Cell Stem Cell
Previews endogenous in vivo counterparts. This point might be relevant if iNCCs will be used to study mechanisms of fate specification and cellular differentiation or to pursue functional cell replacement strategies in the future. In any case, the iNCCs generated by Kim and coauthors constitute a very valuable platform for the detection of new mechanisms underlying NC-related diseases. Indeed, in this study the authors not only showed that FD-patient iNCCs displayed low levels of the IKBAKP transcript most likely due to alternative splicing, but they also identified novel splicing variants in two core NC transcription factors (Figure 1). It will be interesting to use iNCCs in large-scale drug screenings to identify new molecules capable of reversing the phenotype of this
and other diseases. Thus, iNCCs hold great potential for cell-based therapies. To realize this potential, however, the genomic integration of retrovirally transduced Sox10 must be circumvented, for instance with chemical reprogramming. Additionally, it is crucial that the functionality of newly generated cell types will be validated in in vivo disease models.
Dupin, E., and Sommer, L. (2012). Dev. Biol. 366, 83–95. Groves, A.K., and LaBonne, C. (2014). Dev. Biol. 389, 2–12. Kim, Y.J., Lim, H., Li, Z., Oh, Y., Kovlyagina, I., Choi, I.Y., Dong, X., and Lee, G. (2014). Cell Stem Cell 15, this issue, 497–506. Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A., et al. (2009). Nature 461, 402–406.
REFERENCES Britsch, S., Goerich, D.E., Riethmacher, D., Peirano, R.I., Rossner, M., Nave, K.A., Birchmeier, C., and Wegner, M. (2001). Genes Dev. 15, 66–78. Bronner, M.E., and LeDouarin, N.M. (2012). Dev. Biol. 366, 2–9. Chambers, S.M., Qi, Y., Mica, Y., Lee, G., Zhang, X.-J., Niu, L., Bilsland, J., Cao, L., Stevens, E., Whiting, P., et al. (2012). Nat. Biotechnol. 30, 715–720.
Mica, Y., Lee, G., Chambers, S.M., Tomishima, M.J., and Studer, L. (2013). Cell Rep. 3, 1140– 1152. Shakhova, O., Zingg, D., Schaefer, S.M., Hari, L., Civenni, G., Blunschi, J., Claudinot, S., Okoniewski, M., Beermann, F., Mihic-Probst, D., et al. (2012). Nat. Cell Biol. 14, 882–890. Takahashi, Y., Sipp, D., and Enomoto, H. (2013). Science 341, 860–863.
When Old Hematopoietic Stem Cells Get DamAged Jean Soulier1,* 1Institute
of Hematology (IUH), INSERM UMR944/CNRS UMR7212, Saint-Louis Hospital and University Paris Diderot, Sorbonne Paris Cite´, av Claude Vellefaux 75010, Paris, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.09.012
Hematopoietic stem cells (HSCs) functionally decline and are prone to lineage bias and myeloid malignancies upon aging. Two recent studies (Beerman et al., 2014; Flach et al., 2014) investigate the underlying mechanisms associated with aged HSC phenotypes and highlight DNA damage, replication stress, and ribosomal stress in the process. Aging is a complex phenomenon that affects tissue homeostasis and is thought to combine cell-intrinsic and -extrinsic mechanisms (Oh et al., 2014). Old mice develop an aged phenotype in the hematopoietic system that includes a reduced regenerative potential, a lymphoid to myeloid lineage bias, and a tendency of myeloid clones to expand toward malignancies. There is a relative increase of phenotypically defined HSCs in the bone marrow from old mice; however, these cells have less repopulating ability than those from young mice in competitive experiments, which reflects their intrinsic functional decline together with multiple epigenome and transcriptome changes (Sun et al., 2014). HSC aging
coincides with the accumulation of unresolved DNA damage, which may not only reflect but also participate in triggering the cell-intrinsic aged phenotype. The assumption of a driver role for DNA damage accumulation is further supported by the aged phenotype observed in mice with deficiencies in DNA repair (Rossi et al., 2007). All together, the circumstances in which DNA damage occurs in HSCs, the response at the molecular and biochemical levels, and the biological impact unresolved damage has at the cell and tissue levels are fundamental yet unanswered questions. Such answers may reveal strategies for modulating the mechanisms underlying aging.
