Embryonic Stem Cells: Testing the Germ-Cell Theory

Embryonic Stem Cells: Testing the Germ-Cell Theory

Current Biology Vol 21 No 20 R850 instability is merely a side effect of optimizing the mechanical parameters of the cell for otherwise successful cy...

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Current Biology Vol 21 No 20 R850

instability is merely a side effect of optimizing the mechanical parameters of the cell for otherwise successful cytokinesis. However, it is also possible that the small oscillations observed in successfully dividing cells are beneficial. For example, dividing cells must ensure the proper partitioning of cellular components, which is especially critical for low copy number proteins [19]. Oscillations may thus facilitate thorough mixing of the cytoplasm. Alternatively, oscillations could help the cell decide where exactly to put the division plane. Finally, since spindle microtubules can be anchored directly in the cytoplasm [20], cytoplasmic oscillations may have interesting implications for the mechanics of the anaphase spindle. In sum, the new study from Sedzinski et al. [6] greatly expands our knowledge of the mechanics of cell division and beautifully demonstrates the power of combining quantitative cell biology and computational modeling. References 1. Liu, J., Kaksonen, M., Drubin, D.G., and Oster, G. (2006). Endocytic vesicle scission by lipid phase boundary forces. Proc. Natl. Acad. Sci. USA 103, 10277–10282.

2. Liu, J., Sun, Y., Drubin, D.G., and Oster, G.F. (2009). The mechanochemistry of endocytosis. PLoS Biol. 7, e1000204. 3. Shlomovitz, R., and Gov, N.S. (2008). Physical model of contractile ring initiation in dividing cells. Biophys. J. 94, 1155–1168. 4. Zumdieck, A., Kruse, K., Bringmann, H., Hyman, A.A., and Julicher, F. (2007). Stress generation and filament turnover during actin ring constriction. PLoS One 2, e696. 5. Zhang, W., and Robinson, D.N. (2005). Balance of actively generated contractile and resistive forces controls cytokinesis dynamics. Proc. Natl. Acad. Sci. USA 102, 7186–7191. 6. Sedzinski, J., Biro, M., Oswald, A., Tinevez, J.Y., Salbreux, G., and Paluch, E. (2011). Polar actomyosin contractility destabilizes the position of the cytokinetic furrow. Nature 476, 462–466. 7. Dean, S.O., Rogers, S.L., Stuurman, N., Vale, R.D., and Spudich, J.A. (2005). Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis. Proc. Natl. Acad. Sci. USA 102, 13473–13478. 8. Estey, M.P., Di Ciano-Oliveira, C., Froese, C.D., Bejide, M.T., and Trimble, W.S. (2010). Distinct roles of septins in cytokinesis: SEPT9 mediates midbody abscission. J. Cell Biol. 191, 741–749. 9. Straight, A.F., Field, C.M., and Mitchison, T.J. (2005). Anillin binds nonmuscle myosin II and regulates the contractile ring. Mol. Biol. Cell 16, 193–201. 10. Zhao, W.M., and Fang, G. (2005). Anillin is a substrate of anaphase-promoting complex/ cyclosome (APC/C) that controls spatial contractility of myosin during late cytokinesis. J. Biol. Chem. 280, 33516–33524. 11. Madaule, P., Eda, M., Watanabe, N., Fujisawa, K., Matsuoka, T., Bito, H., Ishizaki, T., and Narumiya, S. (1998). Role of citron kinase as a target of the small GTPase Rho in cytokinesis. Nature 394, 491–494. 12. Rankin, K.E., and Wordeman, L. (2010). Long astral microtubules uncouple mitotic spindles from the cytokinetic furrow. J. Cell Biol. 190, 35–43.

Embryonic Stem Cells: Testing the Germ-Cell Theory The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state. Konrad Hochedlinger1,2,3 The study of pluripotent cell lines has captivated researchers for the past five decades because of their enormous developmental and therapeutic potentials [1]. Historically, pluripotent stem cell lines were derived from teratocarcinomas — tumors of germ cell origin, giving rise to so-called embryonic carcinoma cells (ECCs). This discovery subsequently led to the derivation of embryonic stem cells (ESCs) from explanted preimplantation mouse embryos, and of embryonic germ cells (EGCs) from cultured

primordial germ cells (PGCs). Despite their different origins, ESCs, EGCs and ECCs are molecularly and functionally very similar. These observations raise the important question whether ESCs, like ECCs and EGCs, might be derived from early germ cells [2]. Identifying the origins of ESCs is key for understanding the basic biology of existing pluripotent cell lines, as well as for ongoing efforts to derive new ESC and iPSC lines from species and cell types that have thus far been refractory to stem cell isolation. While it has been assumed that ESCs are the direct product of cells from the

