1. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions 
Jose V. Medrano, Ph.D., Charlotte Rombaut, M.Sc., Carlos Simon, M.D., Antonio Pellicer, M.D., Ellen Goossens, Ph.D. 
Fertility and Sterility 
Volume 106, Issue 6, Pages e8 (November 2016)
DOI: /j.fertnstert
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Experimental design and characterization of the phenotype of human testicular cells in vitro. (A) Experimental design of the study with the three main experimental conditions tested: [1] differential plating, [2] whole-cell culture, and [3] fluorescence-activated cell sorting (FACS) enrichment of human spermatogonial stem cells (hSSCs) and coculture with inactivated testicular feeders. (B) Illustrative pictures of the phenotype of human testicular cells in vitro over time. (C) Time-course analysis at day 7 and 14 of culture in both standard medium (SM) and mouse spermatogonial stem cell medium (mSSCM) for the mRNA expression of testicular somatic cell markers. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean ± standard error. Statistically significant differences: *P<.05 and **P<.01. (D) Illustrative pictures of the stainings for αSMA, STAR, and SOX9 on human testicular cells in vitro at day 14.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Identification and characterization of human spermatogonial stem cells (hSSCs) within human testicular cells in vitro. (A) Time-course analysis at day 7 and 14 of culture in both standard medium (SM) and mouse spermatogonial stem cell medium (mSSCM) for the mRNA expression of germ cell markers. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean ± standard error. Statistically significant differences: *P<.05 and **P<.01. (B) Illustrative pictures of the staining colocalizations for vimentin (VIM), VASA, UCHL1, and UTF1 on human testicular cells in vitro at day 14.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Proliferation analysis and quantification of hSSCs in vitro. (A) Illustrative staining colocalizations for Ki67, VASA, and UTF1 at day 14. (B) Time-course quantification at days 7 and 14 of the number of VASA+/UTF1+ cells/cm2 in whole culture and attached cells conditions cultured in mouse spermatogonial stem cell medium (mSSCM). Due to low number of cells at day 7 in differentially plated cells condition, counts at day 7 are missed. (C) Time-course quantification at days 14 and 28 of the number of VASA+/UTF1+ cells/cm2 in HLA−/EPCAM+ and HLA−/EPCAM− sorted cells cocultured in mSSCM with inactivated testicular feeders. (D) Comparison of the number of VASA+/UTF1+ cells/cm2 among culture conditions at day 14. Data are presented as mean ± standard error. Statistically significant differences: *P<.05 and **P<.01.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Results of HLA/EPCAM fluorescence-activated cell sorting (FACS) and coculture with inactivated testicular feeders in mouse spermatogonial stem cell medium (mSSCM). (A) Illustrative graph plots and descriptive statistics for the FACS of human testicular cell suspensions based on HLA/EPCAM double staining. (B) Illustrative picture of the VASA (red)/UTF1 (green) colocalization staining shown in cytospins from HLA−/EPCAM+ sorted cells. (C) Circular graphs showing the percentage of somatic cells (VASA−/UTF1+ and VASA−/UTF1− cells), hSSCs (VASA+/UTF1+ cells), and the rest of germ cells (VASA+/UTF1− cells) within HLA/EPCAM sorted populations. (D) Time-course analysis at day 14 and 28 for the mRNA expression of germ cell markers in coculture conditions. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean ± standard error. Statistically significant differences: *P<.05 and **P<.01.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Results from TUNEL assay on human testicular cells in vitro. Illustrative pictures correspond to day 14 of culture except for spermatogonial stem cell (SSC)-like clusters that correspond to day 3 due to their later disappearance in vitro.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Illustrative pictures of the VASA (red)/UTF1 (green) colocalization staining in cytospins from unsorted and HLA/EPCAM sorted cells.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

8. Supplemental Figure 3
Time-course analysis at days 14 and 28 for the mRNA expression of germ testicular somatic markers in coculture conditions. Relative expression found in frozen/thawed testicular samples is also shown as a reference of physiologic expression. Data are presented as mean ± standard error. Statistically significant differences: *P<.05 and **P<.01.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

9. Supplemental Figure 4
Hormone quantifications in fresh control media and media used in culture conditions for 48 hours. Data are presented as mean ± standard error. Statistically significant difference: *P<.05.
Fertility and Sterility  , e8DOI: ( /j.fertnstert )
Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions