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news and views who later had preeclampsia than in the controls already at 13 to 16 weeks of gestation. The greatest difference occurred during the weeks before the onset of preeclampsia, coincident with the increases in sFlt1. The findings strongly suggested that increased levels of sFlt1 and reduced levels of PlGF predict the subsequent development of preeclampsia. These experimental and clinical observations also quickly established angiogenic regulation as pivotal to preeclampsia and indicated new diagnostic and even therapeutic avenues. However, the story by no means ended there. Karumanchi and his colleagues then pursued other placental-derived molecules that could participate in angiogenic regulation6. They showed that the transforming growth factor-β (TGF-β) co-receptor, endoglin, gives rise to a previously undescribed placenta-derived soluble endoglin (sEng) whose expression is elevated in the sera of preeclamptic women6. sEng is not an Eng splice variant but is instead an N-terminal Eng cleavage product, whose concentrations correlated with disease severity in preeclamptic women and decreased after delivery. sEng inhibited formation of capillary tubes in vitro and induced vascular permeability and hypertension in vivo. sEng was then administered to pregnant rats, where its effects were amplified by sFlt1 co-administration. This combination treatment in pregnant rats induced hemolysis, elevated liver enzyme levels and induced low platelets (HELLP Syndrome), a severe version of preeclampsia in humans.
The team then showed that sEng impairs binding of TGF-β1 to its receptors and therefore the downstream signaling events, including activation of endothelial nitric oxide synthase and vasodilation. These findings suggested that sEng leads to dysregulated TGF-β signaling in the vasculature and thus added sEng to the list of molecules associated with preeclampsia. Indeed, the same cohort of individuals with preeclampsia previously used by Levine et al.5 to validate the association of VEGF, PlGF and sFlt-1 with preeclampsia in humans was also used in a separate study using the same nested case-control design to show the clinical relevance of sEng7. To find other angiogenic regulators, current studies focus on upstream molecular mechanisms that might influence Flt1 splice variant regulation during pregnancy. For example, Kanasaki et al.8 have recently shown that pregnant mice deficient in catechol-Omethyltransferase (COMT) show a preeclampsia–like condition resulting from an absence of 2-methoxyoestradiol, a natural metabolite of estradiol whose expression is elevated during the third trimester of normal human pregnancy and that inhibits hypoxia-inducible factor-1α . The COMT-deficient mice had increased sFlt1 production, though sEng expression was not determined. Exogenous 2-ME administration to Comt−/− pregnant mice ameliorated the preeclampsia–like features without toxicity, suppressed placental hypoxia-inducible factor-1α expression and decreased the elevated sFlt1 levels by as yet unknown mechanisms. The authors
also showed that COMT and 2-ME expression was significantly lower in women with severe preeclampsia8. Functional COMT polymorphisms are associated with endometriosis risk and the timing of menopause, and they could also affect preeclampsia. Nevertheless, much work needs to be done, given that 2-ME is known to inhibit tumor angiogenesis by mechanisms that involve apoptosis9. How a putative angiogenesis inhibitor protects from preeclampsia is not yet clear. The new information on preeclampsia angiogenesis from the last decade has markedly advanced our knowledge of preeclampsia and its pathogenesis. Samples from an earlier pre eclampsia treatment trial showed how rapidly new knowledge about sFlt1 and sEng4,6 can be potentially translated into clinical medicine5,7. These newly appreciated angiogenesismodifying molecules involved in preeclampsia offer diagnostic, and hopefully also therapeutic, avenues that will tame a devastating and barely treatable human disease in the near future. 1. Sibai, B. et al. Lancet 365, 785–799 (2005). 2. Khaliq, A. et al. Lab. Invest. 79, 151–170 (1999). 3. Park, J.E. et al. J. Biol. Chem. 269, 25646–25654 (1994). 4. Maynard, S.E. et al. J. Clin. Invest. 111, 649–658 (2003). 5. Levine, R.J. et al. N. Engl. J. Med. 350, 672–683 (2004). 6. Venkatesha, S. et al. Nat. Med. 12, 642–649 (2006). 7. Levine, R.J. et al. N. Engl. J. Med. 355, 992–1005 (2006). 8. Kanasaki, K. et al. Nature 453, 1117–1121 (2008). 9. Davoodpour, P. & Landström, M. J. Biol. Chem. 280, 14773–14779 (2005).
From stem cells to germ cells and back again Marco Conti & Linda Giudice Producing germ cells in vitro would open important new avenues for regenerative medicine, and obtaining alternative sources of pluripotent stem cells is desirable. In this regard, Geijsen et al. have shown that it is possible to differentiate stem cells into cells similar to male gametes, whereas Guan et al. reported the ability to turn undifferentiated germ cells into pluripotent stem cells. Unlike all other cells of the body, germ cells have the unique property of simultaneously being highly differentiated enough to participate in the complex processes of fertilization but also Marco Conti and Linda Giudice are in the Center for Reproductive Sciences and the Department of Obstetrics and Gynecology and Reproductive Sciences, University of California at San Francisco, 513 Parnassus Avenue, Health Sciences West 1656, Box 0556, San Francisco, California 94143, USA. e-mail:
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retaining pluripotent properties, allowing them to support the development of a new indivi dual. Given these distinctive traits, for decades, scientists have attempted to reproduce germ cell differentiation, or gametogenesis, in a culture dish. Unlimited production of such cells (both sperm and eggs) would have tremendous impact in the most disparate settings. It would provide simple models amenable to dissecting the molecular basis of germ cell differentiation, which in many aspects is still unknown. But also, in the field of medicine, production of an
unlimited number of oocytes would remove one of the major obstacles to therapeutic cloning—namely, the severely limited availability of donated eggs. Likewise, treatment of infertility by transplantation, a need becoming ever more pressing as the number of young cancer survivors with infertility increases, could be another application. Primordial germ cells (PGCs) arise from a set of progenitor cells set aside during early embryonic development1. This tiny population of cells expands while migrating through the
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Figure 1 In vitro differentiation of ES cells into gametes and back. (a) ES cells can differentiate into PGCs and haploid germ cells. Although unable to fertilize an egg, these haploid cells can be injected into oocytes and support embryo differentiation. (b) In the reverse process, testicular SSCs can dedifferentiate into pluripotent ES-like stem cells in vitro. EB, embryoid body.
hindgut to finally colonize the gonads. During the ensuing differentiation in the gonad, germ cells undergo a specialized cell division (meiosis) and acquire morphological traits that set them apart from all other cells. In addition, germ cells require a specialized environment, or niche, provided by the supporting somatic cells in which to differentiate. Given the complex developmental processes involved and the cross talk with supporting somatic cells, generation of gametes in vitro, with few exceptions, has been a disappointing and frustrating endeavor. Culture of testicular explants and attempts to immortalize germ cells are the strategies that have most often been tried, but the outcome has been unclear and often controversial2–4. With new developments in the embryonic stem (ES) cell field, these obstacles to in vitro production of gametes that a few years ago seemed insurmountable are beginning to crumble. ES cells are cultured cells derived from the inner cell mass of a blastocyst (an 80–100-cell embryo). They can be either maintained in an uncommitted and self-perpetuating state or induced to differentiate into any or all of the three germ layers of the early embryo. Publications that have appeared in the last four years provide indisputable evidence that ES cells can also be induced to differentiate toward the germ cell lineage and that cells morphologically, and in some cases functionally, resembling germ cells can be produced in vitro5,6. What is even more compelling, manipulations of immature cells derived from
the mouse testis show that the reverse is also possible7,8. That is, under appropriate culture conditions, adult spermatogonial stem cells (SSCs), the precursors of spermatogenic cells, can be induced to revert to a phenotype that resembles that of ES cells, functionally acquiring most of the ES cell properties, including the ability to differentiate into all cell lineages of the body. The early work of Hans Schöler and his collaborators provided a first hint that germ cell derivation from ES cells is possible, at least for female germ cells9. They showed the development of structures resembling ovarian follicles and oocytes in cultured ES monolayers9. This derivation of oocyte-like cells from ES cells in vitro has been confirmed by others; however, the functionality of these oocytes is yet to be determined10. This initial breakthrough was recently extended to male germ cells. Under standard culture conditions, ES cells form cell aggregates (embryoid bodies) and rapidly lose expression of markers of pluripotency as they differentiate. The strategy followed by Geijsen et al.6 is based on the observation that when cells from embryoid bodies are transferred to culture medium containing retinoic acid, known to stimulate the proliferation of PGCs while leading to the differentiation of ES cells, a substantial number of cells continue to express a surface marker called stage-specific embryonic antigen-1 (SSEA1) (ref. 6) that is shared by PGCs and undifferentiated ES cells (Fig. 1a). In addition, these cells undergo
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changes in DNA methylation and express enzymes that are hallmarks of PGC differentiation1. To extend these findings, Geijsen et al.6 used the expression of SSEA1 to select a more homogenous population that has many of the traits of germ cells. Exploring whether these embryoid body–derived PGCs differentiate into functional gametes, Geijsen et al.6 found that embryoid body–derived germ cells expressed two genes associated with sperm differentiation in vivo. What is remarkable is their observation that markers of supporting somatic cells of the gonad were also detectable after long-term culture of the embryoid bodies, strongly suggesting that germ cell and the supporting somatic cell differentiation were taking place simultaneously in these embryoid body cultures. In addition to these findings, the team also found evidence that a subpopulation of these germ cells underwent meiosis and produced haploid male germ cells6. In particular, a small population of cells had haploid DNA content (1N), as detected by FACS analysis. And when these ‘haploid’ cells were injected into oocytes, they transmitted a GFP transgene into the developing embryo that in some cases allowed progression of the embryo up to the blastocyst stage. This data from George Daley’s team showing that ES cells can be induced to differentiate into PGCs and then into haploid male germ cells6 is consistent with an earlier report by Toshiaki Noce’s group, who also showed differentiation of germ cells from ES cells5. In this earlier study, a different marker of germ cell
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news and views differentiation (the mouse homolog of DDXR (also known as VASA)) and slightly different culture conditions were used. Although no functional data of the germ cells produced in vitro were provided, Toyooka et al.5 showed that these embryoid body–derived germ cells can be transplanted under the adult testis capsule and form tubules containing germ cells at advanced stages of differentiation, including elongated spermatids and spermatozoa. These initial reports did not meet the gold standard of demonstrating that offspring are produced with these in vitro–derived germ cells; however, subsequent publications have shown that this is indeed the case. Notably, Wolfgang Engel and his collaborators were able to produce offspring by oocyte injection of in vitro–derived sperm-like cells, although their viability was compromised11. The ES cell–germ cell transition is plastic, as the reverse transition occurs, as well. For example, adult spermatogonial stem cells isolated from the testis, under appropriate culture conditions, acquire a phenotype similar to that of undifferentiated ES cells7 (Fig. 1b). Guan et al.7 used transgenic mice expressing EGFP under the control of a germ cell–specific promoter (Stra8) to isolate cells similar to SSCs. They then exposed these cells to different culture conditions and followed the appearance of markers of pluripotency7. Several cell lines were derived from these heterogeneous populations of cells, some of them morphologically similar to and expressing many of the markers of ES cells. Functionally, these cells behave