Some important clues have been addressed in two recent papers, which both began by studying the phosphorylated form of the variant histone H2AX (gH2AX) foci (using immunofluorescent detection) and DNA breaks directly (using alkaline comet assay) in highly purified HSCs from young and old mice (Beerman et al., 2014; Flach et al., 2014). It was previously shown that gH2AX foci, which are indicative of DNA damage, increase in number over time. In both papers the accumulation of gH2AX in old HSCs was confirmed, but they were interpreted differently and these differences paved the way to subsequent experiments and conclusions. The Rossi team focused on the discreet increase of DNA breaks, which they
Cell Stem Cell 15, October 2, 2014 ª2014 Elsevier Inc. 399
Cell Stem Cell
Previews detected in old quiescent HSCs. By analyzing the timing of the DNA damage response and repair with the comet assay and gene expression profiling of sorted HSC and progenitor fractions (HSCs, MPPs, GMPs, and CLPs) as well as HSC fate in single-cell culture, the authors found unexpectedly that the HSCs, which are essentially quiescent, respond to and repair DNA damage less efficiently than the downstream progenitors, regardless of age. This decrease was associated with a broad attenuation of the DNA damage response and repair pathways and was dependent upon HSC quiescence. Conversely, these systems were induced in proliferating HSCs, particularly in fetal liver cells that are highly proliferative. These data put forth a model in which old hematopoietic cells uniquely accumulate DNA damage in the quiescent stem cell compartment, with this damage being largely resolved at subsequent stages upon cell cycle entry. Because many HSCs remain quiescent for a very long time, the continuing accumulation of DNA damage in these cells may eventually lead to their impaired function in old age. The Passegue team focused on analyzing what kind of damage, distinct from DNA breaks, could be primarily marked by the gH2AX foci in old HSCs. Exploration of many checkpoint and repair proteins revealed an excess of ATR and single-stranded DNA binding protein signaling in old HSCs, indicative of a high level of replication stress with impaired S phase progression in these cells and subsequent acquisition of chromosomal gaps or breaks in the progeny in culture. Gene expression profiling was performed and identified a selective downregulation of all the Mcm genes in old HSCs as a plausible origin of the replication stress, in line with a protective role of the MCM complex (Ibarra et al., 2008). This relationship was confirmed experimentally by showing that depleting the Mcm proteins impaired stem cell properties in vitro and in vivo. The next step, considering that most HSCs are quiescent and do not replicate, was to investigate which cell structures are marked by gH2AX in noncycling HSCs. Interestingly, the authors found that the gH2AX foci colocalized with nucleolar markers. Nucleoli are primarily sites of rRNA transcription
and ribosome biogenesis, while ribosome stress is central in the pathogenesis of some bone marrow deficiency diseases (Narla and Ebert, 2010). Consistently, rRNA transcripts and ribosome biogenesis were found decreased in quiescent HSCs from old mice, and elegant experiments showed that the nucleolar gH2AX marks disappeared from old HSCs upon cell cycle entry but were readily detected months after transplantation, when HSCs had re-entered quiescence. All together, these data suggest that gH2AX foci can mark replication stress or ribosomal stress at the rRNA genes sites and that decreased ribosome biogenesis is a hallmark of old quiescent HSCs. These papers contribute to a better knowledge of cell-intrinsic mechanisms of the complex HSC aging phenotype. While multiple cytoprotective properties are active in HSCs to prevent cell damage (i.e., low oxygen exposure in the hematopoietic niche, detoxifying systems, etc.), the study of Beerman et al. argues against the usual view that these cells are uniquely genoprotected during aging by enhanced DNA repair systems ability. Moreover, the study of Flach et al. convincingly highlights the replication stress and decreased ribosome biogenesis in the biology of aged HSCs. These works raise many questions, including questions about the nature of the molecular signals that regulate the damage response systems in the HSCs, and the downstream mechanisms by which the damage accrual may contribute to functional decline. More generally, one can ask whether the accumulation of various damage and DNA mutations in the stem cell compartment could be seen as playing a physiological ‘‘integrative’’ role in tuning homeostasis over aging. It is noteworthy that the current studies have been conducted in mouse cells, so human HSCs will have to be investigated, with their inherent practical difficulties. The results of these studies also have implications for the cancer field regarding cancer initiation and progression. Unresolved or misrepaired DNA damage favors the accumulation of mutations. This accumulation can start in utero and increases over the life time, as was shown by exome sequencing of healthy human hematopoietic progenitor cells (including those from cord blood) and the deciphering of
