In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid  Federica Riva, Claudia Omes, Roberto Bassani, Rossella E Nappi, Giuliano Mazzini, Antonia Icaro Cornaglia, Andrea Casasco  Reproductive BioMedicine Online  Volume 29, Issue 4, Pages 457-469 (October 2014) DOI: 10.1016/j.rbmo.2014.06.006 Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Primary cell culture from follicular fluid seeded on coverslip and grown for different times in minimal culture condition. Colonies are composed of heterogeneous cell populations containing at least two cell types, displaying an elongated fibroblast-like shape (A) or a cuboidal epithelial-like phenotype (B) after 24 h in culture. Some cells with cytoplasmic protrusion and a large and spherical cell body, typical of a neural morphology, are already evident at 24 h (C), and even more after 72 h (D). Scale bars: 10 µm. Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Proliferative activity of cell population derived from human ovarian follicular fluid, grown on coverslips at different times in minimal culture conditions and incubated with bromo-deoxyuridine for 1 h (A and B). The panels A and B show immunofluorescence analysis of cells at 3 days: the nucleus of all cells was counterstained with Hoechst 33258 (A, blue fluorescence); the proliferating cells were immunostained for bromo-deoxyuridine with FITC (B, nuclear green fluorescence). Panel C shows the percentage of proliferating cells compared with total counted cells. All data were obtained from 10 samples and were presented as mean ± standard deviation. BrdU, bromo-deoxyuridine; FITC, fluorescein isothiocyanate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Putative mesenchymal stem cells isolated from human follicular fluid and expanded in vitro on coverslip for 3 days in culture medium without leukaemia inhibitory factor. All cells show cytoplasmic immunostaining for vimentin (B) and are negative for pan-cytokeratins (D, green fluorescence). Nuclear deoxyribonucleic acid was stained using Hoechst 33258 (A,C blue fluorescence). Panel E shows the percentage of cell expression of these proteins at different time of growth compared with total counted cells (***P < 0.0001). All data were obtained from 10 samples and were presented as mean ± standard deviation. CK, cytokeratin; FITC, fluorescein isothiocyanate; VIM, vimentin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Cells isolated from human ovarian follicular fluid and seeded on coverslip for 7 days. Cells were immunostained for specific mesenchymal markers (CD44, CD105 and CD73), identified respectively on cell membrane (B, arrows) or in cytoplasm (D, F) as fluorescein isothiocyante-green fluorescence. Cells show a negative expression for CD34 (H). Nuclei were stained with Hoechst 33258 (A, C, E, G, blue fluorescence). Scale bars: 10 µm. FITC, fluorescein isothiocyanate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Flow cytometric analysis of CD105, CD44, CD90, CD45, CD66 and CD34 antigens expression in bone marrow mesenchymal stem cells (A) and in human follicular fluid cells (B) collected at time t 0 h. Histograms show the distribution of different antigen expression as ‘grey curves’ against the ‘white’ curve of the negative control (see details in the ‘Results’). MSC, mesenchymal stem cells. Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Osteogenic, chondrogenic and adipogenic differentiation of human follicular fluid cells. Von Kossa (A, B), Alcian blue (C, D) and Oil Red O (E, F) staining of follicular fluid cells cultured with control medium (–) and with differentiative medium for 2 weeks (+) and observed with light contrast microscopy. Von Kossa revealed osteogenic differentiation, with cytoplasms intensively stained in brown (B). Chondroblast-differentiated cells appear blue with alcian staining (D) and cells differentiated with adipogenic medium were red after specific stain with Red Oil (F). Scale bars: 10 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2014 29, 457-469DOI: (10.1016/j.rbmo.2014.06.006) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Reproductive BioMedicine Online 2014 29, 457-469DOI: (10. 1016/j. rbmo Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions