Cell Stem Cell
Previews Sleeping Beauty Wakes Up the Clonal Succession Model for Homeostatic Hematopoiesis Rong Lu1,* 1Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, 1425 San Pablo Street, Los Angeles, CA 90033, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.11.015
Recently in Nature, Sun et al. (2014) used a sleeping beauty transposon system to demonstrate that natural hematopoiesis is sustained by the successive recruitment of thousands of clones that are mostly lineage restricted. These findings call into question whether homeostatic hematopoiesis is sustained by hematopoietic stem cells traditionally identified by transplantation. Traditionally, hematopoietic stem cells (HSCs) have been identified as cells that can perpetually regenerate all blood cell types in a myeloablated recipient. They are very rare and mostly quiescent (Seita and Weissman, 2010). Two general models have been put forth for how HSCs are recruited into differentiation under normal physiological conditions. The ‘‘clonal succession model’’ proposes that small numbers of HSCs are sequentially recruited to enter the cell cycle and thereby initiate the lineage commitment process (Jordan and Lemischka, 1990; Lemischka et al., 1986). Upon the exhaustion of their productive life spans, they are succeeded by a different group of HSCs. In contrast, the ‘‘clonal stability model’’ suggests that a single, static group of HSCs continuously replenishes blood cells throughout an organism’s lifetime (Abkowitz et al., 1995; McKenzie et al., 2006; Prchal et al., 1996). Previous efforts to discriminate between these two models relied on transplantation experiments. It was observed that after a short period of clonal fluctuation following transplantation, a few HSC clones stably supply blood throughout the recipient’s life span (Abkowitz et al., 1995; McKenzie et al., 2006; Prchal et al., 1996). In these transplantation studies, individual HSCs were tracked using viral infection applied ex vivo prior to transplantation. This approach is not feasible for labeling HSCs in vivo because HSCs are dispersed in bone marrow throughout the body. It is also difficult to employ fluorescent proteins for clonal tracking, a
labeling strategy widely used in solid tissues, because HSCs migrate and their progeny blood cells circulate. To conquer these technical challenges, the Camargo group adopted a creative approach to track cells using a transposon system cloned into mice (Sun et al., 2014). The transposon is activated by a hyperactive ‘‘sleeping beauty’’ transposase whose expression is controlled by doxycycline. During the short time period when doxycycline is applied to a mouse, the transposon can randomly mobilize to a different genomic location. This transposition creates an inheritable genomic DNA insertion that is unique to individual cells and their progenies. Cells originating from a common ancestor can be identified by their common transposon insertion site. The authors have conducted a series of experiments to demonstrate the fidelity and sensitivity of this experimental system. This transposon-based cellular tracking system provides an exciting opportunity to study native hematopoiesis without the use of transplantation. Moreover, the Camargo group used a universal promoter that marks all cells randomly and thereby provides an unbiased method to identify cells that sustain the blood supply. In contrast, transplantation experiments can only track cells that engraft. Engrafted cells may proliferate, and consequently multiple HSCs may share the same marker. This may be partially responsible for the previously observed clonal stability. Indeed, the transposon system has produced strikingly different results from
previous studies. At each of their measured time points, dramatically different clones appear to supply the blood. This phenomenon persists even after 4 months postlabeling when blood is thought to derive only from HSCs. The authors also used single-cell assays to confirm the fast clonal turnover and they estimated that thousands of clones contribute to blood formation at any time point. Thus, their data suggest that longterm hematopoiesis is sustained by the successive recruitment of a large number of clones. While their observed clonal dynamics are consistent with the ‘‘clonal succession model,’’ their estimated clonal complexity is much greater than what could possibly be supported by the small number of HSCs traditionally identified using transplantation methods. This finding raises some key questions: what cells can account for the observed clonal abundances and sustain the longterm blood supply? Are ‘‘traditional’’ HSCs involved at all with homeostatic hematopoiesis? To address these questions, the authors performed two experiments. First, they compared the clonal composition of hematopoietic cells from clonally marked donor mice with that from transplanted recipient mice. They found that donor and recipient mice possessed different clonal repertoires. Second, they compared the clonal composition of HSCs, intermediate progenitors, and mature cells from a single mouse. They found that fewer than 5% of HSC clones are subsequently represented in mature cell populations, whereas approximately half of the
Cell Stem Cell 15, December 4, 2014 ª2014 Elsevier Inc. 677
Cell Stem Cell
Previews multipotent progenitor (MPP) and myeloid progenitor (MyP) clones contribute to mature cell populations. Based on these two experiments, the authors conclude that the cells that supply blood under homeostatic conditions are not transplantable and are not found in the conventionally defined HSC pool. Moreover, they suggest that the large number of progenitor cells, including previously defined MPPs and MyPs, may be the major source of ongoing hematopoiesis. These data do not exclude the possibility that HSCs participate in steady-state blood production, but if they do, they must be quickly depleted from the HSC pool once they have committed to differentiation (known as the ‘‘successive deletion’’ model). It will be interesting to examine in future studies whether the number of HSC clones shrinks over time. The Camargo group also examined another defining characteristic of HSCs, multipotency, by comparing the clonality of distinct differentiated cell types in bone marrow with similar maturation times. They found that around half of the lymphoid cell clones are also present in myeloid cells 10 months after single-cell marking is initiated, indicating the active production of diverse lineages by multipotent stem or progenitor cells. However, most of the myeloid clones are myeloid restricted throughout the observation period for up to 45 weeks. This suggests that lineage-restricted progenitors can also supply blood over the long term, which further challenges the conventional view of how HSCs supply blood under normal physiological conditions. Transplantation allows the study of cells isolated by a combination of
markers. It requires the removal of the recipient’s blood system so that donor hematopoietic cells can engraft. This injury and repair process may involve different stem and progenitor cells from those that maintain homeostatic blood supply. Recent studies of hair follicle stem cells and intestinal stem cells suggest that tissue repair and homeostasis may be sustained by distinct stem and progenitor cells and through different mechanisms (Ito et al., 2005; Tian et al., 2011). In their paper, the Camargo group demonstrated for the first time that different stem and progenitor cells sustain and repair the hematopoietic system. If stem cells are defined as cells with the potential for self-renewal and differentiation, then traditionally identified HSCs are undoubtedly qualified to be called stem cells. However, since engraftment is not a required capacity of stem cells by definition, perhaps our previously identified HSCs are only a small subset of all stem cells in the blood system. Nonetheless, those traditional stem cells possess the capacity for engraftment and longterm blood reconstitution and are therefore the only cells accessible for bone marrow transplantation therapy. The striking clonal dynamics discovered by the Camargo group reveal new regulatory mechanisms unobtainable from population level studies. A similar long-term clonal turnover in blood has recently been reported in nonhuman primates (Kim et al., 2014). These exciting findings bring forth many new questions. For instance, the identities of the cells that sustain homeostatic hematopoiesis remain to be elucidated, and the underlying regulatory systems are
678 Cell Stem Cell 15, December 4, 2014 ª2014 Elsevier Inc.
unknown as well. The dynamic clonal turnover discovered in the study by the Camargo group provides a new basis for studying development and aging of natural hematopoiesis. Future studies need to look beyond traditional HSCs for target cells where oncogenic mutations may arise and accumulate over time. More clonal-level analyses are on their way to resolve these questions and together they may radically change our perspective of hematopoiesis. REFERENCES Abkowitz, J.L., Persik, M.T., Shelton, G.H., Ott, R.L., Kiklevich, J.V., Catlin, S.N., and Guttorp, P. (1995). Proc. Natl. Acad. Sci. USA 92, 2031–2035. Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R.J., and Cotsarelis, G. (2005). Nat. Med. 11, 1351–1354. Jordan, C.T., and Lemischka, I.R. (1990). Genes Dev. 4, 220–232. Kim, S., Kim, N., Presson, A.P., Metzger, M.E., Bonifacino, A.C., Sehl, M., Chow, S.A., Crooks, G.M., Dunbar, C.E., An, D.S., et al. (2014). Cell Stem Cell 14, 473–485. Lemischka, I.R., Raulet, D.H., and Mulligan, R.C. (1986). Cell 45, 917–927. McKenzie, J.L., Gan, O.I., Doedens, M., Wang, J.C., and Dick, J.E. (2006). Nat. Immunol. 7, 1225–1233. Prchal, J.T., Prchal, J.F., Belickova, M., Chen, S., Guan, Y., Gartland, G.L., and Cooper, M.D. (1996). J. Exp. Med. 183, 561–567. Seita, J., and Weissman, I.L. (2010). Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 640–653. Sun, J., Ramos, A., Chapman, B., Johnnidis, J.B., Le, L., Ho, Y.J., Klein, A., Hofmann, O., and Camargo, F.D. (2014). Nature 514, 322–327. Tian, H., Biehs, B., Warming, S., Leong, K.G., Rangell, L., Klein, O.D., and de Sauvage, F.J. (2011). Nature 478, 255–259.
