A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways

Abstract

Embryonic stem (ES) cells share markers with undifferentiated primordial germ cells (PGCs). Here, we discovered that a cellular state with some molecular markers of male gonocyte induction, including a G1/S phase arrest and upregulation of specific genes such as Nanos2, Tdrd1, Ddx4, Zbtb16 and Plk1s1, can be chemically induced in male mouse ES cells in vitro, which we termed gonogenic stimulated transition (GoST). After longer culture of the resulting GoST cells without chemical stimulation, several molecular markers typical for early gonocytes were detected including the early gonocyte marker Tex101. Motivated by previous studies that found multipotency in cell lines derived from neonatal male germ cells in vitro, we then compared the differentiation potential of GoST cells to that of ES cells in vitro. Interestingly, GoST cells showed equal neurogenic, but enhanced cardiogenic and hepatogenic differentiation compared to ES cells in vitro. This work shows for the first time that some important molecular markers of the first developmental sexual differentiation program can be induced in male mouse ES cells in vitro and defines a novel concept to generate cells with enhanced multipotency.

Figure 1. Gonocyte induction during embryogenesis in vivo and GoST induction in vitro.

(A) A schematic representation of germ cell development during mouse embryogenesis in vivo. PGCs are induced in the epiblast by BMP signaling at E6.25 and their specification is completed by E7.25. PGCs migrate from E8.5–11.5 towards the genital ridge, while their genome is demethylated from E8.5–13.5. PGCs arrest and differentiate within the genital ridge into gonocytes, described here as gonocyte induction from E12.5–13.5. Gonocytes mature within the developing ovaries and testis from E13.5 onwards and ultimately differentiate into the primary oocytes and spermatogonial stem cells, respectively. Green arrows indicate the direction of PGC migration. Drawings were created from observation of mouse embryos (http://www.emouseatlas.org). Markers expressed by the germline during male gonocyte development are shown for the time period of E11.5-birth. TRA98 is expressed by the germline at all time points. Expression of Nanos2 and p27 is upregulated in differentiating PGCs during gonocyte induction from E12.5–13.5 onwards. Tex101 is upregulated in gonocytes from E14–16 onwards. Dppa3 is expressed in gonocytes until E15.5 and gradually decreases in male gonocytes afterwards until it becomes no longer detectable at 1 day postpartum (1dpp). SSEA1 is expressed in PGCs and is downregulated from E12.5–15 onwards. Cxcr4 is expressed in migrating PGCs, but is downregulated from E13.5 onwards. (B) A schematic representation of GoST induction and the treatment procedure in vitro. cES (green) cells are obtained by culturing ES cells (white) in the presence of LIF, βME, RG108 (inhibition of DNMTs) and Ex527 (inhibition of Sirt1) while passaging the cells every 2 days for 10 cell passages (20 days). GoST cells (dark blue) are obtained by culturing cES cells without LIF and without βME, but in the presence of tBHQ (induction of ROS and Nrf2), RG108 and Ex527 for 7 days. Further culture of GoST cells resulted in cells with markers of early gonocytes (GoST-GL cells). Markers of the relevant developmental time points listed in Fig. 1A are indicated for the cells obtained during the in vitro process in Fig. 1B. GoST cells could also be induced towards multiple somatic lineages.

Figure 2. GoST cells survive in conditions deprived of LIF and βME and show decreased proliferation with a G1/S phase arrest compared to ES cells.

(A) Flow cytometric analysis of cell viability by 7-AAD/Annexin V staining for cES cells (green), GoST cells (dark blue), ES cells (white) and ES cells subjected to 7 days of LIF and βME withdrawal (white). Viable cells are located in the lower left quadrant of 7-AAD/Annexin V plots. After 7 days, GoST cells showed considerably higher cell viability (61.1%) compared to ES cells (10.8%). (B) Phase contrast images of cell colonies. At day 7 of LIF and βME withdrawal ES cells were mostly dead and only very flat or elongated cells remained, while GoST cells at day 7 could still be observed in cellular colonies with similar morphology to ES cells at day 0. Scalebar = 50 μm. (C) Dual parameter plot of EdU incorporation (6 hour pulse of EdU) and DNA content (DAPI). The fraction of EdU + cells was lower upon GoST induction (61.7%) compared to ES cells and cES cells (98.9%). EdU + cells had a 3times lower mean EdU intensity upon GoST induction, compared to EdU + ES cells or cES cells (shown in the lower right bar chart). (D) Fluorescence images of EdU incorporation (red). DNA was counterstained with DAPI (blue). Upon GoST induction, the EdU signal intensity was decreased, while some cells within as well as outside the colonies showed no EdU signal. Scalebar = 100 μm. (E) Flow cytometric analysis of cell cycle distribution by measurement of DNA content. Upon GoST induction, the G0/G1 phase was increased and the G2/M phase was decreased which implies a G1/S-phase arrest. DNA was stained with 7AAD. (F) Western blot analysis of cell cycle regulatory proteins. Upon GoST induction, expression of Cyclin D1 and Cyclin B1 was decreased. Western blot data were normalized to α-Tubulin. Cell viability, EdU incorporation, Cell cycle and Western blot data were generated from three independent experiments. Error bars correspond to S.E.M. One star represents p < 0.05 and three stars represent p < 0.001. See Figs S1 and S2.

