Embryonic stem cells can form germ cells in vitro

Abstract

Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering.

Fig. 1.

Targeted replacement of the Mvh gene. (a) Genomic organization of the Mvh locus and structure of the targeting vectors used to replace the 3.0-kb region containing the translation initiation site with IRES-lacZ or IRES-GFP and pGK-Neo. Probes used for Southern blots are indicated by shaded bars. (b) Southern blots of HindIII-digested genomic DNA from the ES clones. The wild-type, lacZ knock-in, and GFP knock-in loci gave, respectively, 15.0-, 2.8-, and 2.7-kb bands with the 5′ probe, and 15.0-, 11.1-, and 11.0-kb bands with the 3′ probe. B, BamHI; H, HindIII; N, NotI; P, PstI; Xb, XbaI.

Fig. 2.

In vitro differentiation of knock-in ES cell EBs. Phase contrast (a and b) and fluorescence (c and d) images of GFP knock-in ES cells cultured in the undifferentiated state (a and c), and of EBs after 3 days of culture (b and d; arrow indicates a GFP-positive cell). Shown is a confocal microscopic view merging phase contrast and fluorescence images of EBs after 5 days of culture (e). 5-Bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) staining of lacZ-knock-in ES cells; undifferentiated state (f), 3-day EBs (g; arrow indicates blue-stained cells expressing lacZ), 5-day EBs (h), and 7-day EBs (i). LacZ-positive cells were partially purified from the 5-day EBs and stained with anti-MVH to detect endogenous MVH expression (j and l). Partially purified cells were double-stained with anti-MVH (j) and GCNA1 (k) antibodies, or anti-MVH (l) and anti-SYCP3 (m) antibodies. Anti-MVH staining was detected with horseradish peroxidase-conjugated secondary antibody, and GCNA1 and anti-SYCP3 staining was visualized with FITC-conjugated secondary antibody.

Fig. 3.

MVH expression and RT-PCR analyses of coaggregation cultures of knock-in ES and effector cells. (Upper) Immunoblot with anti-MVH. Extracts (20 μg of protein) of 1-day aggregation culture of ES cells (lane 1), ES cells coaggregated with wild-type M15 cells (lane 2), ES cells coaggregated with BMP4-producing M15 cells (lane 3), ES cells coaggregated with trophoblast cells (lane 4), ES cells coaggregated with STO cells (lane 5), BMP4-producing M15 cells (lane 6), and trophoblast cells (lane 7). (Lower) Poly(A) RNA from undifferentiated ES cells (lanes a) and from coaggregates of ES and BMP-producing cells after 1 day (lanes b) was used for amplification by using primer sets specific for each of the genes indicated below the panels.

Fig. 4.

Coaggregation cultures of knock-in ES and effector cells. GFP-knock-in ES cells were cultured for 1 day as aggregates with trophoblast cells prepared from E7.5 embryos (a and d), BMP4-producing M15 cells (b and e), and the wild-type M15 cells (c and f). (d–f) Phase contrast views of the upper fluorescence images in a–c, respectively. (g–i) Flow cytometric analyses of cells from 1-day-old aggregates of GFP knock-in ES cells mixed with wild-type M15 cells (1:1) (g), the same cells cultured for 1 day with BMP4-producing M15 cells (h), and GFP-positive cells sorted once from the cells in h (i). (j) Inductive effect of BMP4/BMP8b and inhibitory effect of ALK3-IgG fusion protein on germ cell differentiation in vitro. Cell-free supernatants of normal Cos7 cells (open bars) or ALK3-IgG-producing Cos7 cells (filled bars), were added at a 2× dilution to cultures of ES cells coaggregated with BMP4-producing M15 cells for 1 day or to EBs formed by ES cells for 7 days. Values are derived from flow cytometric analyses of four independent experiments.

Fig. 5.

Transplantation of ES-derived germ cells into adult testis. (a) At 5–6 weeks after transplantation, host testes and transplants were stained with 5-Bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal). (b) A magnified view of the transplant. (c) A representative view of seminiferous tubules in a section of the transplant. (d) A section of the transplant involving unpurified ES cells and embryonic gonadal cells. (e) A transplant of gonadal cells on their own. (Scale bars = 100 μm.)

Fig. 6.

(A) Sections of wild-type (a–c) testicular tubules and transplant (d–j) tubules double-stained with anti-β-gal and affinity-purified anti-HSC70t antibodies. Shown is nuclear staining (a, d, and g), anti-β-gal staining (b, e, and h), and anti-HSC70t staining (c, f, and i). (j) Higher magnification (×6) of a stained transplant tubule and merged image of g–i. Arrowheads in g and h indicate positions of Sertoli and myoid cell. (Bar in a for a–f is 50 μm and in j for g–j is 20μm.) (B) Detection of the knock-in allele in genomic DNA extracted from Mvh-lacZ knock-in ES cells (lane 1), sperm from a lacZ-positive transplant (lane 2), and a wild-type seminiferous tubule (lane 3). Primer pairs detecting lacZ (a), Neo (c), and Sry (d) genes were used for PCR (30 cycles; 94°C, 58°C, and 72°C at 1 min each). (b) Southern blot of the PCR product hybridized with labeled lacZ probe. (e) Photograph of sperm purified from transplant seminiferous tubules.