Novel in vivo endometriotic models associated eutopic endometrium by implanting menstrual blood-derived stromal cells from patients with endometriosis

Abstract

The eutopic endometrium provides novel insights into endometriotic pathophysiology and treatment. However, no in vivo models currently available are suitable for eutopic endometrium in endometriosis. In this study, we present new endometriotic in vivo models associated with eutopic endometrium using menstrual blood-derived stromal cells (MenSCs). First, we isolated endometriotic MenSCs (E-MenSCs) and healthy MenSCs (H-MenSCs) from the menstrual blood of patients with endometriosis (n = 6) and healthy volunteers (n = 6). Then, we identified MenSCs' endometrial stromal cell properties using adipogenic and osteogenic differentiation. A cell counting kit-8 and wound healing assay were used to compare the proliferation and migration capability between E-MenSCs and H-MenSCs. Seventy female nude mice were used to prepare endometriotic models related to eutopic endometrium by implanting E-MenSCs relying on three approaches, including surgical implantation using scaffolds seeded with MenSCs, and subcutaneous injection of MenSCs in the abdomen and the back (n = 10). H-MenSCs or scaffolds only were implanted in control groups (n = 10). One month after the surgical implantation and 1 week after the subcutaneous injection, we evaluated modeling by hematoxylin-eosin (H&E) and immunofluorescent staining of human leukocyte antigen α (HLAA). Fibroblast morphology, lipid droplets, and calcium nodules in E-MenSCs and H-MenSCs identified their endometrial stromal cell properties. We noticed that the proliferation and migration of E-MenSCs were considerably enhanced compared to H-MenSCs (P < 0.05). E-MenSCs implanted in nude mice formed ectopic lesions using three approaches (n = 10; lesions formation rate: 90%, 115%, and 80%; average volumes: 123.60, 27.37, and 29.56 mm³), while H-MenSCs in the nude mice shaped nothing at the implantation sites. Endometrial glands, stroma, and HLAA expression in these lesions further verified the success and applicability of the proposed endometriotic modeling. Findings provide in vitro and in vivo models and paired controls associated with eutopic endometrium in women with endometriosis using E-MenSCs and H-MenSCs. The approach of subcutaneous injection of MenSCs in the abdomen is highlighted due to non-invasive, simple, and safe steps, a short modeling period (1 week), and an excellent modeling success rate (115%), which could improve the repeats and success of endometriotic nude mice model and shorten the modeling period. These novel models could nearly intimate human eutopic endometrial mesenchymal stromal cells in the progress of endometriosis, opening a new path for disease pathology and treatment.

Figure 1

Adipogenic and osteogenic differentiation of MenSCs. Adipogenic and osteogenic differentiation was used for identifying H-MenSCs and E-MenSCs. These cells both showed flat and fibroblast-like morphology and grew dispersedly. Images taken before induction illustrate the original state of H-MenSCs and E-MenSCs (2nd to 4th passage). In the figures, NC (negative control) indicates the development of H-MenSCs and E-MenSCs after 21 days without induction, AD (adipogenic differentiation) depicts MenSCs after adipogenic differentiation, and OD shows the cells after osteogenic differentiation (Scale bar: 100 μm).

Figure 2

Comparison between E-MenSCs and H-MenSCs on cell proliferation. (A) The state of the 2nd passage of H-MenSCs and E-MenSCs from 0 to 96 h. II H-MenSCs: the 2nd passage of healthy menstrual blood-derived stromal cells; II E-MenSCs: the 2nd passage of menstrual blood-derived stromal cells with endometriosis. (B) The proliferation of H-MenSCs and E-MenSCs at different time points and cell densities. *P < 0.05, **P < 0.01 vs. H-MenSC group at the same cell density and time point using Student’s t-test (n = 5). Notes: H-MenSCs: healthy menstrual blood-derived stromal cells; E-MenSCs: menstrual blood-derived stromal cells with endometriosis.

