (a–e). The uterus tissue consists of endometrium epithelium, stroma, deep myometrium (DM), superficial myometrium (SM), and serosa, from the inside to the outside (a). CM-Dil-Labeled hUC-MSCs were detected in the serosa and DM (1), in the SM (2), in the stroma (3), but not found in the epithelium of endometrium and gland (4) from the model with hUC group (b). The fluorescent cells were not detected in the same regions of the other groups (c). The number of fluorescent cells in different regions of the uterus in the model with the hUC group was counted. CM-Dil-labeled cells in the stroma region were significantly higher than those in the seroma (*p = 0.000), in the epithelium (*p = 0.000), and in the SM (*p = 0.005). However, there were no differences between the stroma and the DM (∆p = 0.93), or the DM and the SM (∆p = 0.33) (d). Detection of the CXCR4 expression around the damaged uterine tissues in terms of protein level. The protein level of CXCR4 in the model with the hUC group was significantly higher than those in the other groups. *p < 0.05 was considered to be a significant difference (e)
Fig. 4
Fig. 4
Differentiation evaluation of CM-Dil-labeled hUC-MSCs. The target protein color was green, the DAPI with Dil-hUC color was magenta representing the living cells, and the superposition color of green and magenta was white which means differentiated cells (arrow). CK7 was expressed in the cytoplasm of epithelial cells, CD31 was expressed in the cytoplasm of vascular endothelial cells, and ERα was expressed in the nucleus and cytoplasm. From the 2nd week of treatment, hUC-MSCs began to differentiate into epithelial cells, vascular endothelial cells, and a small amount of ER cells
Fig. 5
Fig. 5
Evaluation of the effect of hUC-MSC therapy on IUA model (a–b). Photomicrographs represent the three different morphological evaluation methods about the efficacy: HE, Masson, and CD31 staining (a). The number of fetuses in the model with the hUC group was more than that in the model group (b)
Fig. 6
Fig. 6
The expression of TGF-β1/Smad3 pathway in uterine tissues in different time points (a–b). Quantitative analysis for TGF-β1 and Smad3 gene expression on the 2nd and the 4th week after the indicated treatment of IUAs with hUC-MSCs (a). Data were shown as mean ± SD, n = 4; *p < 0.05, **p < 0.001. Western blot detected TGF-β1 and Smad3 protein expression of different groups on the 2nd and the 4th week after therapy (b)
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