as ES cells because they develop the ability to differentiate into the three major embryonic lineages and to produce teratomas. The developmental potential of these cells was confirmed by injection into blastocysts and detection of a high percentage of chimerism in the offspring. The team concluded that either SSCs are multipotent or, at least, the culture system allows them to revert to an uncommitted, undifferentiated state7. It should be noted that an earlier study in SSC culture from neonatal testis8 was able to identify a population of cells similar to that derived from the adult testis. In this latter experimental paradigm, it could be conclusively determined that SSCs can dedifferentiate to a pluripotent state12. Taken together, these reports demonstrate that gametogenesis can be attained in vitro and, conversely, that committed adult germ cells can revert to a pluripotent, ES-like state— both remarkable accomplishments. But this is only the beginning, as the efficiency of the process is difficult to evaluate but in general seems quite low, and it must be improved for further development for therapeutic application. In addition, a surprising finding is the short time required to recapitulate germ cell differentiation in these in vitro gametogenesis models. At one extreme, only 72 h are sufficient to reach the haploid state in vitro11. Thus, more studies are warranted to clarify whether all the steps in the meiotic division (for example, the correct complement of chromosomes and their segregation in haploid cells, as well as the occurrence of recombination) are correctly executed
in such a short time by these embryoid body– derived germ cells. Indeed, in vivo aneuploidy is often associated with male infertility and recurrent pregnancy loss in females, and this situation must be avoided if this technique is to be developed for reliable fertility therapies. Likewise, epigenetic changes may not be correctly executed in vitro, as suggested by inefficient embryo development and pregnancy failure. Finally, given the finding that human ES cells express some of the germ cell markers13, there is an obvious question to be posed—namely, can these strategies be applied to human ES cells? The bet is that we will not have a long wait to find out. Indeed, the human equivalent of the Guan et al.7 study has now been confirmed and reported14. 1. Seydoux, G. & Braun, R.E. Cell 127, 891–904 (2006). 2. Hofmann, M.C., Hess, R.A., Goldberg, E. & Millan, J.L. Proc. Natl. Acad. Sci. USA 91, 5533–5537 (1994). 3. Feng, L.X. et al. Science 297, 392–395 (2002). 4. Kierszenbaum, A.L., Abdullah, M., Ueda, H. & Tres, L.L. Adv. Exp. Med. Biol. 219, 535–560 (1987). 5. Toyooka, Y., Tsunekawa, N., Akasu, R. & Noce, T. Proc. Natl. Acad. Sci. USA 100, 11457–11462 (2003). 6. Geijsen, N. et al. Nature 427, 148–154 (2004). 7. Guan, K. et al. Nature 440, 1199–1203 (2006). 8. Kanatsu-Shinohara, M. et al. Cell 119, 1001–1012 (2004). 9. Hubner, K. et al. Science 300, 1251–1256 (2003). 10. Novak, I. et al. Stem Cells 24, 1931–1936 (2006). 11. Nayernia, K. et al. Dev. Cell 11, 125–132 (2006). 12. Kanatsu-Shinohara, M. et al. Biol. Reprod. 78, 681– 687 (2008). 13. Clark, A.T. et al. Hum. Mol. Genet. 13, 727–739 (2004). 14. Conrad, S. et al. Nature published online, doi:10.1038/ nature07404 (8 October 2008).
Making eggs: is it now or later? Teresa K Woodruff Although it has been thought that female mammals develop all the eggs they will ever have by the time they are born, new research suggesting otherwise has now sparked a debate. Although long held as true, the notion that females are born with a finite number of nonreplenishable oocytes is somewhat remarkable. How do eggs, made during embryonic life, ‘last’ so long? This traditional narrative of oogenesis was recently challenged; indeed, Teresa K. Woodruff is the Watkins Professor of Obstetrics and Gynecology in the Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, 303 East Superior, Lurie 10-117, Chicago, Illinois 606113015, USA. e-mail:
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the paper by Johnson et al.1 highlighted here suggests the radical notion that the adult ovary must and does have the capacity to create new oocytes. After it was published, the reproductive science community collectively asked, “How did we miss this?,” and thus new studies were launched to validate these findings. To date, however, the overall evidence still favors the established dogma. Nonetheless, the field has been energized by this vigorous debate. The purpose of germ cells, from pollen grains to mouse oocytes, is to produce future progeny and recreate the ‘self ’, not necessar-
ily for the individual but for the species as a whole. Unraveling the mechanisms that drive germ cell development and persistence in mammals is fascinating and challenging. The prevailing dogma of mammalian reproduction states that females lose the capacity to produce germ cells during fetal development and are born with a finite number of follicle-enclosed oocytes, only a small number of which will be ovulated after puberty (Fig. 1). Several recent studies from the laboratory of Jonathan Tilly at Harvard led to the hypothesis that adult mice are capable of regenerating oocytes, suggesting the
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