1. Mesenchymal stem cells derived from Wharton jelly of the human umbilical cord ameliorate damage to human endometrial stromal cells 
Xiaoqing Yang, M.S., Mu Zhang, B.S., Yuquan Zhang, Ph.D., Wei Li, Ph.D., Bing Yang, M.S. 
Fertility and Sterility 
Volume 96, Issue 4, Pages e4 (October 2011)
DOI: /j.fertnstert
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Growth inhibition and the proliferation assay of mifepristone treatment with endometrial stromal cells (ESCs). (A, B) Passage 2 ESCs were treated with various concentrations of mifepristone (1, 10, 100, and 1,000 μmol/L). The results indicated that concentrations of mifepristone >100 μmol/L and treatment after 96 hours reduced cell proliferation by >95% compared with control samples. In contrast, at concentrations of ≤10 μmol/L, the inhibition was weak. (C, D) 10, 20, 40, 60, and 80 μmol/L mifepristone treatment with ESCs for 24, 48, 72, and 96 hours. The results showed that with 40 or 60 μmol/L mifepristone treatment with ESCs after 48 hours, inhibition percentage (IC) was ∼50%. The result also indicated that cell proliferation decreased with mifepristone treatment in a dose-dependent fashion. (E, F) ESCs were treated with various concentrations of mifepristone (1, 10, 20, 40, 60, 80, 100, and 1,000 μmol/L) or vehicle 48 hours after withdrawal of mifepristone for 24, 48, 72, and 96 hours and the IC was assessed. The concentration and duration to achieve ∼50% growth inhibition (IC50) in both phase ESCs was estimated to be 60 μmol/L and withdrawal after 48 hours. (G, H) ESCs were treated with 60 μmol/L mifepristone for 48 hours and withdraw for 0, 24, 48, and 72 hours. The proliferation of ESCs was examined in mifepristone-treated (damaged but without coculture), untreated (without mifepristone), and damaged cells cocultured with WJ-MSCs (damaged and coculture) (A, C, E, G: ESCs in the proliferative phase; B, D, F, H: ESCs in the secretory phase). The results were representative of three separate experiments conducted in triplicate using samples from different patients. Each column represents the mean of acquired data ± SE. ∗P<.05; ∗∗P<.01.
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3. Figure 2
Apoptosis of endometrial stromal cells (ESCs) was measured by flow cytometric analysis. (A, C) Representative flow cytometric analysis of annexin V–FITC (AV) staining of ESCs with or without 60 μmol/L mifepristone treatment for 48 hours, after withdrawal of mifepristone from the coculture or without coculture after 48 hours. The proportion of AV+/propidium iodide (PI)− (in the top left corners) and AV+/PI+ (in the bottom left corners) cells represent the number of early-apoptotic cells and late-apoptotic/necrotic cells. (B, D) The bar graph summarizes the mean ± SE of three independent experiments which were done in triplicate. ESCs of early-apoptotic and late-apoptotic/necrotic were significantly increased in the group of treatment with 60 μmol/L mifepristone (damaged but without coculture; B: ± 11.87%; D: ± 6.14%) compared with the untreated group (without mifepristone; B: ± 5.09%; D: ± 3.21%) and the groups of WJ-MSCs cocultured with damaged cells (damaged and coculture; B: ± 2.01%; D: 25.95% ± 5.34%). A, B: ESCs in the proliferative phase. C, D: ESCs in the secretory phase. ∗∗P<.01.
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Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Expression of vascular endothelial growth factor (VEGF) and caspases 3, 8, and 9 mRNA. Qualitative and quantitative analysis of the mRNA expression of VEGF and caspases 3, 8, and 9 of endometrial stromal cells (ESCs). ESCs were treated with (damaged but without coculture, damaged and coculture) or without 60 μmol/L mifepristone (without mifepristone) for 48 hours, with coculture (damaged and cocultured) or without coculture (damaged but without coculture) after the withdrawal of mifepristone after 48 hours. The expression levels of all genes’ mRNAs were normalized by the group untreated with mifepristone (without mifepristone). (A–D) ESCs in the proliferative phase. (E–H) ESCs in the secretory phase. Bar graph summarizes the mean ± SE of three independent experiments which were done in triplicate. ∗P<.05; ∗∗P<.01.
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Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Western blot analysis of VEGF and caspases 3, 8, and 9 protein levels in ESCs. ESCs were treated with (damaged but without coculture, damaged and cocultured) or without 60 μM mifepristone(without mifepristone) for 48 hours, with coculture (damaged and coculture) or without coculture (damaged but without coculture) after the withdrawal of mifepristone after 48 h. (A, B) β-Actin was used as an internal control. Densitometry of immunoreactive bands on Western blot was performed with Bio-Rad Quality One Software (Bio-Rad Laboratories. (C, D) The bar graph summarizes the quantitative analysis of the protein expression of VEGF and caspases 3, 8, and 9 of ESCs and normalized to β-actin. (A, C) ESCs in the proliferative phase. (B, D) ESCs in the secretory phase. All of the Western blot experiments were performed at least twice to confirm the reproducibility. Each column represents the mean of acquired data plus SE. ∗P<.05; ∗∗P<.01. Abbreviations as in Figure 3.
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6. Supplemental Figure 1
Observation of endometrial cells under phase-contrast microscope and after immunofluorescent staining. (A) Primarily cultured endometrial cells: (a, b) Endometrial stromal cells (ESCs) mostly remained round and grew as spindle-shaped cells with long cytoplasmic processes; (c) Endometrial epithelial cells grew as swirling and a wire mesh connection between each cell under phase-contrast microscope. (B) ESCs in primary culture were positively stained by vimentin: (a) Cytoplasm staining with Texas red; (b) Nuclei counterstaining with Hoechst 33342; (c) Merger of a and b. (C) ESCs in primary culture were negatively stained by cytokeratin: (a) Cytoplasm staining with Texas red; (b) Nuclei counterstaining with Hoechst 33342; (c) Merger of a and b.
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7. Supplemental Figure 2
The surface antigens of Wharton jelly–derived mesenchymal stem cells (WJ-MSCs): (A–C) Positive for CD73, CD90, and CD105; (D–H) negative for CD14, CD34,CD38,,CD45, CD79a, and HLA-DR. The results confirmed that the cells were a kind of MSC but nonhematopoietic.
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8. Supplemental Figure 3
Osteogenic and adipogenic differentiation. Osteogenic differentiation was assayed by the von Kossa procedure. (A) No mineralized matrix formation was found in Wharton jelly–derived mesenchymal stem cells (WJ-MSCs) maintained in regular growth medium. (B, C) Osteogenic differentiation was evidenced by staining alizarin red in WJ-MSCs after osteogeneic induction. (E) Adipogenic differentiation was evidenced by the formation of lipid vacuoles after adipogenic induction. (F) Adipogenic differentiation was detected by oil red O staining. (D) No lipid vacuoles were found in WJ-MSCs maintained in the regular medium.
Fertility and Sterility  , e4DOI: ( /j.fertnstert )
Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions