A novel therapeutic approach for endometriosis using adipose-derived stem cell-derived conditioned medium- A new hope for endometriotic patients in improving fertility

Abstract

Introduction: Endometriosis is defined as the growth of endometrial glands and stromal cells in a heterotopic location with immune dysregulation. It usually leads to chronic pelvic pain and subfertility. Although various treatments are available, the recurrence rate remains high. Adipose tissue is an abundant source of multipotent mesenchymal adipose-derived stem cells (ADSCs). ADSCs display effects on not only tissue regeneration, but also immune regulation. Thus, the current study aims to test the effects of ADSCs on the growth of endometriosis.

Methods: ADSCs isolated from lipoaspiration-generated adipose tissue and their conditioned medium (ADSC-CM) were subjected to quality validation, including karyotyping as well as growth promotion and sterility tests for microbial contamination under Good Tissue Practice and Good Manufacturing Practice regulations. An autologous endometriosis mouse model was established by suturing endometrial tissue to peritoneal wall followed by treating with DMEM/F12 medium, ADSC-CM, ADSCs or ADSC-CM+ADSCs for 28 days. The area of endometriotic cysts and the degree of pelvic adhesion were measured. ICAM-1, VEGF and caspase 3 expression was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Moreover, the mice were allowed to mate and deliver. The pregnancy outcomes were recorded. The ADSC-CM was subjected to proteomics analysis with further data mining with Ingenuity Pathway Analysis (IPA).

Results: Both ADSC-CM and ADSCs passed quality validation. ADSC-CM reduced the area of endometriotic cysts. The inhibition by ADSC-CM was obliterated by adding ADSCs. The presence of ADSCs with or without ADSC-CM increased the peritoneal adhesion. ADSC-CM inhibited ICAM-1 and VEGF mRNA and protein expression, whereas the addition of ADSCs not only did not inhibit by itself, but also blocked the inhibition by ADSC-CM. The resorption rate was reduced by ADSC-CM. The number of live birth/dam and the survival rate of pup at 1 week-old were both increased by ADSC-CM in mice with endometriosis. IPA demonstrated that PTX3 was potentially critical for the inhibition of endometriosis by ADSC-CM due to its anti-inflammatory and antiangiogenic properties as well as its importance in implantation.

Conclusion: ADSC-CM inhibited endometriosis development and improved pregnancy outcomes in mice. Potential translation to clinical treatment for human endometriosis is expected.

Keywords: PTX3; adipose-derived stem cell; conditioned medium; endometriosis; proteomics.

Figure 1

Experimental design Four pieces of uterine tissue were generated from the uterus of a female C57BL/6JNarl mice using a disposable 2-mm dermal biopsy punch and sutured to the peritoneal wall with two pieces on each side. Then, the culture medium, ADSC, ADSC-CM or ADSC + ADSC-CM was added into the peritoneal cavity. The endometriotic lesions were harvested at 28 days. To examine the pregnancy outcomes, the mouse was mated with a male C57BL/6JNarl at day 28 and allowed to deliver at term. The percentage of vaginal plug, resorption rate, live births per dam, and pup survival rate at 1 week-old were recorded.

Figure 2

Characterization of ADSCs (A) Cells isolated from adipose tissue were stained with various markers, including CD29, CD44, CD73, CD90 and CD105 for MSC, CD14 for monocytes, CD19 for B-cells, CD31 for endothelial cells, CD34 for hematopoietic cells, and pan-leukocyte marker, CD45. Representative histograms of flow cytometric analysis are shown. (B) Representative karyotyping of cells isolated from adipose tissue.

Figure 3

ADSC-CM inhibits the development of endometriosis Uterine tissue was sutured to the peritoneal wall of female C57B/6 mice and treated with (A) Culture medium, (B) ADSC-CM, (C) ADSC, (D) ADSC-CM + ADSC. (E) Lesion size and (F) grade of adhesion were measured. The data were reported as mean ± SEM. n=6; *p < 0.05.; **p < 0.01.

Figure 4

qRT-PCR of endometriosis-associated molecule expression The expression of (A) ICAM-1, (B) VEGF and (C) Caspase-3 in the endometriotic lesions were examined by qRT-PCR. The data were reported as mean ± SEM. n=6; *p < 0.05.; **p < 0.01.

Figure 5

Immunohistochemical staining of endometriosis-associated molecules The expression of (A) ICAM-1, VEGF and caspase-3 in endometriotic lesions was demonstrated by immunohistochemistry. The immunoreactivity of (B) ICAM-1, (C) VEGF and (D) Caspase-3 in the tissue were semi-quantified by HSCORE. The data were reported as mean ± SEM. n=6; **p < 0.01.

Figure 6

ADSC-CM Improves Pregnancy Outcomes in Mice with Endometriosis Fecundity and pregnancy outcomes represented by (A) numbers of vaginal plug, however, (B) resorption rate, (C) live births per dam, (D) pup survival rate at 1 week-old were recorded in mice complicated with endometriosis treated with or without ADSC-CM. The data were reported as mean ± SEM. n=4; *p < 0.05.; **p < 0.01.

Figure 7

qRT-PCR of receptivity markers The expression of (A) LIF, (B) HOXA10 and (C) HOXA11 in the eutopic endometrium of mice with endometriosis at GD4 was examined by qRT-PCR. The data were reported as mean ± SEM. n=6; *p < 0.05.

Figure 8

Histology and immunochemical staining of receptivity markers Eutopic endometria of mice with endometriosis at GD4 were subjected to (A) H & E stain. Arrows: lymphocytes; Arrow heads: polymorphonuclear cells. (B) Immunohistochemistry was used to assess the expression of (C) LIF, (D) HOXA10 and (E) HOXA11. The data were reported as mean ± SEM. n=6; *p < 0.05.

Figure 9

Network of molecules interacting with PTX3 One hundred and fourteen molecules interact with PTX3 in the ADSC-CM.