Different therapeutic effects of cells derived from human amniotic membrane on premature ovarian aging depend on distinct cellular biological characteristics

Abstract

Background: Many reports have shown that various kinds of stem cells have the ability to recover premature ovarian aging (POA) function. Transplantation of human amniotic epithelial cells (hAECs) improves ovarian function damaged by chemotherapy in a mice model. Understanding of how to evaluate the distinct effects of adult stem cells in curing POA and how to choose stem cells in clinical application is lacking.

Methods: To build a different degrees of POA model, mice were administered different doses of cyclophosphamide: light dose (70 mg/kg, 2 weeks), medium dose (70 mg/kg, 1 week; 120 mg/kg, 1 week), and high dose (120 mg/kg, 2 weeks). Enzyme-linked immunosorbent assay detected serum levels of sex hormones, and hematoxylin and eosin staining allowed follicle counting and showed the ovarian tissue structure. DiIC18(5)-DS was employed to label human amniotic mesenchymal stem cells (hAMSCs) and hAECs for detecting the cellular retention time in ovaries by a live imaging system. Proliferation of human ovarian granule cells (ki67, AMH, FSHR, FOXL2, and CYP19A1) and immunological rejection of human peripheral blood mononuclear cells (CD4, CD11b, CD19, and CD56) were measured by flow cytometry (fluorescence-activated cell sorting (FACS)). Distinction of cellular biological characteristics between hAECs and hAMSCs was evaluated, such as collagen secretory level (collagen I, II, III, IV, and VI), telomerase activity, pluripotent markers tested by western blot, expression level of immune molecules (HLA-ABC and HLA-DR) analyzed by FACS, and cytokines (growth factors, chemotactic factors, apoptosis factors, and inflammatory factors) measured by a protein antibody array methodology.

Results: After hAMSCs and hAECs were transplanted into a different degrees of POA model, hAMSCs exerted better therapeutic activity on mouse ovarian function in the high-dose administration group, promoting the proliferation rate of ovarian granular cells from premature ovarian failure patients, but also provoking immune rejection. Meanwhile, our results showed that the biological characteristics of hAMSCs were superior to hAECs, but not to expression of immune molecules.

Conclusions: These results suggest that hAMSCs are a more effective cell type to improve ovarian function than hAECs. Meanwhile, this distinct effect is attributable to cellular biological characteristics of hAMSCs (telomerase activity, expression level of pluripotent markers, cytokine and collagen secretion) that are superior to hAECs, except for immunological rejection. Sufficient consideration of cell properties is warranted to move forward to more effective clinical therapy.

Keywords: Cellular biological characteristics; Human amniotic epithelial cells; Human amniotic mesenchymal stem cells; Premature ovarian aging.

Figures

Fig. 1

Established mice model with different degrees of POA. a Numbers of four stages of follicles (primordial, primary, secondary, and antral follicles) counted from 0 to 4 weeks after different doses of CTX treatment. b Weight of the ovary measured after different doses of CTX treatment. c Serum hormone levels of E2, AMH, and FSH measured by ELISA after different doses of CTX treatment. d Litter size from the mice model with different degrees of POA. All experiments were carried out three times. Error bars indicate SD. *p < 0.05, ***p < 0.001, compared to control group; #p < 0.05, ##p < 0.01, compared with light-dose group

Fig. 2

hAMSCs improved the function of the ovary more forcefully than hAECs. a Grafted (a) hAMSCs and (b) hAECs detected in vivo separately. Sterilized mice after tail-vein cell transplantation detected by live imaging for the identification of Dil-labeled cells in vivo. b Number of four-period follicles counted during 4 weeks after (a–d) hAMSC and (e–h) hAEC transplantation respectively. c Levels of E2, AMH, and FSH measured by ELISA at week 4 after hAMSC and hAEC transplantation respectively. d In the high-dose group, the weight of the ovary after hAEC transplantation was significantly lower than after hAMSC transplantation in the fourth week. All experiments were carried out three times. Error bars indicate SD. *p < 0.05, **p < 0.01,***p < 0.001, compared with medium-dose group; #p < 0.05, ##p < 0.01, compared with light-dose group. hAEC human amniotic epithelial cell, hAMSC human amniotic mesenchymal stem cell