400 Cell Stem Cell 15, October 2, 2014 ª2014 Elsevier Inc.
the age-dependent mutational history captured in leukemia cells (Alexandrov et al., 2013; Welch et al., 2012). Although the vast majority of the mutations in HSCS are likely to be neutral (passenger), the occurrence of driver mutations could favor a progressive clonal dominance over the surrounding aged (declining) cells (Busque et al., 2012). It is also conceivable that when a driver mutation occurs, the quiescent state intrinsic to HSCs (or the quiescence conferred by the mutation to a more differentiated progenitor) might be permissive, in relation to an attenuated DNA damage and repair response, to allow the cell to survive and additional mutations to occur toward eventual expansion of a successfully transformed clone. This scenario would be consistent with certain mutations that are observed in myeloid malignancies that act as drivers of oncogenesis, although they are apparently ‘‘counterproductive’’ when observed in isolation. REFERENCES Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C., Aparicio, S.A., Behjati, S., Biankin, A.V., Bignell, G.R., Bolli, N., Borg, A., Børresen-Dale, A.L., et al.; Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain (2013). Nature 500, 415–421. Beerman, I., Seita, J., Inlay, M.A., Weissman, I.L., and Rossi, D.J. (2014). Cell Stem Cell 15, 37–50. Busque, L., Patel, J.P., Figueroa, M.E., Vasanthakumar, A., Provost, S., Hamilou, Z., Mollica, L., Li, J., Viale, A., Heguy, A., et al. (2012). Nat. Genet. 44, 1179–1181. Flach, J., Bakker, S.T., Mohrin, M., Conroy, P.C., Pietras, E.M., Reynaud, D., Alvarez, S., Diolaiti, M.E., Ugarte, F., Forsberg, E.C., et al. (2014). Nature 512, 198–202. Ibarra, A., Schwob, E., and Me´ndez, J. (2008). Proc. Natl. Acad. Sci. USA 105, 8956–8961. Narla, A., and Ebert, B.L. (2010). Blood 115, 3196– 3205. Oh, J., Lee, Y.D., and Wagers, A.J. (2014). Nat. Med. 20, 870–880. Rossi, D.J., Bryder, D., Seita, J., Nussenzweig, A., Hoeijmakers, J., and Weissman, I.L. (2007). Nature 447, 725–729. Sun, D., Luo, M., Jeong, M., Rodriguez, B., Xia, Z., Hannah, R., Wang, H., Le, T., Faull, K.F., Chen, R., et al. (2014). Cell Stem Cell 14, 673–688. Welch, J.S., Ley, T.J., Link, D.C., Miller, C.A., Larson, D.E., Koboldt, D.C., Wartman, L.D., Lamprecht, T.L., Liu, F., Xia, J., et al. (2012). Cell 150, 264–278.
