Granulosa cells and retinoic acid co-treatment enrich potential germ cells from manually selected Oct4-EGFP expressing human embryonic stem cells  Hsin-Fu Chen, Pey-Shynan Jan, Hung-Chih Kuo, Fang-Chun Wu, Chen-Wei Lan, Mei-Chi Huang, Chung-Liang Chien, Hong-Nerng Ho  Reproductive BioMedicine Online  Volume 29, Issue 3, Pages 319-332 (September 2014) DOI: 10.1016/j.rbmo.2014.05.009 Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Representative images for the undifferentiated H9-Oct4-enhanced green fluorescence protein (EGFP) human embryonic stem (HES) cells (A, B), HES-derived embryoid bodies (C, D), HES cells after further differentiation (E, F), mouse ovarian stromal cells (G-H), and human granulosa cells (I, J), as described in the Materials and methods section. (A and B), a bright-field and a fluorescence image of the undifferentiated H9-Oct4-EGFP HES cells, respectively; (C and D), a bright-field and a fluorescence image of the embryoid bodies (day 5 differentiation), respectively; (E and F), further adherent culture of embryoid bodies for 7 days (E) and for 35 days (F); (G and H), mouse ovarian stromal cells at passage 0 and 2, respectively; (I and J), human granulosa cells at passage 0 and 2, respectively. Scale bars are 1000 μm for A, B, E, F, 200 μm for C, D, and 500 μm for G–J. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Fluorescence-activated cell sorting (FACS) and analysis of cells expressing SSEA1 (SSEA1[+]), Oct4-EGFP (Oct4-EGFP[+]) in the differentiated H9 Oct4-EGFP HES cells, and subsequent real-time reverse transcription PCR (RT-PCR) analysis of the germ cell marker expression of the sorted cell populations, as described in the Materials and methods section. A number of co-treatments were used to culture H9 Oct4-EGFP HES cells, and were collected after 14 days. Harvested cells were sorted for SSEA1-PE by FACS. (A) Representative plots of FACS analysis and sorting of SSEA1-PE, Oct4-EGFP populations, or both; (B) percentages of SSEA1[+] cells, Oct4-EGFP[+] cells, and SSEA1[+]/Oct4-EGFP[+] doubly positive cells under different culture environments; (C) real-time RT-PCR analysis of the germ cell marker (VASA, GDF9) expression in the FACS-sorted populations. GADPH was used as the internal control for quantitation. Gra-CM, culture in conditioned medium obtained from human granulosa cells; Gra-Co, human granulosa cell co-culture; La, culture on laminin-coated dishes; Ova-CM, culture in conditioned medium obtained from mouse ovarian stromal cells; Ova-Co, mouse ovarian stromal cell co-culture. hES are undifferentiated H9 Oct4-EGFP HES cells. The P-values (statistical significance) between treatments are shown for some points of comparison. *P < 0.05. **P < 0.01. ***P < 0.001. Bars represent mean ± SD of data. Each experiment was repeated at least three to five times. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Fluorescence-activated cell sorting and analysis of cells expressing early germ cell marker SSEA1 (SSEA1[+]) or not expressing SSEA1 (SSEA1[-]) in the differentiated (for 14 days) NTU1 HES cells using a number of co-treatments (A). Subsequently, real-time reverse transcription (RT-PCR) analysis for the germ cell markers of the sorted cell populations after differentiation for 14 days (B) or 21 days (C). The co-treatments for differentiation include the followings: Gra-CM, culture in conditioned medium obtained from human granulosa cells; Gra-Co, human granulosa cell co-culture; La, culture on laminin-coated dishes; Ova-CM, culture in conditioned medium obtained from mouse ovarian stromal cells; Ova-Co, mouse ovarian stromal cell co-culture; hES, undifferentiated HES cells; spermatozoon, human spermatozoon as the positive control for quantitation of VASA and GDF9 expression; SSEA1[+], sorted cells expressing SSEA1. SSEA1[−], sorted cells not expressing SSEA1. GADPH was used as the internal control for quantitation in real-time PCR. *P < 0.05 (statistical significance). Bars represent mean ± SD of data. Each experiment was repeated for at least three to five times. (D) Harvested cells were sorted for SSEA1-PE by fluorescence activated cell sorting into cells expressing SSEA1 (SSEA1[+]) and not expressing SSEA1 (SSEA1[−]). The cells were then re-examined by immunofluorescence for SSEA1 protein using SSEA1-FITC (green fluorescence). The cell nuclei were co-stained with Hoechst 33342. Scale bars were 100 μm. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Representative images of immunofluorescence study of SSEA1 and VASA expression in the NTU1 HES cells after differentiation for 14 days (A) or 21 days (B), as described in the Materials and methods section. The cells were examined by immunofluorescence for SSEA1 (green fluorescence) and VASA (red fluorescence). The cell nuclei were co-stained with Hoechst 33342. Scale bars were 20 μm for (A), and 50 μm for (B). Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Manual selection of Oct4-EGFP expressing (Oct4-EGFP[+]) cells during differentiation of H9 Oct4-EGFP HES cells, and real-time RT-PCR analysis of the germ cell marker expression of the manually selected cells, as described in the Materials and methods section. H9 Oct4-EGFP HES cells were put into adherent culture for differentiation up to 42 days. (A), dissection and collection of EGFP[+] cells under fluorescence stereomicroscopy; (B–G), real-time RT-PCR analysis of germ cell markers (c-Kit, DAZL, VASA, GDF9, SYCP3, and ZP3) expression. hES was undifferentiated HES cells. GADPH was used as the internal control for quantitation. Bars represent mean ± SD of data. Each experiment was repeated at least three to five times. (H–I), ovarian follicle-like structures viewed under fluorescence microscope in the differentiated H9 Oct4-EGFP cells. EGFP[+], cells showing EGFP under fluorescence stereomicroscope. EGFP[−], cells not showing EGFP under fluorescence stereomicroscope. *P < 0.05. **P < 0.01. ***P < 0.001. Scale bars: 100 μm for A, and 200 μm for H and I. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Real-time RT-PCR analysis of the expression of germ cell markers (VASA and GDF9) in NTU1 HES cells after in vitro differentiation for 21 days by co-treatment with growth factors, as described in the Materials and methods section. (A), using 15% ES (DMEM medium plus 15% fetal bovine serum [FBS]) as the base medium; (B), using Ova-CM (conditioned medium obtained from mouse ovarian stromal cells) as the base medium; (C), using Gra-CM (conditioned medium obtained from human granulosa cells) as the base medium. The growth factor co-treatments include the following: B4, bone morphogenic protein 4 (BMP4); RA, retinoic acid; BSR, combination of BMP4, stem cell factor and retinoic acid; growth factor (−), no growth factor supplement; SCF, stem cell factor. hES, undifferentiated HES cells; GAPDH was used as the internal control for quantitation. Bars represent mean ± SD of data. *P < 0.05; **P < 0.01; ***P < 0.001. Each experiment was repeated at least three to five times. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Real-time RT-PCR analysis of the expression of germ cell markers (Blimp1, VASA, GDF9 and SCP3) in the differentiated NTU1 human embryonic stem cells after in-vitro culture for up to 35 days, as described in the Materials and methods section. The growth factor co-treatments include: BMPs, combination of human bone morphogenic proteins BMP4, BMP7 and BMP8b; GF(−), no growth factor supplement. GAPDH was used as the internal control for quantitation; RA, retinoic acid. Bars represent mean ± SD of data. *P < 0.05 (statistical significance). **P < 0.01. ***P < 0.001. Each experiment was repeated at least three to five times. Reproductive BioMedicine Online 2014 29, 319-332DOI: (10.1016/j.rbmo.2014.05.009) Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions

Reproductive BioMedicine Online 2014 29, 319-332DOI: (10. 1016/j. rbmo Copyright © 2014 Reproductive Healthcare Ltd. Terms and Conditions