13. Rappaport, R. (1996). Cytokinesis in Animal Cells (Cambridge, UK: Cambridge University Press). 14. O’Connell, C.B., Warner, A.K., and Wang, Y. (2001). Distinct roles of the equatorial and polar cortices in the cleavage of adherent cells. Curr. Biol. 11, 702–707. 15. Thery, M., and Bornens, M. (2008). Get round and stiff for mitosis. HFSP J. 2, 65–71. 16. Robinett, C.C., Giansanti, M.G., Gatti, M., and Fuller, M.T. (2009). TRAPPII is required for cleavage furrow ingression and localization of Rab11 in dividing male meiotic cells of Drosophila. J. Cell Sci. 122, 4526–4534. 17. Szafer-Glusman, E., Giansanti, M.G., Nishihama, R., Bolival, B., Pringle, J., Gatti, M., and Fuller, M.T. (2008). A role for very-long-chain fatty acids in furrow ingression during cytokinesis in Drosophila spermatocytes. Curr. Biol. 18, 1426–1431. 18. Charras, G.T. (2008). A short history of blebbing. J. Microsc. 231, 466–478. 19. Huh, D., and Paulsson, J. (2011). Random partitioning of molecules at cell division. Proc. Natl. Acad. Sci. USA 108, 15004–15009. 20. Kimura, K., and Kimura, A. (2011). Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo. Proc. Natl. Acad. Sci. USA 108, 137–142.

1Institute for Research in Immunology and Cancer (IRIC), Universite´ de Montre´al P.O. Box 6128, Station Centre-Ville Montre´al QC, H3C 3J7 Canada. 2Department of Pathology and Cell Biology, Universite´ de Montre´al P.O. Box 6128, Station Centre-Ville Montre´al QC, H3C 3J7 Canada. E-mail: [email protected], [email protected]

DOI: 10.1016/j.cub.2011.09.012

pluripotent inner cell mass (ICM) of the blastocyst, several observations are consistent with the idea that they may in fact originate from primitive germ cells. For example, expression of the essential epiblast and germ-cell gene Oct4 becomes confined to a few cells in explanted ICM outgrowths [3], which is reminiscent of the emergence of rare Oct4-expressing PGCs from proximal epiblast cells soon after implantation. In agreement, only a small fraction of singly plated epiblast cells yields ESC colonies in conventional culture conditions, suggesting that these may represent rare germ-cell precursors [4]. Moreover, PGCs are the only postimplantation cell type that continues to express several pluripotency genes such as Oct4, Nanog and Sox2 [5]. In this issue of Current Biology, Zwaka and colleagues [6] revisit this important question by deriving ESCs from blastocysts in which the nascent germ-cell lineage has been genetically tagged.

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ESCs and the Germ-Cell State To genetically mark germ cells, the authors utilized an elegant fate mapping system that permanently activates a red fluorescent RFP-reporter gene (ROSA26-loxSTOPlox (lsl)-RFP) in most cells that express the germ-cell specification factor Blimp1 and their progeny (Blimp1-Cre). While no RFP-positive cells were detectable in blastocysts isolated from double transgenic mice, rare RFP-positive cells emerged after their explantation and amplified when cultured in regular ESC-conditions (serum and leukemia inhibitory factor (Lif)). Single-cell expression analysis of RFP-positive vs. RFP-negative cells from early ICM outgrowths confirmed that other germ-cell markers, such as Stella and Prdm14, had also been activated in those cells. Collectively, these findings suggested that explanted blastocysts transiently activate a transcriptional program specific for PGCs. To assess whether Blimp1-positive blastocyst-derived cells are functional germ cells, the authors transplanted Blimp1-positive ICM outgrowth cells into E8.5 germ-cell-deficient embryos. Indeed, Blimp1-positive cells migrated to the genital ridges and upregulated the germ line maturation marker Mvh, indicating that Blimp1-positive cells have migratory and differentiation potential akin to that of endogenous PGCs. When blastocysts containing Blimp1-Cre and the RFP reporter were put in culture containing serum and Lif for ESC derivation, around 80% of the resultant ESC lines were RFP-positive, indicating that transit through a germ-cell-like state may be obligatory for ESC derivation. In support of this interpretation, Zwaka and colleagues [6] showed that the sorting and explantation of RFP-positive cells from early ICM outgrowths gives rise to ESC lines nine times more efficiently than bulk ICM cells do. Taken together, these results show that activation of Blimp1 predicts successful ESC derivation from blastocysts. In an effort to genetically test whether activation of a germ-cell program is required for ESC derivation, the authors attempted to derive ESC lines from blastocysts deficient for Blimp1. Loss of Blimp1 in development results in defects in PGC migration and specification. Surprisingly, however, Blimp1-null ESCs were received at