Previews endogenous in vivo counterparts. This point might be relevant if iNCCs will be used to study mechanisms of fate specification and cellular differentiation or to pursue functional cell replacement strategies in the future. In any case, the iNCCs generated by Kim and coauthors constitute a very valuable platform for the detection of new mechanisms underlying NC-related diseases. Indeed, in this study the authors not only showed that FD-patient iNCCs displayed low levels of the IKBAKP transcript most likely due to alternative splicing, but they also identified novel splicing variants in two core NC transcription factors (Figure 1). It will be interesting to use iNCCs in large-scale drug screenings to identify new molecules capable of reversing the phenotype of this
and other diseases. Thus, iNCCs hold great potential for cell-based therapies. To realize this potential, however, the genomic integration of retrovirally transduced Sox10 must be circumvented, for instance with chemical reprogramming. Additionally, it is crucial that the functionality of newly generated cell types will be validated in in vivo disease models.
Dupin, E., and Sommer, L. (2012). Dev. Biol. 366, 83–95. Groves, A.K., and LaBonne, C. (2014). Dev. Biol. 389, 2–12. Kim, Y.J., Lim, H., Li, Z., Oh, Y., Kovlyagina, I., Choi, I.Y., Dong, X., and Lee, G. (2014). Cell Stem Cell 15, this issue, 497–506. Lee, G., Papapetrou, E.P., Kim, H., Chambers, S.M., Tomishima, M.J., Fasano, C.A., Ganat, Y.M., Menon, J., Shimizu, F., Viale, A., et al. (2009). Nature 461, 402–406.
REFERENCES Britsch, S., Goerich, D.E., Riethmacher, D., Peirano, R.I., Rossner, M., Nave, K.A., Birchmeier, C., and Wegner, M. (2001). Genes Dev. 15, 66–78. Bronner, M.E., and LeDouarin, N.M. (2012). Dev. Biol. 366, 2–9. Chambers, S.M., Qi, Y., Mica, Y., Lee, G., Zhang, X.-J., Niu, L., Bilsland, J., Cao, L., Stevens, E., Whiting, P., et al. (2012). Nat. Biotechnol. 30, 715–720.
Mica, Y., Lee, G., Chambers, S.M., Tomishima, M.J., and Studer, L. (2013). Cell Rep. 3, 1140– 1152. Shakhova, O., Zingg, D., Schaefer, S.M., Hari, L., Civenni, G., Blunschi, J., Claudinot, S., Okoniewski, M., Beermann, F., Mihic-Probst, D., et al. (2012). Nat. Cell Biol. 14, 882–890. Takahashi, Y., Sipp, D., and Enomoto, H. (2013). Science 341, 860–863.
When Old Hematopoietic Stem Cells Get DamAged Jean Soulier1,* 1Institute
of Hematology (IUH), INSERM UMR944/CNRS UMR7212, Saint-Louis Hospital and University Paris Diderot, Sorbonne Paris Cite´, av Claude Vellefaux 75010, Paris, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.09.012
Hematopoietic stem cells (HSCs) functionally decline and are prone to lineage bias and myeloid malignancies upon aging. Two recent studies (Beerman et al., 2014; Flach et al., 2014) investigate the underlying mechanisms associated with aged HSC phenotypes and highlight DNA damage, replication stress, and ribosomal stress in the process. Aging is a complex phenomenon that affects tissue homeostasis and is thought to combine cell-intrinsic and -extrinsic mechanisms (Oh et al., 2014). Old mice develop an aged phenotype in the hematopoietic system that includes a reduced regenerative potential, a lymphoid to myeloid lineage bias, and a tendency of myeloid clones to expand toward malignancies. There is a relative increase of phenotypically defined HSCs in the bone marrow from old mice; however, these cells have less repopulating ability than those from young mice in competitive experiments, which reflects their intrinsic functional decline together with multiple epigenome and transcriptome changes (Sun et al., 2014). HSC aging
coincides with the accumulation of unresolved DNA damage, which may not only reflect but also participate in triggering the cell-intrinsic aged phenotype. The assumption of a driver role for DNA damage accumulation is further supported by the aged phenotype observed in mice with deficiencies in DNA repair (Rossi et al., 2007). All together, the circumstances in which DNA damage occurs in HSCs, the response at the molecular and biochemical levels, and the biological impact unresolved damage has at the cell and tissue levels are fundamental yet unanswered questions. Such answers may reveal strategies for modulating the mechanisms underlying aging.
