The restorative effects of adipose-derived mesenchymal stem cells on damaged ovarian function

Yuji Takehara, Akiko Yabuuchi, Kenji Ezoe, Tomoko Kuroda, Rie Yamadera, Chiaki Sano, Nana Murata, Takuya Aida, Ken Nakama, Fumihito Aono, Naoki Aoyama, Keiich Kato, Osamu Kato, Yuji Takehara, Akiko Yabuuchi, Kenji Ezoe, Tomoko Kuroda, Rie Yamadera, Chiaki Sano, Nana Murata, Takuya Aida, Ken Nakama, Fumihito Aono, Naoki Aoyama, Keiich Kato, Osamu Kato

Abstract

The clinical application of human adipose-derived mesenchymal stem cells (MSCs) as treatment for intractable diseases or traumatic tissue damage has attracted attention. To address the ability of reactivating injured ovaries, we prepared a rat model with damaged ovaries by using an anticancer agent, cyclophosphamide (CTX). We then investigated the restorative effects on ovarian function and the safety of adipose-derived MSCs (A-MSCs). MSCs were shown to be capable of inducing angiogenesis and restoring the number of ovarian follicles and corpus lutea in ovaries. No deformities, tumor formation or deaths were observed in F1 and F2 rats, indicating that the local injection of MSCs into the ovary did not have any obvious side effects. In addition, the localization of the Y chromosome was investigated using the fluorescent in situ hybridization method by injecting male A-MSCs into the ovaries; as a result, the Y chromosomes were localized not in the follicles, but in the thecal layers. ELISA revealed that A-MSCs secreted higher levels of vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) than tail fibroblast cells. Quantitative real-time PCR and immunohistochemistry showed that higher expression levels of VEGF, IGF-1 and HGF were observed in CTX-treated ovaries after A-MSC transplantation. These findings suggest that MSCs may have a role in restoring damaged ovarian function and could be useful for regenerative medicine.

