Elsevier

Cytotherapy

Volume 10, Issue 4, 2008, Pages 353-363
Cytotherapy

Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage

https://doi.org/10.1080/14653240802035926Get rights and content

Background

Many investigations have reported that mesenchymal stem cell (MSC) transplantation can ameliorate the structure and function of injured tissues. The purpose of this study was to explore the therapeutic potency of MSC transplantation for chemotherapy-induced ovarian damage.

Methods

MSC were isolated and cultured in vitro. The cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1), were detected in the MSC cultures using enzyme-linked immunosorbent assay (ELISA). Phosphoramide mustard (PM) was added to the media of granulosa cells (GC) cultured alone or co-cultured with MSC. GC apoptosis was assayed by Annexin-V and DNA fragmentation analysis. Chemotherapy-induced ovarian damage was induced in rats by intraperitoneal injection of cyclophosphamide (CTX). After the injection, MSC labeled with green fluorescent protein (GFP) were transplanted directly into bilateral ovaries. The rats were killed at 2, 4, 6 and 8 weeks after transplantation. Ovarian function was evaluated by estrous cycle changes and sexual hormone levels. The follicle number was counted, and GC apoptosis was analyzed by TUNEL. The expressions of Bcl-2 and Bax proteins were detected by Western blotting.

Results

MSC released VEGF, HGF and IGF-1 in vitro. The GC apoptosis was diminished by co-culture with MSC, which also resulted in increased Bcl-2 expression. The ovarian function of the rats exposed to CTX injection was improved after MSC transplantation. MSC reduced apoptosis of GC and induced up-regulation of Bcl-2 in vivo.

Discussion

MSC transplantation can improve ovarian function and structure damaged by chemotherapy. The paracrine mediators secreted by MSC might be involved in the repair of damaged ovaries.

Introduction

Chemotherapy has been used widely in the treatment of various malignancies and autoimmune diseases, and recent advances in chemotherapy have resulted in significant long-term survival improvements for these patients [1]. However, patients undergoing chemotherapy have to face some severe adverse effects and, in female patients, chemotherapy-induced irreversible damage to the ovarian tissues remains of great concern. Hot flashes, osteoporosis, sexual dysfunction and infertility are all the consequences of premature ovarian failure (POF) induced by chemotherapy [2., 3., 4.].

Chemotherapy-induced ovarian damage is directly correlated with the dose, kind of therapeutic agents used, duration of therapy and age of the patient undergoing treatment [5]. Among the commonly used chemotherapeutic medications, alkylating agents are shown to be the most gonadotoxic. Recent studies both in vitro and in vivo have proved that these agents can induce abnormal apoptosis of oocytes and granulosa cells (GC) and consequently loss of follicles [6., 7., 8.].

Mesenchymal stem cells (MSC) residing within the bone marrow (BM) microenvironment are pluripotent adult stem cells, whose multipotency, easy isolation and culture as well as high ex vivo expansive potential make them attractive candidates for stem cell therapies [9]. Successful attempts of BM-derived MSC transplantation for repairing spinal, cardiac and cutaneous injuries, for instance, have been reported [10, 11]. MSC can differentiate into osteoblasts, chondrocytes, adipocytes, cardiocytes, neural cells and hematopoietic-supporting stroma, and may therefore partly replace the impaired cells [12]. The mechanisms of repair effects also involves their supplying cytokines for angiogenesis, anti-apoptosis and mitogenesis, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (b-FGF) [13]. However, currently little is known about the therapeutic potential of MSC for chemotherapy-induced ovarian damage. We conducted this study to investigate the following topics: whether (1) MSC inhibit chemotherapy-induced apoptosis of ovarian GC in vitro; (2) MSC ameliorate the structural and functional damage of ovaries as a result of chemotherapy in a rat model; and (3) the therapeutic effects of MSC are mediated by secreted anti-apoptotic cytokines including VEGF, IGF-1 and HGF.

Section snippets

Experimental animals

All animal procedures were conducted in accordance with the institutional guidelines of the Southern Medical University (Guangzhou, P. R. China) for the care and use of laboratory animals. Adult female Wistar rats, weighing 180–210 g, were fed rat chow and water ad libitum under controlled temperature (30 ±; 2°C) and light (14 h light, 10 h dark) conditions. Vaginal smears were obtained daily. Only those showing at least two consecutive normal 4-day vaginal estrus cycles were included in the

Characterization and labeling of cultured MSC

Cells of the third passage of MSC in culture were adherent and morphologically resembled fibroblasts (Figure 1A). Most of the cultured MSC expressed CD29 and CD44 but were negative for markers of the hematopoietic lineage, including CD34 and CD45, consistent with previously published data on MSC surface markers [16] (Figure 1C).

A MOI of 100 appeared to be optimal for Ad-GFP transfection of MSC, resulting in a transfection efficiency of 95%. The transfected MSC showed bright green fluorescence

Discussion

MSC have attracted interest for their possible use for both cell and gene therapies because of their capacity for self-renewal and multipotentiality for differentiation [17]. These cells can be induced, either in vitro or in vivo, to differentiate terminally into osteoblasts, chondrocytes, adipocytes, tenocytes, cardiomyoctyes, neural cells and hematopoietic-supporting stroma. Transplantation of MSC directly into adult rat brain and heart reduces functional deficits resulting from stroke [18]

Acknowledgements

This work was supported by a grant from the Science and Technology Project of Guangdong Province (2006B35901003).

References (34)

  • ItoM. et al.

    Hepatocyte growth factor and stem cell factor involvement in paracrine interplays of theca and granulosa cells in the human ovary

    Fertil Steril

    (2001)
  • DengY.B. et al.

    Implantation of BM mesenchymal stem cells into injured spinal cord elicits de novo neurogenesis and functional recovery: evidence from a study in rhesus monkeys

    Cytotherapy

    (2006)
  • JemalA. et al.

    Cancer statistics, 2007

    CA Cancer J Clin

    (2007)
  • StearnsV. et al.

    Breast cancer treatment and ovarian failure: risk factors and emerging genetic determinants

    Nat Rev Cancer

    (2006)
  • BeerendonkC.C. et al.

    Present and future options for the preservation of fertility in female adolescents with cancer

    Endocr Dev

    (2005)
  • DesmeulesP. et al.

    Characterizing the ovotoxicity of cyclophosphamide metabolites on cultured mouse ovaries

    Toxicol Sci

    (2006)
  • RazA. et al.

    Possible direct cytoxicity effects of cyclophosphamide on cultured human follicles: an electron microscopy study

    J Assist Repro Gen

    (2002)
  • Cited by (167)

    • Endoplasmic reticulum stress is involved in small white follicular atresia in chicken ovaries

      2022, Theriogenology
      Citation Excerpt :

      Previous research showed that GCs death dominates the progression of atretic follicles degeneration [8]. It is demonstrated that abnormal apoptosis of GCs results in the atresia of follicles, and follicles become atretic when 10% of GCs have undergone apoptosis [9]. Previous research on mice showed that GCs apoptosis induced by regulation of EGR1 [10], Slit/Robo pathway [11], and BRE caused follicular atresia [12].

    View all citing articles on Scopus
    View full text