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F-Class Cells: New Routes and Destinations for Induced Pluripotency
Cell Stem Cell
Previews F-Class Cells: New Routes and Destinations for Induced Pluripotency Simon E. Vidal,1 Matthias Stadtfeld,1,* and Eftychia Apostolou2,* 1The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA 2Division of Hematology/Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10021, USA *Correspondence: [email protected] (M.S.), [email protected] (E.A.) http://dx.doi.org/10.1016/j.stem.2014.12.007
A series of five related publications describe an alternative pluripotent state that is dependent on continuous high levels of exogenous reprogramming factor expression. A comprehensive effort to molecularly compare the acquisition of this state to induced pluripotency aims at providing new insights into the mechanisms underlying cellular reprogramming.
The ectopic expression of transcription factors (TFs) associated with the embryonic state in somatic cells affords researchers an accessible source of pluripotent stem cells. The question of how TFs achieve this remarkable conversion has fueled increasingly sophisticated analyses of reprogramming intermediates (Papp and Plath, 2013). While aspects of the reprogramming process—such as the starting somatic cell type, reprogramming factor stoichiometry, and culture conditions—can influence the properties of induced pluripotent stem cells (iPSCs), it is generally assumed that reprogramming yields stem cell lines that are highly similar to each other. In fact, the desire to generate iPSCs that are as close as possible to fertilization-derived embryonic stem cells (ESCs) has been a driving force behind iPSC research ever since the discovery of induced pluripotency. In a series of noteworthy publications (Benevento et al., 2014; Clancy et al., 2014; Hussein et al., 2014; Lee et al., 2014; Tonge et al., 2014), a consortium of international research teams challenges the notion of a singular end-point of reprogramming and takes an ambitious approach to understand an alternative pluripotent state and its acquisition. Using an inducible transposon system, Andras Nagy and colleagues from the University of Toronto report in Nature that very high (5- to 100-fold of the levels seen in ESCs) and persistent expression of the ‘‘Yamanaka factors’’ Oct4, Klf4, Sox2, and c-Myc (OKSM) in mouse fibroblasts reproducibly establish a pluripotent state that is different from that of conven-
tional iPSCs in several aspects (Tonge et al., 2014) (Figure 1). Cells generated in this manner—referred to as F-class cells due to the fuzzy borders of colonies obtained—express Nanog and endogenous Oct4, but not most other ESC-associated genes. Instead, F-class cells express high levels of genes associated with cell fate commitment, suggesting that they are primed for differentiation. Additional noteworthy features of these cells are an ultrafast cell cycle transition, the capacity to grow in absence of leukemia inhibitory factor (LIF), and the propensity to not aggregate in suspension culture. Maybe most importantly, F-class cells require continuous high levels of exogenous OKSM for their propagation, thereby deviating from the concept of transgene independence that has been established as a hallmark of induced pluripotency. Nevertheless, upon shutdown of exogenous factors, F-class cells appear to undergo multilineage differentiation in vitro and in the context of teratomas, but they fail in stricter pluripotency assays such as chimera formation. Intriguingly, F-class cells treated with histone deacetylase inhibitors convert into transgene-independent cells similar to ESCs that are capable of germline transmission within days of treatment (Tonge et al., 2014). This transition suggests that F-class reprogramming does not negatively impact upon genomic integrity. Formation of F-class cells normally requires 2–3 weeks of high levels of OKSM expression, representing conditions under which iPSCs form very infrequently. However, when exogenous OKSM levels
are experimentally lowered at an arbitrary point during reprogramming, fully pluripotent transgene-independent cell lines appear more frequently. In a second paper in Nature, the same group exploits this observation to conduct an exceptionally deep analysis of the transcriptome, proteome, and epigenome of bulk cell populations at various points along these two routes to pluripotency (Hussein et al., 2014). In addition, three papers in Nature Communications dissect in great detail specific aspects of F-class and iPSC reprogramming, in particular dynamic changes of small RNAs (Clancy et al., 2014), protein abundance (Benevento et al., 2014), and DNA methylation (Lee et al., 2014). Fittingly, the authors refer to their combined efforts as Project Grandiose. A corresponding online portal (http://www.stemformatics.org) will be a versatile resource for future research into the biology of reprogramming. The authors’ own analyses largely confirm previous genome-wide studies of prospectively isolated reprogramming intermediates and ‘‘pre-iPSC’’ lines (Koche et al., 2011; Polo et al., 2012), although a number of molecular differences exist that might be explained by the particularities of their reprogramming system. This includes the rapid and global disappearance of the repressive H3K27me3 histone mark upon OKSM expression, which is reacquired during the transition to the F-state. DNA methylation at somatic loci occurs gradually during both F-class and iPSC reprogramming, while demethylation of ESC-associated loci is impaired in the F-class trajectory (Hussein et al., 2014).