Previews Sleeping Beauty Wakes Up the Clonal Succession Model for Homeostatic Hematopoiesis Rong Lu1,* 1Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, 1425 San Pablo Street, Los Angeles, CA 90033, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.11.015
Recently in Nature, Sun et al. (2014) used a sleeping beauty transposon system to demonstrate that natural hematopoiesis is sustained by the successive recruitment of thousands of clones that are mostly lineage restricted. These findings call into question whether homeostatic hematopoiesis is sustained by hematopoietic stem cells traditionally identified by transplantation. Traditionally, hematopoietic stem cells (HSCs) have been identified as cells that can perpetually regenerate all blood cell types in a myeloablated recipient. They are very rare and mostly quiescent (Seita and Weissman, 2010). Two general models have been put forth for how HSCs are recruited into differentiation under normal physiological conditions. The ‘‘clonal succession model’’ proposes that small numbers of HSCs are sequentially recruited to enter the cell cycle and thereby initiate the lineage commitment process (Jordan and Lemischka, 1990; Lemischka et al., 1986). Upon the exhaustion of their productive life spans, they are succeeded by a different group of HSCs. In contrast, the ‘‘clonal stability model’’ suggests that a single, static group of HSCs continuously replenishes blood cells throughout an organism’s lifetime (Abkowitz et al., 1995; McKenzie et al., 2006; Prchal et al., 1996). Previous efforts to discriminate between these two models relied on transplantation experiments. It was observed that after a short period of clonal fluctuation following transplantation, a few HSC clones stably supply blood throughout the recipient’s life span (Abkowitz et al., 1995; McKenzie et al., 2006; Prchal et al., 1996). In these transplantation studies, individual HSCs were tracked using viral infection applied ex vivo prior to transplantation. This approach is not feasible for labeling HSCs in vivo because HSCs are dispersed in bone marrow throughout the body. It is also difficult to employ fluorescent proteins for clonal tracking, a
labeling strategy widely used in solid tissues, because HSCs migrate and their progeny blood cells circulate. To conquer these technical challenges, the Camargo group adopted a creative approach to track cells using a transposon system cloned into mice (Sun et al., 2014). The transposon is activated by a hyperactive ‘‘sleeping beauty’’ transposase whose expression is controlled by doxycycline. During the short time period when doxycycline is applied to a mouse, the transposon can randomly mobilize to a different genomic location. This transposition creates an inheritable genomic DNA insertion that is unique to individual cells and their progenies. Cells originating from a common ancestor can be identified by their common transposon insertion site. The authors have conducted a series of experiments to demonstrate the fidelity and sensitivity of this experimental system. This transposon-based cellular tracking system provides an exciting opportunity to study native hematopoiesis without the use of transplantation. Moreover, the Camargo group used a universal promoter that marks all cells randomly and thereby provides an unbiased method to identify cells that sustain the blood supply. In contrast, transplantation experiments can only track cells that engraft. Engrafted cells may proliferate, and consequently multiple HSCs may share the same marker. This may be partially responsible for the previously observed clonal stability. Indeed, the transposon system has produced strikingly different results from
previous studies. At each of their measured time points, dramatically different clones appear to supply the blood. This phenomenon persists even after 4 months postlabeling when blood is thought to derive only from HSCs. The authors also used single-cell assays to confirm the fast clonal turnover and they estimated that thousands of clones contribute to blood formation at any time point. Thus, their data suggest that longterm hematopoiesis is sustained by the successive recruitment of a large number of clones. While their observed clonal dynamics are consistent with the ‘‘clonal succession model,’’ their estimated clonal complexity is much greater than what could possibly be supported by the small number of HSCs traditionally identified using transplantation methods. This finding raises some key questions: what cells can account for the observed clonal abundances and sustain the longterm blood supply? Are ‘‘traditional’’ HSCs involved at all with homeostatic hematopoiesis? To address these questions, the authors performed two experiments. First, they compared the clonal composition of hematopoietic cells from clonally marked donor mice with that from transplanted recipient mice. They found that donor and recipient mice possessed different clonal repertoires. Second, they compared the clonal composition of HSCs, intermediate progenitors, and mature cells from a single mouse. They found that fewer than 5% of HSC clones are subsequently represented in mature cell populations, whereas approximately half of the