Figure 3

Comparison between E-MenSCs and H-MenSCs on cell migration and wound-healing capability. (A) Migration states of H-MenSCs and E-MenSCs. The same scratched area of H-MenSCs and E-MenSCs at 0 h, 24 h, and 48 h was captured by light microscopy. The migration area was highlighted using yellow curves and a scale bar of 100 μm. (B) Analysis of wound width and migration area. The wound width (pixels) and migration area (%) were analyzed via ImageJ software. * P < 0.05 vs. H-MenSCs at the same time using Student’s t-test (n = 5). H-MenSCs healthy menstrual blood-derived stromal cells, E-MenSCs menstrual blood-derived stromal cells with endometriosis.

Figure 4

Implants at the beginning and finishing time point after the implantation in seven groups of mice. (A) implants in the mice using approach 1 (surgical implantation using scaffolds seeded with MenSCs). The experiments involved three groups: SEM, SHM, and S. The red arrow shows the bulge of implant directly after the implanting operation. The red rectangle highlights the lesion 1 month after the implantation, and the two white arrows mark the surrounded blood vessels. (B) Implants in the mice using approaches 2 and 3 (subcutaneous injection of MenSCs in the abdomen and back). Groups were divided into SCEA, SCHA, SCEB, and SCHB. The red circle shows the protrusion hours after the injection. The red rectangle highlights the lesion 1 week after the injection, and the two white arrows mark the surrounded blood vessels. SEM scaffolds seeded with E-MenSCs, SHM scaffolds seeded with H-MenSCs, S scaffolds, SCEA subcutaneous injection of E-MenSCs in the abdomen, SCHA subcutaneous injection of H-MenSCs in the abdomen, SCEB subcutaneous injection of H-MenSCs in the back, SCHB subcutaneous injection of H-MenSCs in the back, E-MenSCs menstrual blood-derived stromal cells with endometriosis, H-MenSCs healthy menstrual blood-derived stromal cells.

Figure 5

Pathological structure of lesions in E-MenSCs implanting groups: SEM (A), SCEA (B), and SCEB (C) groups. Paraffin-embedded sections of lesions in nude mice of the three groups after hematoxylin and eosin (H&E) staining (Scale bar: 50 μm, 25 μm). Green arrows are pointing to the stromal cells, blue arrows are pointing to glandular epithelial cells (columnar and sponge-like), and red arrows show blood vessels. SEM scaffolds seeded with E-MenSCs, SCEA subcutaneous injection of E-MenSCs in the abdomen, SCEB subcutaneous injection of E-MenSCs in the back.

Figure 6

HLAA immunofluorescent staining of ectopic lesions from SEM (A), SCEA (B), and SCEB (C) groups. DAPI (blue fluorescent) represents the nucleus in the tissues. The images confirm that HLAA (red fluorescent) was expressed in the lesions of all three groups. The last pictures in the row is a merging process of both in a scale bar of 100 μm. SEM scaffolds seeded with E-MenSCs, SCEA subcutaneous injection of E-MenSCs in the abdomen, SCEB subcutaneous injection of E-MenSCs in the back, DAPI diamidino-phenyl-indole, HLAA human leukocyte antigen α.

Figure 7

Graphic protocols for the three approaches of in vivo endometriotic model by MenSCs implantation. H-MenSCs menstrual blood-derived stromal cells from healthy volunteers, E-MenSCs menstrual blood-derived stromal cells with endometriosis, Ap. 1 approach 1—surgical implantation using scaffolds seeded with MenSCs, Ap. 2 approach 2—subcutaneous injection of MenSCs in the abdomen, Ap. 3 approach 3—subcutaneous injection of MenSCs in the back, S scaffolds, SEM scaffolds seeded with E-MenSCs, SHM scaffolds seeded with H-MenSCs, SCEA subcutaneous injection of E-MenSCs in the abdomen, SCHA subcutaneous injection of H-MenSCs in the abdomen, SCEB subcutaneous injection of E-MenSCs in the back, SCHB subcutaneous injection of H-MenSCs in the back, sc subcutaneous injection, HE staining hematoxylin–eosin staining, HLAA human leukocyte antigen α.