Cell Stem Cell 16, January 8, 2015 ª2015 Elsevier Inc. 9
Cell Stem Cell
Previews
to not aggregate in suspension culture opens up the possibility of large-scale expansion in stirring incubators. However, it remains to be seen whether this particular F-class feature is compatible with differentiation protocols that require the formation of embryoid bodies as an intermediate step. As it is, the discovery of Fclass cells offers a new and conceptually stimulating approach to study transcription-factor-induced pluripotency. REFERENCES
Figure 1. Illustration of the Reprogramming Routes Leading to Conventional iPSCs and Newly Discovered F-Class Cells Note the importance of different levels of Oct4, Klf4, Sox2, and c-Myc (OKSM) expression. Key cellular and functional differences of the two cell types are shown on the right (see the text for molecular differences). F-class cells can be converted into iPSCs by exposure to histone deacetylase inhibitors (HDACi), while at least some F-class features can be established in iPSCs by high OKSM levels in the presence of Janus kinase inhibitors (JAKi) and the absence of leukemia inhibitory factors (LIF).
Additional important observations are the intriguing discovery of new proteincoding transcripts and unexpected layers of post-transcriptional gene regulation, such as intron retention for many highly transcribed genes in the F-state (Hussein et al., 2014). The description of an alternative, albeit transgene-dependent, pluripotent state is provocative and raises several questions. For example, can additional pluripotent states be obtained upon expression of OKSM or other factor combinations able to drive iPSC formation? Of note, Tonge et al. themselves mention the generation of a second class of cells with similar morphology but different gene expression from that of ESCs (referred to as C-class) in high OKSM conditions. Determining DNA occupancy of reprogramming factors likely will shed light on why alternative types of pluripotent cells emerge, as it has for iPSC reprogramming (Papp and Plath, 2013). The observation
that many ESC-associated genes remain silent in F-class cells points toward a repressive role of OKSM when expressed at high levels, which supports a documented inhibitory effect of these factors on iPSC formation late in reprogramming (Golipour et al., 2012). Histone deacetylase inhibitors can overcome this repression, raising the question of whether OKSM in F-class cells interacts with the same repressive complexes that have been proposed to constitute reprogramming roadblocks (Rais et al., 2013). Will some of the unique properties of F-class cells facilitate the use of pluripotent cells in downstream applications? Because mouse pluripotent cells can be easily and quickly expanded to large numbers, the ultrafast cycling time of Fclass cells may not be all too relevant for practical purposes. This might be different if human F-class cells with similar properties are discovered. As suggested by Tonge et al., the propensity of F-class cells