Figures

Figure 1
Figure 1
Characterization of adipose-derived mesenchymal stem cells (A-MSCs) and bone marrow-derived MSCs (BM-MSCs) and the procedure for MSC transplantation into ovaries. (a) The procedure for MSC transplantation into ovaries. (A) A sufficient visual field was secured by observing the ovarian blood vessels using a stereomicroscope and the removal of excess fat. (B) An A-MSC/BM-MSC suspension (2 × 106 cells/10 μl) was injected into the ovary, which was held in place with forceps. (The above figure shows the indigo carmine injection.) (C) No leakage was visually observed after the injection. (b) The morphology of cultured A-MSCs and BM-MSCs. Adipose: A-MSCs; BM: BM-MSCs. Scale bar=40 μm. (c) Fluorescence cell sorting (FACS) analysis of A-MSCs, BM-MSCs and tail fibroblast cells stained with cell surface antigen markers of MSCs. BM: BM-MSCs; adipose: A-MSCs; fibroblast: tail fibroblast cells.
Figure 1
Figure 1
Characterization of adipose-derived mesenchymal stem cells (A-MSCs) and bone marrow-derived MSCs (BM-MSCs) and the procedure for MSC transplantation into ovaries. (a) The procedure for MSC transplantation into ovaries. (A) A sufficient visual field was secured by observing the ovarian blood vessels using a stereomicroscope and the removal of excess fat. (B) An A-MSC/BM-MSC suspension (2 × 106 cells/10 μl) was injected into the ovary, which was held in place with forceps. (The above figure shows the indigo carmine injection.) (C) No leakage was visually observed after the injection. (b) The morphology of cultured A-MSCs and BM-MSCs. Adipose: A-MSCs; BM: BM-MSCs. Scale bar=40 μm. (c) Fluorescence cell sorting (FACS) analysis of A-MSCs, BM-MSCs and tail fibroblast cells stained with cell surface antigen markers of MSCs. BM: BM-MSCs; adipose: A-MSCs; fibroblast: tail fibroblast cells.
Figure 2
Figure 2
Histological analysis of rat ovary after the administration of cyclophosphamide (CTX). (a) Comparison of the numbers of follicles and corpus lutea (CL) in ovaries between CTX-non-treated rats and CTX-treated rats (*significantly different, P<0.05). (b) Hematoxylin- and eosin- (HE) stained ovaries of CTX-non-treated rats (A, C) and CTX-treated rats (C, D). (A, B) Scale bar=1 mm and (C, D) scale bar=200 μm.
Figure 3
Figure 3
Angiogenesis on cyclophosphamide-(CTX)-treated ovaries after adipose-derived mesenchymal stem cell (A-MSC) and tail fibroblast transplantation. (a) Immunohistochemistry (IHC) of CD34 on rat ovaries. Left, is the cell or saline administration; right, non-injected control. Fibroblast: tail fibroblast cells; adipose: A-MSCs. Scale bar=200 μm. (b) The number of CD34-positive cells in corpus lutea (CL) in ovaries. The number of CD34-positive cells per 1 mm2 of CL was examined. Significant differences between each character is represented by a, b and c (P<0.05). Fibroblast: tail fibroblast cells; adipose: A-MSCs.
Figure 4
Figure 4
Analysis of ovaries after adipose-derived mesenchymal stem cell (A-MSC) transplantation. (a) Histological analysis of follicles in ovaries and after A-MSC transplantation. (A, C) A-MSC non-transplanted and (B, D) A-MSC transplanted. (A, B) Scale bar=1 mm and (C, D) scale bar=200 μm. (b) Ratio of number of follicles and corpus lutea (CL) in MSC-transplanted left ovary vs MSC-non-transplanted right ovary. BM: bone marrow-derived MSCs (BM-MSCs); adipose: A-MSCs; fibroblast: tail fibroblast cells. (c) Ratio of number of antral follicles in MSC-transplanted left ovary vs MSC-non-transplanted right ovary. BM: BM-MSCs; adipose: A-MSCs; fibroblast: tail fibroblast cells.
Figure 5
Figure 5
F1 litters and F2 litters obtained by mating after adipose-derived mesenchymal stem cell (A-MSC) transplantation into the ovary. (a) The photographs of F1 and F2 litters. (A) Immediately after birth (F1); (B) 1-week-old rats (F1); (C) 3-week-old rats (F1); (D) 5-week-old rats (F1); (E) immediately after birth (F2); and (F) 5-week-old rats (F2). (b) The number of F1 litters. BM: bone marrow-derived MSCs (BM-MSCs); adipose: A-MSCs (*significantly different, P<0.05). (c) The number of F2 litters. BM: BM-MSCs; adipose: A-MSCs.
Figure 6
Figure 6
Cytokine secretion from adipose-derived mesenchymal stem cells (A-MSCs), cytokine expression in A-MSC-transplanted ovaries and the localization of A-MSCs in ovaries after transplantation. (a) Cytokine secretion (vascular endothelial cell growth factor (VEGF), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF) and estradiol) from A-MSCs. (A) VEGF; (B) IGF-1; (C) HGF; and (D) estradiol. Each cytokine was measured by enzyme immunoassay (EIA) analysis. Significantly higher levels of VEGF, IGF-1 and HGF (*P<0.05) were secreted from A-MSCs (n=7), compared with fibroblasts (n=9). No significant difference in estradiol levels was observed. Adipose: A-MSCs; fibroblast: tail fibroblast cells. (b) Quantitative real-time polymerase chain reaction (qRT-PCR) on ovaries after A-MSC and tail fibroblast cell transplantation. (A) VEGF; (B) IGF-1; (C) HGF; and (D) StAR. Adipose: A-MSCs; fibroblast: tail fibroblast cells (*significantly different, P<0.05). (c) Immunohistochemistry (IHC) of cytokines (VEGF, IGF-1 and HGF) in ovaries after A-MSC transplantation. Top panels: sham operation; middle panels: cyclophosphamide (CTX) injected; bottom panels: CTX and A-MSC injected. BM: bone marrow-derived MSCs; adipose: A-MSCs. Scale bar=200 μm. (d) Localization of A-MSCs after transplantation into ovaries by fluorescent in situ hybridization (FISH). Male A-MSCs were injected into ovaries and detected by FISH using the Y-chromosome-specific probe. Red: X chromosome conjugated with Texas Red; green: Y chromosome conjugated with FITC. (A) AF: antral follicles, GC: granulosa cells; (B) CL: corpus luteum; (C) T: thecal cells. Cells containing XY karyotype, indicating male-derived ASCs, were observed only in thecal layers.
Figure 7
Figure 7
Growth factors administration into mouse ovaries. (a) Cytokine secretion from mouse adipose-derived mesenchymal stem cells (A-MSCs) measured by enzyme immunoassay (EIA). Secretion of mouse vascular endothelial cell growth factor (VEGF), hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) were measured by enzyme-linked immunosorbent assay (ELISA). (b) Fluorescence cell sorting (FACS) analysis of mouse A-MSCs stained with cell surface antigen markers of MSCs. (c) Ratio of number of follicles and corpus lutea (CL) in A-MSC and growth factor-transplanted left ovary vs -non-transplanted right ovary. V: VEGF; H: HGF; I: IGF-1; 1: 10 × dose; and 10: 10 × doses. Significant differences between each character each character is represented by a, b, c, d, e and f (P<0.05).

