The slippery slope of hematopoietic stem cell aging
2017; Elsevier BV; Volume: 56; Linguagem: Inglês
10.1016/j.exphem.2017.09.008
ISSN1873-2399
AutoresMartin Wahlestedt, David Bryder,
Tópico(s)Hematopoietic Stem Cell Transplantation
Resumo•Changes in hematopoietic stem cells (HSCs) have been proposed to underlie hematopoietic aging.•We previously generated induced pluripotent stem cells (iPSCs) from individual and functionally defined aged HSCs.•These iPSCs generated hematopoietic systems indistinguishable from those associated with young age.•HSC aging and the potential for reversal are discussed. The late stages of life, in most species including humans, are associated with a decline in the overall maintenance and health of the organism. This applies also to the hematopoietic system, where aging is not only associated with an increased predisposition for hematological malignancies, but also identified as a strong comorbidity factor for other diseases. Research during the last two decades has proposed that alterations at the level of hematopoietic stem cells (HSCs) might be a root cause for the hematological changes observed with age. However, the recent realization that not all HSCs are alike with regard to fundamental stem cell properties such as self-renewal and lineage potential has several implications for HSC aging, including the synchrony and the stability of the aging HSC state. To approach HSC aging from a clonal perspective, we recently took advantage of technical developments in cellular barcoding and combined this with the derivation of induced pluripotent stem cells (iPSCs). This allowed us to selectively approach HSCs functionally affected by age. The finding that such iPSCs were capable of fully regenerating multilineage hematopoiesis upon morula/blastocyst complementation provides compelling evidence that many aspects of HSC aging can be reversed, which indicates that a central mechanism underlying HSC aging is a failure to uphold the epigenomes associated with younger age. Here we discuss these findings in the context of the underlying causes that might influence HSC aging and the requirements and prospects for restoration of the aging HSC epigenome. The late stages of life, in most species including humans, are associated with a decline in the overall maintenance and health of the organism. This applies also to the hematopoietic system, where aging is not only associated with an increased predisposition for hematological malignancies, but also identified as a strong comorbidity factor for other diseases. Research during the last two decades has proposed that alterations at the level of hematopoietic stem cells (HSCs) might be a root cause for the hematological changes observed with age. However, the recent realization that not all HSCs are alike with regard to fundamental stem cell properties such as self-renewal and lineage potential has several implications for HSC aging, including the synchrony and the stability of the aging HSC state. To approach HSC aging from a clonal perspective, we recently took advantage of technical developments in cellular barcoding and combined this with the derivation of induced pluripotent stem cells (iPSCs). This allowed us to selectively approach HSCs functionally affected by age. The finding that such iPSCs were capable of fully regenerating multilineage hematopoiesis upon morula/blastocyst complementation provides compelling evidence that many aspects of HSC aging can be reversed, which indicates that a central mechanism underlying HSC aging is a failure to uphold the epigenomes associated with younger age. Here we discuss these findings in the context of the underlying causes that might influence HSC aging and the requirements and prospects for restoration of the aging HSC epigenome. In most species including humans, the late stages of life are associated with a decline in overall organism maintenance and health. At a population level, one dominating evolutionary theory proposes that aging is the result of selective benefits for individuals in their reproductive prime at the expense of those available to nonreproductive, aged individuals. When applied to the level of the individual, a priority of energy expenditure might thus primarily be aimed at preserving the longevity of the germline as opposed to maintenance processes that are unrelated to reproduction (the soma) [1Kirkwood T.B. Holliday R. The evolution of ageing and longevity.Proc R Soc Lond B Biol Sci. 1979; 205: 531-546Crossref PubMed Scopus (613) Google Scholar]. Although the disposable soma theory is popular among gerontologists, it could be argued that, at least to some extent, it fails to consider adult- or tissue-specific stem cells, which, due to their multipotency and self-renewal capabilities and above all their extensive longevity, function actively to maintain tissue homeostasis of the soma throughout life [2Bryder D. Rossi D.J. Weissman I.L. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell.Am J Pathol. 2006; 169: 338-346Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar] and beyond an organism's reproductive prime. In fact, as has been suggested for the blood system, adult stem cells can function well beyond the lifespan of an organism [3Harrison D.E. Long-term erythropoietic repopulating ability of old, young, and fetal stem cells.J Exp Med. 1983; 157: 1496-1504Crossref PubMed Scopus (116) Google Scholar]. As a consequence, the activity of adult stem cells opposes the degeneration of the disposable soma and adult stem cells thus can be regarded as an intermediate and/or a parallel to the immortal germline and the soma. Conversely, we must conclude that there is little evidence that (any) adult or somatic stem cell populations can be regarded as truly immortal. Therefore, it might well be that many aspects of aging are propelled by alterations at the level of tissue-specific stem cell populations, leading ultimately to a failure to uphold organ and tissue function later in life [4Rossi D.J. Jamieson C.H. Weissman I.L. Stems cells and the pathways to aging and cancer.Cell. 2008; 132: 681-696Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar]. Like most other tissues, the hematopoietic system is not spared from alterations with advancing age. In the blood, aging is commonly associated with a decreased capacity to mount adaptive immune responses effectively [5Linton P.J. Dorshkind K. Age-related changes in lymphocyte development and function.Nat Immunol. 2004; 5: 133-139Crossref PubMed Scopus (916) Google Scholar], increased incidences of anemia with unknown origins [6Beghe C. Wilson A. Ershler W. 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Human and murine hematopoietic stem cell aging is associated with functional impairments and intrinsic megakaryocytic/erythroid bias.PLoS ONE. 2016; 11Google Scholar, 9Elias H.K. Bryder D. Park C.Y. Molecular mechanisms underlying lineage bias in aging hematopoiesis.Semin Hematol. 2017; 54: 4-11Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. These changes, which can be traced to what appears to be rather stable intrinsic changes in the differentiation capacity of hematopoietic stem cells (HSCs) [10Wahlestedt M. Norddahl G.L. Sten G. et al.An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state.Blood. 2013; 121: 4257-4264Crossref PubMed Scopus (72) Google Scholar], proposes that, as downstream progenitor fractions eventually become depleted and replenished from HSCs, age-associated alterations in HSCs might have critical influences on hematopoiesis. Such a "lineage bias" in blood cell differentiation might have direct connections to the increased incidence in myelogenous diseases with age, although direct experimental data on this issue are rather limited to date, not the least for the human system [11Signer R.A. Montecino-Rodriguez E. Witte O.N. McLaughlin J. Dorshkind K. Age-related defects in B lymphopoiesis underlie the myeloid dominance of adult leukemia.Blood. 2007; 110: 1831-1839Crossref PubMed Scopus (58) Google Scholar, 12Henry C. Marusyk A. Zaberezhnyy V. Adane B. DeGregori J. Declining lymphoid progenitor fitness promotes aging-associated leukemogenesis.Proc Natl Acad Sci USA. 2010; 107: 21713-21718Crossref PubMed Scopus (60) Google Scholar]. In fact, many of these questions are in general difficult to approach in the human system due to issues of longevity and interindividual genetic heterogeneity, which has positioned animal models and inbred mice in particular as particularly tractable model systems. Focusing more directly on the consequence of HSC aging, it can be said that, in general, most or all of the early studies addressed the HSC compartment as a pool composed of functionally homogenous units [3Harrison D.E. Long-term erythropoietic repopulating ability of old, young, and fetal stem cells.J Exp Med. 1983; 157: 1496-1504Crossref PubMed Scopus (116) Google Scholar, 13Morrison S.J. Wandycz A.M. Akashi K. Globerson A. Weissman I.L. The aging of hematopoietic stem cells.Nat Med. 1996; 2: 1011-1016Crossref PubMed Scopus (655) Google Scholar, 14Van Zant G. Holland B.P. Eldridge P.W. Chen J.J. Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice.J Exp Med. 1990; 171: 1547-1565Crossref PubMed Scopus (89) Google Scholar]. Not until later was it discovered that the HSC compartments of both young and aged individuals are made up of cells that differ both in the magnitude by which they support blood formation and also in what mature cell types they give rise to preferentially [15Dykstra B. Olthof S. Schreuder J. Ritsema M. de Haan G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.J Exp Med. 2011; 208: 2691-2703Crossref PubMed Scopus (301) Google Scholar, 16Beerman I. Bhattacharya D. Zandi S. et al.Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.Proc Natl Acad Sci USA. 2010; 107: 5465-5470Crossref PubMed Scopus (455) Google Scholar, 17Cho R.H. Sieburg H.B. Muller-Sieburg C.E. A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells.Blood. 2008; 111: 5553-5561Crossref PubMed Scopus (251) Google Scholar, 18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar, 19Challen G.A. Boles N.C. Chambers S.M. Goodell M.A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.Cell Stem Cell. 2010; 6: 265-278Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 20Dykstra B. Kent D. Bowie M. et al.Long-term propagation of distinct hematopoietic differentiation programs in vivo.Cell Stem Cell. 2007; 1: 218-229Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, 21Grover A. Sanjuan-Pla A. Thongjuea S. et al.Single-cell RNA sequencing reveals molecular and functional platelet bias of aged haematopoietic stem cells.Nat Commun. 2016; 7: 11075Crossref PubMed Scopus (156) Google Scholar]. Taking this perspective, accumulating data have proposed that HSC clones with a propensity toward myeloid cell development increase in frequency and numbers at the expense of HSCs with lymphoid potential [15Dykstra B. Olthof S. Schreuder J. Ritsema M. de Haan G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.J Exp Med. 2011; 208: 2691-2703Crossref PubMed Scopus (301) Google Scholar, 16Beerman I. Bhattacharya D. Zandi S. et al.Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.Proc Natl Acad Sci USA. 2010; 107: 5465-5470Crossref PubMed Scopus (455) Google Scholar, 17Cho R.H. Sieburg H.B. Muller-Sieburg C.E. A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells.Blood. 