10 Cell Stem Cell 16, January 8, 2015 ª2015 Elsevier Inc.
Benevento, M., Tonge, P.D., Puri, M.C., Hussein, S.M., Cloonan, N., Wood, D.L., Grimmond, S.M., Nagy, A., Munoz, J., and Heck, A.J. (2014). Nat. Commun. 5, 5613. Clancy, J.L., Patel, H.R., Hussein, S.M., Tonge, P.D., Cloonan, N., Corso, A.J., Li, M., Lee, D.S., Shin, J.Y., Wong, J.J., et al. (2014). Nat. Commun. 5, 5522. Golipour, A., David, L., Liu, Y., Jayakumaran, G., Hirsch, C.L., Trcka, D., and Wrana, J.L. (2012). Cell Stem Cell 11, 769–782. Hussein, S.M., Puri, M.C., Tonge, P.D., Benevento, M., Corso, A.J., Clancy, J.L., Mosbergen, R., Li, M., Lee, D.S., Cloonan, N., et al. (2014). Nature 516, 198–206. Koche, R.P., Smith, Z.D., Adli, M., Gu, H., Ku, M., Gnirke, A., Bernstein, B.E., and Meissner, A. (2011). Cell Stem Cell 8, 96–105. Lee, D.S., Shin, J.Y., Tonge, P.D., Puri, M.C., Lee, S., Park, H., Lee, W.C., Hussein, S.M., Bleazard, T., Yun, J.Y., et al. (2014). Nat. Commun. 5, 5619. Papp, B., and Plath, K. (2013). Cell 152, 1324– 1343. Polo, J.M., Anderssen, E., Walsh, R.M., Schwarz, B.A., Nefzger, C.M., Lim, S.M., Borkent, M., Apostolou, E., Alaei, S., Cloutier, J., et al. (2012). Cell 151, 1617–1632. Rais, Y., Zviran, A., Geula, S., Gafni, O., Chomsky, E., Viukov, S., Mansour, A.A., Caspi, I., Krupalnik, V., Zerbib, M., et al. (2013). Nature 502, 65–70. Tonge, P.D., Corso, A.J., Monetti, C., Hussein, S.M., Puri, M.C., Michael, I.P., Li, M., Lee, D.S., Mar, J.C., Cloonan, N., et al. (2014). Nature 516, 192–197.
Previews F-Class Cells: New Routes and Destinations for Induced Pluripotency Simon E. Vidal,1 Matthias Stadtfeld,1,* and Eftychia Apostolou2,* 1The Helen L. and Martin S. Kimmel Center for Biology and Medicine, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA 2Division of Hematology/Oncology, Department of Medicine and Meyer Cancer Center, Weill Cornell Medical College, New York, NY 10021, USA *Correspondence: [email protected] (M.S.), [email protected] (E.A.) http://dx.doi.org/10.1016/j.stem.2014.12.007
A series of five related publications describe an alternative pluripotent state that is dependent on continuous high levels of exogenous reprogramming factor expression. A comprehensive effort to molecularly compare the acquisition of this state to induced pluripotency aims at providing new insights into the mechanisms underlying cellular reprogramming.
The ectopic expression of transcription factors (TFs) associated with the embryonic state in somatic cells affords researchers an accessible source of pluripotent stem cells. The question of how TFs achieve this remarkable conversion has fueled increasingly sophisticated analyses of reprogramming intermediates (Papp and Plath, 2013). While aspects of the reprogramming process—such as the starting somatic cell type, reprogramming factor stoichiometry, and culture conditions—can influence the properties of induced pluripotent stem cells (iPSCs), it is generally assumed that reprogramming yields stem cell lines that are highly similar to each other. In fact, the desire to generate iPSCs that are as close as possible to fertilization-derived embryonic stem cells (ESCs) has been a driving force behind iPSC research ever since the discovery of induced pluripotency. In a series of noteworthy publications (Benevento et al., 2014; Clancy et al., 2014; Hussein et al., 2014; Lee et al., 2014; Tonge et al., 2014), a consortium of international research teams challenges the notion of a singular end-point of reprogramming and takes an ambitious approach to understand an alternative pluripotent state and its acquisition. Using an inducible transposon system, Andras Nagy and colleagues from the University of Toronto report in Nature that very high (5- to 100-fold of the levels seen in ESCs) and persistent expression of the ‘‘Yamanaka factors’’ Oct4, Klf4, Sox2, and c-Myc (OKSM) in mouse fibroblasts reproducibly establish a pluripotent state that is different from that of conven-