Cell Stem Cell 15, December 4, 2014 ª2014 Elsevier Inc. 677
Cell Stem Cell
Previews multipotent progenitor (MPP) and myeloid progenitor (MyP) clones contribute to mature cell populations. Based on these two experiments, the authors conclude that the cells that supply blood under homeostatic conditions are not transplantable and are not found in the conventionally defined HSC pool. Moreover, they suggest that the large number of progenitor cells, including previously defined MPPs and MyPs, may be the major source of ongoing hematopoiesis. These data do not exclude the possibility that HSCs participate in steady-state blood production, but if they do, they must be quickly depleted from the HSC pool once they have committed to differentiation (known as the ‘‘successive deletion’’ model). It will be interesting to examine in future studies whether the number of HSC clones shrinks over time. The Camargo group also examined another defining characteristic of HSCs, multipotency, by comparing the clonality of distinct differentiated cell types in bone marrow with similar maturation times. They found that around half of the lymphoid cell clones are also present in myeloid cells 10 months after single-cell marking is initiated, indicating the active production of diverse lineages by multipotent stem or progenitor cells. However, most of the myeloid clones are myeloid restricted throughout the observation period for up to 45 weeks. This suggests that lineage-restricted progenitors can also supply blood over the long term, which further challenges the conventional view of how HSCs supply blood under normal physiological conditions. Transplantation allows the study of cells isolated by a combination of
markers. It requires the removal of the recipient’s blood system so that donor hematopoietic cells can engraft. This injury and repair process may involve different stem and progenitor cells from those that maintain homeostatic blood supply. Recent studies of hair follicle stem cells and intestinal stem cells suggest that tissue repair and homeostasis may be sustained by distinct stem and progenitor cells and through different mechanisms (Ito et al., 2005; Tian et al., 2011). In their paper, the Camargo group demonstrated for the first time that different stem and progenitor cells sustain and repair the hematopoietic system. If stem cells are defined as cells with the potential for self-renewal and differentiation, then traditionally identified HSCs are undoubtedly qualified to be called stem cells. However, since engraftment is not a required capacity of stem cells by definition, perhaps our previously identified HSCs are only a small subset of all stem cells in the blood system. Nonetheless, those traditional stem cells possess the capacity for engraftment and longterm blood reconstitution and are therefore the only cells accessible for bone marrow transplantation therapy. The striking clonal dynamics discovered by the Camargo group reveal new regulatory mechanisms unobtainable from population level studies. A similar long-term clonal turnover in blood has recently been reported in nonhuman primates (Kim et al., 2014). These exciting findings bring forth many new questions. For instance, the identities of the cells that sustain homeostatic hematopoiesis remain to be elucidated, and the underlying regulatory systems are
678 Cell Stem Cell 15, December 4, 2014 ª2014 Elsevier Inc.
unknown as well. The dynamic clonal turnover discovered in the study by the Camargo group provides a new basis for studying development and aging of natural hematopoiesis. Future studies need to look beyond traditional HSCs for target cells where oncogenic mutations may arise and accumulate over time. More clonal-level analyses are on their way to resolve these questions and together they may radically change our perspective of hematopoiesis. REFERENCES Abkowitz, J.L., Persik, M.T., Shelton, G.H., Ott, R.L., Kiklevich, J.V., Catlin, S.N., and Guttorp, P. (1995). Proc. Natl. Acad. Sci. USA 92, 2031–2035. Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R.J., and Cotsarelis, G. (2005). Nat. Med. 11, 1351–1354. Jordan, C.T., and Lemischka, I.R. (1990). Genes Dev. 4, 220–232. Kim, S., Kim, N., Presson, A.P., Metzger, M.E., Bonifacino, A.C., Sehl, M., Chow, S.A., Crooks, G.M., Dunbar, C.E., An, D.S., et al. (2014). Cell Stem Cell 14, 473–485. Lemischka, I.R., Raulet, D.H., and Mulligan, R.C. (1986). Cell 45, 917–927. McKenzie, J.L., Gan, O.I., Doedens, M., Wang, J.C., and Dick, J.E. (2006). Nat. Immunol. 7, 1225–1233. Prchal, J.T., Prchal, J.F., Belickova, M., Chen, S., Guan, Y., Gartland, G.L., and Cooper, M.D. (1996). J. Exp. Med. 183, 561–567. Seita, J., and Weissman, I.L. (2010). Wiley Interdiscip. Rev. Syst. Biol. Med. 2, 640–653. Sun, J., Ramos, A., Chapman, B., Johnnidis, J.B., Le, L., Ho, Y.J., Klein, A., Hofmann, O., and Camargo, F.D. (2014). Nature 514, 322–327. Tian, H., Biehs, B., Warming, S., Leong, K.G., Rangell, L., Klein, O.D., and de Sauvage, F.J. (2011). Nature 478, 255–259.
![[Is Sleeping Beauty really asleep?].](/assets/img/hold.png)