References

    1. Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet. 1978;2:366.
    1. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet. 1992;340:17–18.
    1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature. 1983;305:707–709.
    1. Zeilmaker GH, Alberda AT, van Gent I, et al. Two pregnancies following transfer of intact frozen–thawed embryos. Fertil Steril. 1984;42:293–296.
    1. Cohen J, Simons RF, Edwards RG, et al. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert Embryo Transf. 1985;2:59–64.
    1. Kuwayama M, Kato O. All round vitrification of human oocytes and embryos. J Assist Reprod Genet. 2000;17:477.
    1. Kuwayama M, Vajta G, Kato O, et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online. 2005;11:300–308.
    1. Cohen J, Elsner C, Kort H, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod. 1990;5:7–13.
    1. Handyside AH, Kontogianni EH, Hardy K, et al. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature. 1990;344:768–770.
    1. Levin HS. Testicular biopsy in the study of male infertility: its current usefulness, histologic techniques, and prospects for the future. Hum Pathol. 1979;10:569–584.
    1. Schoysman R, Vanderzwalmen P, Nijs M, et al. Pregnancy after fertilisation with human testicular spermatozoa. Lancet. 1993;342:1237.
    1. Craft I, Bennett V, Nicholson N. Fertilising ability of testicular spermatozoa. Lancet. 1993;342:864.
    1. Tournaye H, Devroey P, Liu J, et al. Microsurgical epididymal sperm aspiration and intracytoplasmic sperm injection: a new effective approach to infertility as a result of congenital bilateral absence of the vas deferens. Fertil Steril. 1994;61:1045–1051.
    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292:154–156.
    1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–1147.
    1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676.
    1. Nayernia K, Nolte J, Michelmann HW, et al. In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell. 2006;11:125–132.
    1. Nishiyama N, Miyoshi S, Hida N, et al. The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro. Stem Cells. 2007;25:2017–2024.
    1. Okamoto K, Miyoshi S, Toyoda M, et al. ‘Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells. Exp Cell Res. 2007;313:2550–2562.
    1. Hida N, Nishiyama N, Miyoshi S, et al. Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells. Stem Cells. 2008;26:1695–1704.
    1. Ikegami Y, Miyoshi S, Nishiyama N, et al. Serum-independent cardiomyogenic transdifferentiation in human endometrium-derived mesenchymal cells. Artif Organs. 2010;34:280–288.
    1. Tsuji H, Miyoshi S, Ikegami Y, et al. Xenografted human amniotic membrane-derived mesenchymal stem cells are immunologically tolerated and transdifferentiated into cardiomyocytes. Circ Res. 2010;106:1613–1623.
    1. Fu X, He Y, Xie C, et al. Bone marrow mesenchymal stem cell transplantation improves ovarian function and structure in rats with chemotherapy-induced ovarian damage. Cytotherapy. 2008;10:353–363.
    1. Housman TS, Lawrence N, Mellen BG, et al. The safety of liposuction: results of a national survey. Dermatol Surg. 2002;28:971–978.
    1. Schreml S, Babilas P, Fruth S, et al. Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction. Cytotherapy. 2009;11:947–957.
    1. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211–228.
    1. De Ugarte DA, Morizono K, Elbarbary A, et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174:101–109.
    1. Rodbell M. Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem. 1964;239:375–380.
    1. Xie LW, Fang H, Chen AM, et al. Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro. Chin J Traumatol. 2009;12:98–103.
    1. Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med. 2005;54:132–141.
    1. Baglioni S, Francalanci M, Squecco R, et al. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J. 2009;23:3494–3505.
    1. Plowchalk DR, Mattison DR. Reproductive toxicity of cyclophosphamide in the C57BL/6N mouse: 1. Effects on ovarian structure and function. Reprod Toxicol. 1992;6:411–421.
    1. Plowchalk DR, Mattison DR. Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide. Toxicol Appl Pharmacol. 1991;107:472–481.
    1. Choi MR, Kim HY, Park JY, et al. Selection of optimal passage of bone marrow-derived mesenchymal stem cells for stem cell therapy in patients with amyotrophic lateral sclerosis. Neurosci Lett. 2010;472:94–98.
    1. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cells in vitro: effects of dexamethasone and IL-1 alpha. J Cell Physiol. 1996;166:585–592.
    1. Liu CH, Hwang SM. Cytokine interactions in mesenchymal stem cells from cord blood. Cytokine. 2005;32:270–279.
    1. Shin SY, Lee JY, Lee E, et al. Protective effect of vascular endothelial growth factor (VEGF) in frozen–thawed granulosa cells is mediated by inhibition of apoptosis. Eur J Obstet Gynecol Reprod Biol. 2006;125:233–238.
    1. Mao J, Smith MF, Rucker EB, et al. Effect of epidermal growth factor and insulin-like growth factor I on porcine preantral follicular growth, antrum formation, and stimulation of granulosal cell proliferation and suppression of apoptosis in vitro. J Anim Sci. 2004;82:1967–1975.
    1. Uzumcu M, Pan Z, Chu Y, et al. Immunolocalization of the hepatocyte growth factor (HGF) system in the rat ovary and the anti-apoptotic effect of HGF in rat ovarian granulosa cells in vitro. Reproduction. 2006;132:291–299.
    1. D'Andrea F, De Francesco F, Ferraro GA, et al. Large-scale production of human adipose tissue from stem cells: a new tool for regenerative medicine and tissue banking. Tissue Eng Part C. 2008;14:233–242.
    1. Manger K, Wildt L, Kalden JR, et al. Prevention of gonadal toxicity and preservation of gonadal function and fertility in young women with systemic lupus erythematosus treated by cyclophosphamide: the PREGO-Study. Autoimmun Rev. 2006;5:269–272.
    1. Del Mastro L, Catzeddu T, Venturini M. Infertility and pregnancy after breast cancer: current knowledge and future perspectives. Cancer Treat Rev. 2006;32:417–422.

Source: PubMed

スポンサーと協力者

医学的状態

薬物療法

3
購読する