Fig. 3

hAMSCs increased the number of offspring more than hAECs in the medium-dose and high-dose treated mice model. a Litter sizes counted after hAMSC transplantation into the mice model with different levels of ovarian aging. b Litter sizes counted after hAEC transplantation into the mice model with different levels of ovarian aging. hAEC human amniotic epithelial cell, hAMSC human amniotic mesenchymal stem cell

Fig. 4

hAMSCs improved the proliferation rate of hGCs and upregulated the expression of hGC markers more forcefully than hAECs. a Schematic diagram of different degrees of ovarian aging mice model and patients. b Schematic overview of hGC filtered procedures. c Expression levels of ki67+FSHR+ hGCs tested after coculture with hAECs and hAMSCs respectively. d Number of ki67+AMH+ hGCs evaluated after coculture with hAECs and hAMSCs respectively. e Expression level of ki67+FOXL2+ hGCs tested after coculture with hAECs and hAMSCs respectively. f Number of ki67+CYP19A1+ hGCs evaluated after coculture with hAECs and hAMSCs respectively. Experiments were carried out after 7 days of coculture, n = 3. Error bars indicate SD. *p < 0.05, ***p < 0.001, compared with control group; #p < 0.05, ##p < 0.01, compared with hAEC group. DOR decreased ovarian reserve, POF premature ovarian failure, Sal saline, hGC human ovarian granulosa cell, hAEC human amniotic epithelial cell, hAMSC human amniotic mesenchymal stem cell

Fig. 5

hAMSCs upregulated the expression level of immune molecules in hPBMCs more forcefully than hAECs. a Schematic overview of hPBMC filter procedures. b Expression level of CD8 in hPBMCs evaluated by FACS after coculture with hAECs and hAMSCs respectively. c Expression level of CD4 in hPBMCs evaluated by FACS after coculture with hAECs and hAMSCs respectively. d Expression level of CD11b in hPBMCs evaluated by FACS after coculture with hAECs and hAMSCs respectively. e Expression level of CD19 in hPBMCs evaluated by FACS after coculture with hAECs and hAMSCs respectively. f Expression level of CD56 in hPBMCs evaluated by FACS after coculture with hAECs and hAMSCs respectively. Experiments were carried out after 7 days of coculture, n = 3. Error bars indicate SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with control group; #p < 0.05, ##p < 0.01, compared with hAEC group. Sal saline, DOR decreased ovarian reserve, POF premature ovarian failure, hPBMC human peripheral blood mononuclear cell, hAEC human amniotic epithelial cell, hAMSC human amniotic mesenchymal stem cell, ​MFI mean fluorescence intensity

Fig. 6

Distinction of cellular biological characteristics between hAMSCs and hAECs. a Expression of ovarian markers (AMH, FSH, inhibin α, and inhibin β) and proliferation marker (BrdU) in ovarian tissue measured after hAEC and hAMSC transplantation respectively. b Secretory level of collagen (I, II, III, and IV) from hAECs and hAMSCs estimated by western blot analysis respectively. c Activity of telomerase in hAECs and hAMSCs tested by western blot assay at passage 1 and 5 respectively. d Expression level of HLA-ABC and HLA-DR in hAECs and hAMSCs tested by FACS respectively. e Expression level of pluripotency markers (OCT4, NANOG, SSEA4, and TRA-1-81) in hAECs and hAMSCs measured by western blot analysis. f Growth factor derived from hAECs and hAMSCs estimated by antibody microarray respectively (a–c). fa heatmap exhibited the secretory level of growth facors between hAMSCs and hAECs; fb distribution of 52 growth factors were demonstrated after secretory level of hAMSCs compared to hAECs; fc in accordance with standard criteria of fold change ≥ 8 and statistical significance (p < 0.01), six growth factors were selected: osteoprotegerin, HGF, BDNF, TGF-β2, EGF, and FGF-7. All experiments were carried three times. Error bars indicate SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with hAECs group. hAEC human amniotic epithelial cell, hAMSC human amniotic mesenchymal stem cell