tional iPSCs in several aspects (Tonge et al., 2014) (Figure 1). Cells generated in this manner—referred to as F-class cells due to the fuzzy borders of colonies obtained—express Nanog and endogenous Oct4, but not most other ESC-associated genes. Instead, F-class cells express high levels of genes associated with cell fate commitment, suggesting that they are primed for differentiation. Additional noteworthy features of these cells are an ultrafast cell cycle transition, the capacity to grow in absence of leukemia inhibitory factor (LIF), and the propensity to not aggregate in suspension culture. Maybe most importantly, F-class cells require continuous high levels of exogenous OKSM for their propagation, thereby deviating from the concept of transgene independence that has been established as a hallmark of induced pluripotency. Nevertheless, upon shutdown of exogenous factors, F-class cells appear to undergo multilineage differentiation in vitro and in the context of teratomas, but they fail in stricter pluripotency assays such as chimera formation. Intriguingly, F-class cells treated with histone deacetylase inhibitors convert into transgene-independent cells similar to ESCs that are capable of germline transmission within days of treatment (Tonge et al., 2014). This transition suggests that F-class reprogramming does not negatively impact upon genomic integrity. Formation of F-class cells normally requires 2–3 weeks of high levels of OKSM expression, representing conditions under which iPSCs form very infrequently. However, when exogenous OKSM levels
are experimentally lowered at an arbitrary point during reprogramming, fully pluripotent transgene-independent cell lines appear more frequently. In a second paper in Nature, the same group exploits this observation to conduct an exceptionally deep analysis of the transcriptome, proteome, and epigenome of bulk cell populations at various points along these two routes to pluripotency (Hussein et al., 2014). In addition, three papers in Nature Communications dissect in great detail specific aspects of F-class and iPSC reprogramming, in particular dynamic changes of small RNAs (Clancy et al., 2014), protein abundance (Benevento et al., 2014), and DNA methylation (Lee et al., 2014). Fittingly, the authors refer to their combined efforts as Project Grandiose. A corresponding online portal (http://www.stemformatics.org) will be a versatile resource for future research into the biology of reprogramming. The authors’ own analyses largely confirm previous genome-wide studies of prospectively isolated reprogramming intermediates and ‘‘pre-iPSC’’ lines (Koche et al., 2011; Polo et al., 2012), although a number of molecular differences exist that might be explained by the particularities of their reprogramming system. This includes the rapid and global disappearance of the repressive H3K27me3 histone mark upon OKSM expression, which is reacquired during the transition to the F-state. DNA methylation at somatic loci occurs gradually during both F-class and iPSC reprogramming, while demethylation of ESC-associated loci is impaired in the F-class trajectory (Hussein et al., 2014).
Cell Stem Cell 16, January 8, 2015 ª2015 Elsevier Inc. 9
Cell Stem Cell
Previews
to not aggregate in suspension culture opens up the possibility of large-scale expansion in stirring incubators. However, it remains to be seen whether this particular F-class feature is compatible with differentiation protocols that require the formation of embryoid bodies as an intermediate step. As it is, the discovery of Fclass cells offers a new and conceptually stimulating approach to study transcription-factor-induced pluripotency. REFERENCES
Figure 1. Illustration of the Reprogramming Routes Leading to Conventional iPSCs and Newly Discovered F-Class Cells Note the importance of different levels of Oct4, Klf4, Sox2, and c-Myc (OKSM) expression. Key cellular and functional differences of the two cell types are shown on the right (see the text for molecular differences). F-class cells can be converted into iPSCs by exposure to histone deacetylase inhibitors (HDACi), while at least some F-class features can be established in iPSCs by high OKSM levels in the presence of Janus kinase inhibitors (JAKi) and the absence of leukemia inhibitory factors (LIF).