Some important clues have been addressed in two recent papers, which both began by studying the phosphorylated form of the variant histone H2AX (gH2AX) foci (using immunofluorescent detection) and DNA breaks directly (using alkaline comet assay) in highly purified HSCs from young and old mice (Beerman et al., 2014; Flach et al., 2014). It was previously shown that gH2AX foci, which are indicative of DNA damage, increase in number over time. In both papers the accumulation of gH2AX in old HSCs was confirmed, but they were interpreted differently and these differences paved the way to subsequent experiments and conclusions. The Rossi team focused on the discreet increase of DNA breaks, which they
Cell Stem Cell 15, October 2, 2014 ª2014 Elsevier Inc. 399
Cell Stem Cell
Previews detected in old quiescent HSCs. By analyzing the timing of the DNA damage response and repair with the comet assay and gene expression profiling of sorted HSC and progenitor fractions (HSCs, MPPs, GMPs, and CLPs) as well as HSC fate in single-cell culture, the authors found unexpectedly that the HSCs, which are essentially quiescent, respond to and repair DNA damage less efficiently than the downstream progenitors, regardless of age. This decrease was associated with a broad attenuation of the DNA damage response and repair pathways and was dependent upon HSC quiescence. Conversely, these systems were induced in proliferating HSCs, particularly in fetal liver cells that are highly proliferative. These data put forth a model in which old hematopoietic cells uniquely accumulate DNA damage in the quiescent stem cell compartment, with this damage being largely resolved at subsequent stages upon cell cycle entry. Because many HSCs remain quiescent for a very long time, the continuing accumulation of DNA damage in these cells may eventually lead to their impaired function in old age. The Passegue team focused on analyzing what kind of damage, distinct from DNA breaks, could be primarily marked by the gH2AX foci in old HSCs. Exploration of many checkpoint and repair proteins revealed an excess of ATR and single-stranded DNA binding protein signaling in old HSCs, indicative of a high level of replication stress with impaired S phase progression in these cells and subsequent acquisition of chromosomal gaps or breaks in the progeny in culture. Gene expression profiling was performed and identified a selective downregulation of all the Mcm genes in old HSCs as a plausible origin of the replication stress, in line with a protective role of the MCM complex (Ibarra et al., 2008). This relationship was confirmed experimentally by showing that depleting the Mcm proteins impaired stem cell properties in vitro and in vivo. The next step, considering that most HSCs are quiescent and do not replicate, was to investigate which cell structures are marked by gH2AX in noncycling HSCs. Interestingly, the authors found that the gH2AX foci colocalized with nucleolar markers. Nucleoli are primarily sites of rRNA transcription
and ribosome biogenesis, while ribosome stress is central in the pathogenesis of some bone marrow deficiency diseases (Narla and Ebert, 2010). Consistently, rRNA transcripts and ribosome biogenesis were found decreased in quiescent HSCs from old mice, and elegant experiments showed that the nucleolar gH2AX marks disappeared from old HSCs upon cell cycle entry but were readily detected months after transplantation, when HSCs had re-entered quiescence. All together, these data suggest that gH2AX foci can mark replication stress or ribosomal stress at the rRNA genes sites and that decreased ribosome biogenesis is a hallmark of old quiescent HSCs. These papers contribute to a better knowledge of cell-intrinsic mechanisms of the complex HSC aging phenotype. While multiple cytoprotective properties are active in HSCs to prevent cell damage (i.e., low oxygen exposure in the hematopoietic niche, detoxifying systems, etc.), the study of Beerman et al. argues against the usual view that these cells are uniquely genoprotected during aging by enhanced DNA repair systems ability. Moreover, the study of Flach et al. convincingly highlights the replication stress and decreased ribosome biogenesis in the biology of aged HSCs. These works raise many questions, including questions about the nature of the molecular signals that regulate the damage response systems in the HSCs, and the downstream mechanisms by which the damage accrual may contribute to functional decline. More generally, one can ask whether the accumulation of various damage and DNA mutations in the stem cell compartment could be seen as playing a physiological ‘‘integrative’’ role in tuning homeostasis over aging. It is noteworthy that the current studies have been conducted in mouse cells, so human HSCs will have to be investigated, with their inherent practical difficulties. The results of these studies also have implications for the cancer field regarding cancer initiation and progression. Unresolved or misrepaired DNA damage favors the accumulation of mutations. This accumulation can start in utero and increases over the life time, as was shown by exome sequencing of healthy human hematopoietic progenitor cells (including those from cord blood) and the deciphering of