Transient activation of germ-cell program PGC-like state

Serum Lif

Blastocyst explantation


Selection of Blimp1+ outgrowth cells Most mouse strains refractory to ESC derivation Naive ESCs Suppression of germ-cell program Maintenance of epiblast state Facilitates derivation of ESC lines from non-permissive strains and rats Current Biology

Figure 1. The germ-cell program and ESC derivation. This scheme summarizes the two different approaches to ESC derivation in serum/Lif and 2i, respectively, and their effect on transient activation of a germ-cell program.

expected Mendelian ratios from heterozygous mutant intercrosses. While this result clearly documents that Blimp1 is not required for ESC derivation, it does not unequivocally show that transit through a PGC state per se is not essential as Blimp1 deficiency does not entirely deplete PGCs in vivo [7]. It should be informative to assess whether EGCs can be derived from the residual PGCs present in Blimp1–/– embryos. Bypassing the Germ-Cell Program ESCs have originally been derived and maintained in media containing serum or Bmp4 and Lif. More recently, Austin Smith’s lab has described more defined culture conditions, which comprise two chemical inhibitors of the Fgf/Erk and Gsk3 kinases, — dubbed ‘2i’ — that are thought to counteract ESC differentiation [8]. Importantly, 2i media gives rise to ESC lines more reproducibly and efficiently than serum/Lif culture with almost every ICM cell acquiring the potential to give rise to an ESC line [9]. In further contrast to serum/Lif, 2i facilitates the derivation of ESC lines from mouse strains that have previously been considered recalcitrant to ESC isolation [8] as well as from rats [10]. Intriguingly, the majority of ESC lines recovered by Zwaka and colleagues [6] in 2i from blastocysts carrying Blimp1-Cre and the RFP reporter were RFP-negative. This unexpected result suggests that blocking Fgf/Erk

and/or Gsk3 signaling may suppress a germ-cell program and directly endow epiblast cells with self-renewal potential, thus circumventing the need to pass through a germ-cell state. Taken together, these observations demonstrate that mouse ESC derivation can be achieved via different routes: through a germ-cell-like intermediate in serum/Lif and directly from epiblast in 2i conditions [9] (Figure 1). The finding that a pluripotent ground state can be attained from epiblast cells via different routes, depending on the choice of culture conditions, may explain the previous failure to derive ESC lines from certain mouse strains and other animal species; that is, epiblasts from non-permissive mouse strains, like NOD, or other rodent species, such as rats, may have been unsuccessful in Lif/Bmp because no germ-cell program was activated. 2i treatment presumably bypasses this requirement by directly stabilizing a self-renewing epiblast state. This notion could be easily tested by assessing Blimp1 activation in ICM outgrowths from NOD blastocysts and rats upon exposure to Lif/Bmp. If induction of pluripotency by defined transcription factors follows similar principles as ESC derivation, one might expect that somatic cells from some strains of mice and other species should also be differentially amenable to reprogramming into induced pluripotent stem cells (iPSCs) [11]. In

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agreement with this notion, Silva et al. [12] observed that reprogramming of fibroblasts from a 129/MF1 hybrid strain of mice gives rise mostly to partially reprogrammed iPSCs that rarely progress to pluripotency unless treated with 2i. Similarly, the establishment of rat iPSCs requires 2i culture. What consequences might these findings have for human ESC/iPSC research? Human ESCs are fundamentally different from mouse ESCs in that they require bFgf and Activin A for their stable propagation [1]. Interestingly, exposure of mouse blastocysts, ESCs or postimplantation embryos to bFgf and Activin A gives rise to so-called epiblast stem cells (EpiSCs) [13,14], which are very similar to human ESCs and seem to represent a developmentally more advanced or ‘primed’ state compared with the more primitive or ‘naive’ state of mouse ESCs [15]. The findings by Chu et al. [6] thus raise the interesting possibility that progression of epiblast cells towards a germ-cell fate, either by enforced expression of certain transcription factors [1,9] or by exposure of cells to germ-cell-inducing cytokines, might be sufficient to derive stable naive ESC/iPSC lines in humans and other species. Recent exciting progress in