Additional important observations are the intriguing discovery of new proteincoding transcripts and unexpected layers of post-transcriptional gene regulation, such as intron retention for many highly transcribed genes in the F-state (Hussein et al., 2014). The description of an alternative, albeit transgene-dependent, pluripotent state is provocative and raises several questions. For example, can additional pluripotent states be obtained upon expression of OKSM or other factor combinations able to drive iPSC formation? Of note, Tonge et al. themselves mention the generation of a second class of cells with similar morphology but different gene expression from that of ESCs (referred to as C-class) in high OKSM conditions. Determining DNA occupancy of reprogramming factors likely will shed light on why alternative types of pluripotent cells emerge, as it has for iPSC reprogramming (Papp and Plath, 2013). The observation
that many ESC-associated genes remain silent in F-class cells points toward a repressive role of OKSM when expressed at high levels, which supports a documented inhibitory effect of these factors on iPSC formation late in reprogramming (Golipour et al., 2012). Histone deacetylase inhibitors can overcome this repression, raising the question of whether OKSM in F-class cells interacts with the same repressive complexes that have been proposed to constitute reprogramming roadblocks (Rais et al., 2013). Will some of the unique properties of F-class cells facilitate the use of pluripotent cells in downstream applications? Because mouse pluripotent cells can be easily and quickly expanded to large numbers, the ultrafast cycling time of Fclass cells may not be all too relevant for practical purposes. This might be different if human F-class cells with similar properties are discovered. As suggested by Tonge et al., the propensity of F-class cells
10 Cell Stem Cell 16, January 8, 2015 ª2015 Elsevier Inc.
Benevento, M., Tonge, P.D., Puri, M.C., Hussein, S.M., Cloonan, N., Wood, D.L., Grimmond, S.M., Nagy, A., Munoz, J., and Heck, A.J. (2014). Nat. Commun. 5, 5613. Clancy, J.L., Patel, H.R., Hussein, S.M., Tonge, P.D., Cloonan, N., Corso, A.J., Li, M., Lee, D.S., Shin, J.Y., Wong, J.J., et al. (2014). Nat. Commun. 5, 5522. Golipour, A., David, L., Liu, Y., Jayakumaran, G., Hirsch, C.L., Trcka, D., and Wrana, J.L. (2012). Cell Stem Cell 11, 769–782. Hussein, S.M., Puri, M.C., Tonge, P.D., Benevento, M., Corso, A.J., Clancy, J.L., Mosbergen, R., Li, M., Lee, D.S., Cloonan, N., et al. (2014). Nature 516, 198–206. Koche, R.P., Smith, Z.D., Adli, M., Gu, H., Ku, M., Gnirke, A., Bernstein, B.E., and Meissner, A. (2011). Cell Stem Cell 8, 96–105. Lee, D.S., Shin, J.Y., Tonge, P.D., Puri, M.C., Lee, S., Park, H., Lee, W.C., Hussein, S.M., Bleazard, T., Yun, J.Y., et al. (2014). Nat. Commun. 5, 5619. Papp, B., and Plath, K. (2013). Cell 152, 1324– 1343. Polo, J.M., Anderssen, E., Walsh, R.M., Schwarz, B.A., Nefzger, C.M., Lim, S.M., Borkent, M., Apostolou, E., Alaei, S., Cloutier, J., et al. (2012). Cell 151, 1617–1632. Rais, Y., Zviran, A., Geula, S., Gafni, O., Chomsky, E., Viukov, S., Mansour, A.A., Caspi, I., Krupalnik, V., Zerbib, M., et al. (2013). Nature 502, 65–70. Tonge, P.D., Corso, A.J., Monetti, C., Hussein, S.M., Puri, M.C., Michael, I.P., Li, M., Lee, D.S., Mar, J.C., Cloonan, N., et al. (2014). Nature 516, 192–197.