CD44+/CD105+ human amniotic fluid mesenchymal stem cells survive and proliferate in the ovary long-term in a mouse model of chemotherapy-induced premature ovarian failure

Abstract

Objectives: Stem cell transplantation has been reported to rescue ovarian function in a preclinical mouse model of chemotherapy-induced premature ovarian failure (POF); however, maintaining the survival and self-renewal of transplanted seed cells in ovarian tissues over the long-term remains a troublesome issue. In this study we aimed to determine whether the CD44+/CD105+ human amniotic fluid cell (HuAFCs) subpopulation represent potential seed cells for stem cell transplantation treatments in POF.

Materials and methods: The CD44+/CD105+ subpopulation were isolated from HuAFCs, cultured in vitro, and injected into a cyclophosphamide-induced mouse model of POF.

Results: Under continuous subculture in vitro, CD44+/CD105+ cells proliferated rapidly and expressed high levels of the proliferative markers Ki67 and survivin, as well as high levels of a number of mesenchymal stem cell biomarkers. Moreover, when red fluorescence protein (RFP)-transduced CD44+/CD105+ HuAFCs were transplanted into the ovaries of POF mice, the cells could be detected by fluorescence microscopy up to three weeks after injection. Furthermore, the BrdUrd incorporation assay and immunofluorescent staining demonstrated that CD44+/CD105+ HuAFCs underwent normal cycles of cell proliferation and self-renewal in the ovarian tissues of POF mice over the long-term.

Conclusions: The mesenchymal stem cell properties and long-term in vivo survival of CD44+/CD105+ HuAFCs make them ideal seed cells for stem cell transplantation to treat POF.

Keywords: Mouse premature ovarian failure model; cell transplantation; human amniotic fluid cells; long-term survival; mesenchymal stem cell like cells; proliferation..

Figure 1

Isolation and characterisation of the different subpopulations of cells in human amniotic fluid. (A) Multiple cell populations exist in human amniotic fluid. (a), (b) and (c) illustrate the morphology of different cell subpopulations in human primary amniotic fluid. Original magnification, 200×. (B) Morphology of the CD44+/CD105+ subpopulation isolated from human primary amniotic fluid. Original magnification, 200×. (C) Morphology of the CD44-/CD105- subpopulation isolated from human primary amniotic fluid. Original magnification, 200×. (D) Isolation of CD44+/CD105+ HuAFCs from amniotic fluid. The cells were detected by FCM; CD44+/CD105+ cells represented 1.75% ± 0.25% of the HuAFC population.

Figure 2

Analysis of the proliferation and survival of CD44+/CD105+ HuAFCs in vitro. (A) MTT assays indicated that the viability of CD44-/CD105- HuAFCs significantly reduced at both 2 d and 5 d, compared to CD44+/CD105+ HuAFCs. **P < 0.01 vs. CD44-/CD105- HuAFCs; *P < 0.05 vs. CD44-/CD105- HuAFCs; #P > 0.05 vs. CD44-/CD105- HuAFCs; n = 3. (B) qRT-PCR analysis of Ki67 and survivin mRNA expression in CD44+/CD105+ and CD44-/CD105- HuAFCs; *P < 0.05 vs. CD44-/CD105- HuAFCs; #P > 0.05 vs. CD44-/CD105- HuAFCs; n = 3. (C) Western blotting analysis of Ki67 and survivin protein expression in CD44+/CD105+ and CD44-/CD105- HuAFCs; **P < 0.01 vs. CD44+/CD105+ HuAFCs; * P< 0.05 vs. CD44+/CD105+ HuAFCs; #P > 0.05 vs. CD44+/CD105+ HuAFCs; n = 3. (D) Flow cytometric cell cycle analysis of CD44+/CD105+ and CD44-/CD105- HuAFCs. The majority of CD44-/CD105- HuAFCs were arrested in the G2/M phase with a reduced percentage of S phase cells; **P < 0.01 vs. CD44+/CD105+ HuAFCs; *P < 0.05 vs. CD44+/CD105+ HuAFCs; #P > 0.05 vs. CD44+/CD105+ HuAFCs; n = 3. (E) FCM analysis of human mesenchymal stem cell marker expression in CD44+/CD105+ and CD44-/CD105- HuAFCs in vitro. Expression of the "stemness" markers was higher in CD44+/CD105+ HuAFCs than CD44-/CD105- HuAFCs.

Figure 3

Establishment of a mouse model of POF. (A, B) Ovarian pathology of the wt group (A) and POF group (B) at 2 weeks after injection of cyclophosphamide. The ovaries of the wt group and vehicle group contained a large number of follicles at all stages of immaturity or maturation; whereas the atrophied ovaries of the POF model mice were mostly composed of interstitial cells in a fibrous matrix, with a reduced number of follicles at each stage and an increased number of collapsed oocytes. Original magnification, 100×. (C, D) Plasma E₂ levels (C) and FSH levels (D) as determined by ELISA at various time points after the injection of cyclophosphamide; **P < 0.01 vs. WT; #P > 0.05 vs. WT.

Figure 4

HuAFCs survived in vivo in the ovary in a mouse model of POF. Immunofluorescence (IF) demonstrated that Dil, a marker of HuAFCs, was expressed at high levels in grafted CD44+/CD105+ HuAFCs transplanted into POF mouse ovaries; the transplanted cells survived for at least three weeks in vivo. In addition, BrdUrd incorporation and IF staining indicated that transplanted CD44+/CD105+ HuAFCs underwent normal cell division. CD44-/CD105- HuAFCs could not be detected along the injection tract in POF mouse ovaries. Original magnification: ×200.

Figure 5

HuAFCs proliferated in vivo in the ovary in a mouse model of POF. Immunofluorescent staining and FCM indicated that the number of Dil positive (red, a marker of HuAFCs) CD44+/CD105+ HuAFCs increased from the time of the graft up to three weeks after transplantation. RFP-transduced CD44-/CD105- HuAFCs could not be detected. **P < 0.01 vs. CD44-/CD105- HuAFCs; *P < 0.05 vs. CD44-/CD105- HuAFCs; n = 3.