Figure 3. GoST cells upregulate expression of p27, but retain expression of core pluripotency markers compared to ES cells.

(A) Real-Time PCR analysis of CDK inhibitor markers. Upon GoST induction, expression of p15, p16, p21 and p27 was increased. (B) Western blot analysis of p27. Upon GoST induction, expression of p27 was increased. (C) Immunofluorescence of p27 (red). DNA was counterstained with DAPI (blue). Upon GoST induction, expression of p27 was increased within cells growing in colonies. Scalebar = 50 μm. (D) Real-Time PCR analysis of core pluripotency markers. Upon GoST induction, expression of core pluripotency markers remained mostly unchanged while expression of Nanog and Tbx3 was increased. (E) Immunofluorescence of Nanog, Oct4, Sox2 and ALP (red). DNA was counterstained with DAPI (blue). Upon GoST induction, cells continued to express Nanog, Oct4, Sox2 and ALP. Scalebar = 50 μm. (F) Western blot analysis of Nanog, Oct4 and Sox2. Upon GoST induction, expression of Nanog, Oct4 and Sox2 showed only a slight decrease and remained at similar levels. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. Two stars represent p < 0.01 and three stars represent p < 0.001. See Figure S3.

Figure 4. The expression of germ cell-specific genes is modulated in GoST cells, indicative of gonocyte induction.

(A) Real-Time PCR analysis of naive pluripotency markers. Upon GoST induction, expression of Dazl and Piwil2 was increased, while expression of all other markers remained unchanged. (B) Real-Time PCR analysis of germ cell markers. Upon GoST induction, expression of germ cell markers increased except for expression of Tex101, which remained unchanged. (C) Immunofluorescence of Dazl and Nanos2 (red). DNA was counterstained with DAPI (blue). ES cells and cES cells showed a nuclear and cytoplasmic expression of Dazl within cell colonies, while some cells within the colonies expressed Dazl very strongly in the cytoplasm. Upon GoST induction, all cells located within multicellular colonies continued to express Dazl at the same level as ES cells with low Dazl expression in the nucleus and cytoplasm, while very large cells with strongly increased expression of cytoplasmic Dazl appeared around the cell colonies. White arrowheads indicate cells with strong cytoplasmic Dazl expression. Nanos2 was expressed in ES cells, cES cells and GoST cells and appeared in the form of cytoplasmic round bodies as shown in the zoom images (from white squares). The size of Nanos2 bodies was decreased in cES cells compared to ES cells, but increased in GoST cells compared to ES cells. Yellow arrowheads indicate Nanos2 bodies. Scalebar = 50 μm for upper Dazl and Nanos2 images and scalebar = 5 μm for zoom images. (D) Western blot analysis of Dazl and Nanos2. Upon GoST induction, expression of Dazl and Nanos2 was increased. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Western blot data were normalized to α-Tubulin and generated from three independent experiments. Error bars correspond to S.E.M. One star represents p < 0.05, two stars represent p < 0.01 and three stars represent p < 0.001. See Fig. S4.

Figure 5. Emergence of cells expressing the gonocyte-specific marker Tex101 after release from GoST induction.