identifying molecules that coax pluripotent cells into germ cells may aid in these efforts [16]. References 1. Stadtfeld, M., and Hochedlinger, K. (2010). Induced pluripotency: history, mechanisms, and applications. Genes Dev. 24, 2239–2263. 2. Zwaka, T.P., and Thomson, J.A. (2005). A germ cell origin of embryonic stem cells? Development 132, 227–233. 3. Buehr, M., and Smith, A. (2003). Genesis of embryonic stem cells. Phil. Trans. R. Soc. Lond. 358, 1397–1402, discussion 1402. 4. Brook, F.A., and Gardner, R.L. (1997). The origin and efficient derivation of embryonic stem cells in the mouse. Proc. Natl. Acad. Sci. USA 94, 5709–5712. 5. Durcova-Hills, G., Tang, F., Doody, G., Tooze, R., and Surani, M.A. (2008). Reprogramming primordial germ cells into pluripotent stem cells. PLoS One 3, e3531. 6. Chu, L.F., Surani, M.A., Jaenisch, R., and Zwaka, T.P. (2011). Blimp1 expression predicts embryonic stem cell development in vitro. Curr. Biol. 21, 1759–1765. 7. Saitou, M., Payer, B., O’Carroll, D., Ohinata, Y., and Surani, M.A. (2005). Blimp1 and the emergence of the germ line during development in the mouse. Cell Cycle 4, 1736–1740. 8. Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., and Smith, A. (2008). The ground state of embryonic stem cell self-renewal. Nature 453, 519–523. 9. Nichols, J., and Smith, A. (2011). The origin and identity of embryonic stem cells. Development 138, 3–8. 10. Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., and Smith, A. (2008). Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298. 11. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse

Phagocytosis: Coupling of Mitochondrial Uncoupling and Engulfment Clearance of apoptotic cells by phagocytes avoids triggering an inflammatory response. A new study reveals that phagocytes dissipate their mitochondrial proton electrochemical gradient to allow for the ingestion of more apoptotic corpses. Mitochondria are therefore involved in all aspects of apoptosis, from its activation through to the phagocytosis of dead cells. Grazia M. Cereghetti and Luca Scorrano Sustained cell proliferation during development, tissue renewal or in the course of the immune response is accompanied by the production of excess or damaged cells that die by apoptosis. The accumulation of these cells may lead to tissue damage and inflammation: specialized systems therefore efficiently remove them [1,2].

Phagocytes are deputed to the clearance of apoptotic cells and are able to engulf multiple cells in order to adapt their ‘cleaning efficiency’ to the rate of apoptotic cell accumulation. In recent years, some of the crucial steps in the phagocytosis of apoptotic cells, as well as the principal players in the phagocytic process, have been elucidated [3,4]. Dying cells release signals to attract the motile phagocytes. The two cells make






embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676. Silva, J., Barrandon, O., Nichols, J., Kawaguchi, J., Theunissen, T.W., and Smith, A. (2008). Promotion of reprogramming to ground state pluripotency by signal inhibition. PLoS Biol. 6, e253. Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195. Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., and McKay, R.D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196–199. Nichols, J., and Smith, A. (2009). Naive and primed pluripotent states. Cell Stem Cell 4, 487–492. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S., and Saitou, M. (2011). Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532.

1Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA. 2Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA. 3Harvard Stem Cell Institute, 42 Church Street, Cambridge, MA 02138, USA. E-mail: [email protected] edu

DOI: 10.1016/j.cub.2011.09.024

physical contact via markers that are released from the apoptotic cell and bind to receptors on the phagocyte, inducing a signaling cascade that prepares the phagocyte membrane for the internalization of the dead cell. Several molecules are involved in engulfment by phagocytes, including: brain angiogenesis inhibitor 1 (BAI1), a transmembrane protein highly expressed in the brain; Rac GTPases, which remodel the cytoskeleton; ELMO, an evolutionarily conserved cytoplasmic engulfment protein; and the unconventional guanine nucleotide exchange factor and Rho GTPase activator Dock180 [5]. Despite our knowledge of some key molecular steps in the engulfment cascade, how a single phagocyte can serially internalize many apoptotic cells is unclear. Park et al. [6] have now shown, in a recent issue of Nature, that serial internalization of apoptotic cells unexpectedly depends on