400 Cell Stem Cell 15, October 2, 2014 ª2014 Elsevier Inc.
the age-dependent mutational history captured in leukemia cells (Alexandrov et al., 2013; Welch et al., 2012). Although the vast majority of the mutations in HSCS are likely to be neutral (passenger), the occurrence of driver mutations could favor a progressive clonal dominance over the surrounding aged (declining) cells (Busque et al., 2012). It is also conceivable that when a driver mutation occurs, the quiescent state intrinsic to HSCs (or the quiescence conferred by the mutation to a more differentiated progenitor) might be permissive, in relation to an attenuated DNA damage and repair response, to allow the cell to survive and additional mutations to occur toward eventual expansion of a successfully transformed clone. This scenario would be consistent with certain mutations that are observed in myeloid malignancies that act as drivers of oncogenesis, although they are apparently ‘‘counterproductive’’ when observed in isolation. REFERENCES Alexandrov, L.B., Nik-Zainal, S., Wedge, D.C., Aparicio, S.A., Behjati, S., Biankin, A.V., Bignell, G.R., Bolli, N., Borg, A., Børresen-Dale, A.L., et al.; Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain (2013). Nature 500, 415–421. Beerman, I., Seita, J., Inlay, M.A., Weissman, I.L., and Rossi, D.J. (2014). Cell Stem Cell 15, 37–50. Busque, L., Patel, J.P., Figueroa, M.E., Vasanthakumar, A., Provost, S., Hamilou, Z., Mollica, L., Li, J., Viale, A., Heguy, A., et al. (2012). Nat. Genet. 44, 1179–1181. Flach, J., Bakker, S.T., Mohrin, M., Conroy, P.C., Pietras, E.M., Reynaud, D., Alvarez, S., Diolaiti, M.E., Ugarte, F., Forsberg, E.C., et al. (2014). Nature 512, 198–202. Ibarra, A., Schwob, E., and Me´ndez, J. (2008). Proc. Natl. Acad. Sci. USA 105, 8956–8961. Narla, A., and Ebert, B.L. (2010). Blood 115, 3196– 3205. Oh, J., Lee, Y.D., and Wagers, A.J. (2014). Nat. Med. 20, 870–880. Rossi, D.J., Bryder, D., Seita, J., Nussenzweig, A., Hoeijmakers, J., and Weissman, I.L. (2007). Nature 447, 725–729. Sun, D., Luo, M., Jeong, M., Rodriguez, B., Xia, Z., Hannah, R., Wang, H., Le, T., Faull, K.F., Chen, R., et al. (2014). Cell Stem Cell 14, 673–688. Welch, J.S., Ley, T.J., Link, D.C., Miller, C.A., Larson, D.E., Koboldt, D.C., Wartman, L.D., Lamprecht, T.L., Liu, F., Xia, J., et al. (2012). Cell 150, 264–278.