(A) A schematic representation of the release from GoST induction, leading to a gonogenic differentiation of GoST cells (dark blue) into GoST-GL cells (light blue) with molecular markers of gonocytes at E13.5–14 (see Fig. 1A for markers). GoST cells were cultured in the presence of βME without additional stimulation for 12 days. (B) Real-Time PCR analysis of naive pluripotency markers after release from GoST induction. After release from GoST induction, expression of Dppa3, Stra8 and Fbxo15 was increased, expression of Dazl remained unchanged and only was increased on day 12, expression of Piwil2, Prdm14 and Rex1 was slightly decreased and expression of Dax1 was strongly decreased. (C) Real-Time PCR analysis of germ cell markers after release from GoST induction. After release from GoST induction, expression of Tex101 was increased, expression of Cxcr4 remained unchanged and only was increased on day 12, expression of Zbtb16 was first decreased and increased again on day 12, expression of Plk1s1 and Ddx4 remained unchanged and expression of Nanos2 and Tdrd1 was decreased. (D) Immunofluorescence of Tex101 (red, white arrowheads) and Dppa3 (green) after release from GoST induction. DNA was counterstained with DAPI (blue). Cells expressing Tex101 were detected at 4, 8 and 12 days after release from GoST induction, but could not be detected in ES cells, cES cells or GoST cells. Zoom images (from white squares) showed that Tex101 was associated with the cell membrane. The simultaneous detection of Tex101 and Dppa3 showed that Dppa3 was expressed in ES cells, cES cells, GoST cells and in GoST-GL cells expressing Tex101. Scalebar = 50 μm for upper Tex101 images and scalebar = 5 μm for Tex101 zoom images and Tex101-Dppa3 simultaneous detection images. Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Error bars correspond to S.E.M. Two stars represent p < 0.01 and three stars represent p < 0.001. Day 7 data served as reference and is identical to that of charts of GoST induction (Fig. 4). See Figure S5.

Figure 6. Downregulation of Cxcr4, SSEA1 and expression of TRA98 after release from GoST induction.

(A) Immunofluorescence of Cxcr4 (white) and SSEA1 (green) after release from GoST induction. Cxcr4 and SSEA1 were detected simultaneously. DNA was counterstained with DAPI (blue). Cxcr4 and SSEA1 were expressed in ES, cES and GoST cells. Cxcr4 was downregulated at 4, 8 and 12 days after release from GoST induction, while SSEA1 remained expressed until 4 days after release from GoST induction and was downregulated afterwards at 8 and 12 days after release. Scalebar = 50 μm. (B) Immunofluorescence of TRA98 (white) and SSEA1 (green) after release from GoST induction. TRA98 and SSEA1 were detected simultaneously. DNA was counterstained with DAPI (blue). TRA98 and SSEA1 were expressed in ES, cES and GoST cells. Expression of TRA98 slightly decreased but remained detectable in the nucleus at 4, 8 and 12 days after release from GoST induction, while SSEA1 remained expressed until 4 days after release from GoST induction and was downregulated afterwards at 8 and 12 days after release. Scalebar = 50 μm. See Fig. S6.

Figure 7. GoST cells exhibit an equal or better in vitro multilineage differentiation potential compared to ES cells.

(A) A schematic representation of the neurogenic differentiation of GoST cells (dark blue) and ES cells (white) into GoST-NL cells (orange) and ES-NL cells (white) for 12 days, respectively. (B) Real-Time PCR analysis of neurogenic markers. Expression of Nes, Chrna2, Zcchc12, Eno2 and Npy was increased in GoST-NL and ES-NL cells. (C) Immunofluorescence of Nestin (red). Cells expressing Nestin were detected in GoST-NL cells and ES-NL cells. Undifferentiated ES cells (day 0) already showed a strong staining for Nestin, while undifferentiated GoST cells growing in colonies (day 7) showed no Nestin signal. Some large cells surrounding GoST cell colonies stained positive for Nestin. Scalebar = 50 μm. (D) A schematic representation of the cardiogenic differentiation of GoST cells (dark blue) and ES cells (white) into GoST-CL cells (pink) and ES-CL cells (white) for 12 days, respectively. (E) Real-Time PCR analysis of cardiogenic markers. Expression of Gata4, Nkx2.5, Mef2c, Mlc2v and αMhc was increased in GoST-CL cells, but only expression of Gata4 and Nkx2.5 was increased in ES-CL cells. (F) Immunofluorescence of cardiac Mhc (red). Cells expressing Mhc were detected in GoST-CL cells, but not in ES-CL cells. Scalebar = 50 μm. (G) A schematic representation of the hepatogenic differentiation of GoST cells (dark blue) and ES cells (white) into GoST-HL cells (brown) and ES-HL cells (white) for 12 days, respectively. (H) Real-Time PCR analysis of hepatogenic markers. Expression of Afp, Hnf4a, Alb and Cyp1a1 was increased in GoST-HL cells, but only expression of Afp, Cyp1a1 and to a lesser degree Hnf4a was increased in ES-HL cells. (I) Immunofluorescence of Hnf4a (red). Cells expressing Hnf4a localized to cell nuclei were detected in GoST-HL cells, but not in ES-HL cells. Scalebar = 50 μm. DNA was counterstained with DAPI (blue). Real-Time PCR data were normalized to Gapdh and generated from duplicates of two independent experiments. Error bars correspond to S.E.M. One star represents p < 0.05, two stars represent p < 0.01 and three stars represent p < 0.001. See Fig. S7.