2008; 111: 5553-5561Crossref PubMed Scopus (251) Google Scholar, 18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar, 19Challen G.A. Boles N.C. Chambers S.M. Goodell M.A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.Cell Stem Cell. 2010; 6: 265-278Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar]. In fact, attempts have been made to approach this concept more directly and the prospective isolation of candidate HSCs based on differential cell surface expression patterns and/or different dye-efflux abilities seem to provide conclusive support to this notion [16Beerman I. Bhattacharya D. Zandi S. et al.Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion.Proc Natl Acad Sci USA. 2010; 107: 5465-5470Crossref PubMed Scopus (455) Google Scholar, 19Challen G.A. Boles N.C. Chambers S.M. Goodell M.A. Distinct hematopoietic stem cell subtypes are differentially regulated by TGF-beta1.Cell Stem Cell. 2010; 6: 265-278Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 22Morita Y. Ema H. Nakauchi H. Heterogeneity and hierarchy within the most primitive hematopoietic stem cell compartment.J Exp Med. 2010; 207: 1173-1182Crossref PubMed Scopus (313) Google Scholar]. Recently, we detailed the composition of the aging HSC compartment carefully using a genetic (lentiviral) barcoding approach [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. This allowed us to track retrospectively the blood-cell–forming potential of a relatively large number of candidate young and aged HSC clones. In agreement with previous studies, we found that the frequency of HSC clones with myeloid potential indeed did increase with age. However, and perhaps more unexpectedly, we also found that the frequency of HSC clones that harbored B-cell and/or erythroid potential did not decrease appreciably with age. Because the barcoding approach also provides (semi)quantitative data, our data strongly imply an overall reduced blood-forming ability of most aged HSC clones such that more aged HSCs are required to participate actively in hematopoiesis with advancing age. We also observed that the frequency of aged HSCs with T-cell potential was hugely diminished with age. In fact, the T-cell output from aged HSCs derived from only 3% of the evaluated clones, which was in sharp contrast to the young scenario, in which 45% of the clones contributed actively to T-cell lymphopoiesis. Furthermore, as much as one-third of the T-cell output of aged HSCs derived from clones with the potential to give rise to all (evaluated) lineages despite the fact that this fraction constituted only 1% of all aged HSCs. In the young setting, such HSCs were more than tenfold more prevalent. Nevertheless, the vast majority of candidate HSC clones evaluated, even from young mice, failed to associate with the classically defining criteria of HSCs as being able to generate offspring for all (evaluated) hematopoietic lineages. Rather, our work revealed clones with more or less every possible combination of differentiation potential [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. A broader implication of these findings is therefore how we should relate to clones that present with more restricted lineage potentials. Ignoring stochastic explanations, restrictions in lineage potential has more traditionally been assigned to that of more committed progenitors [2Bryder D. Rossi D.J. Weissman I.L. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell.Am J Pathol. 2006; 169: 338-346Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar]. However, as opposed to HSCs, a defining hallmark of such progenitors is their limited self-renewal capacity, which seems to be at odds with our findings. Rather, we observed lineage-restricted clones that persisted and gave rise continuously to mature progeny for up to 5 months [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. Such behavior might not be totally surprising when evaluating lymphopoiesis, in which mature cells can be very long-lived and also maintain themselves by homeostatic proliferation [23Boyman O. Letourneau S. Krieg C. Sprent J. Homeostatic proliferation and survival of naive and memory T cells.Eur J Immunol. 2009; 39: 2088-2094Crossref PubMed Scopus (177) Google Scholar, 24Woodland R. Schmidt M. Homeostatic proliferation of B cells.Semin Immunol. 2005; 17: 209-217Crossref PubMed Scopus (36) Google Scholar]. However, our observations included many clone types that also encompassed very short-lived granulocytic and erythroid lineages, which are in need of continuous regeneration. This implies that they should be included in our definition of HSCs because they associate with extensive self-renewal and appear to be preset toward producing specific lineages at an early age or to represent HSCs that have lost their broader differentiation potential with age. Importantly, because HSCs with full differentiation potential constituted only 13% of the young HSC compartment, HSCs with more restricted differentiation potential make up the bulk of HSC clones also in the young bone marrow (BM) [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. HSC aging has been proposed to result from perturbations involving several molecular mechanisms, including acquisition of DNA damage and/or telomere shortening, loss of cell polarity, epigenetic and transcriptional alterations, and other more specific alterations that appear secondary to the above-mentioned mechanisms [25Wahlestedt M. Pronk C.J. Bryder D. Concise review: hematopoietic stem cell aging and the prospects for rejuvenation.Stem Cells Transl Med. 2015; 4: 186-194Crossref PubMed Scopus (25) Google Scholar]. This applies also to potential alterations in the supportive BM microenvironment [25Wahlestedt M. Pronk C.J. Bryder D. Concise review: hematopoietic stem cell aging and the prospects for rejuvenation.Stem Cells Transl Med. 2015; 4: 186-194Crossref PubMed Scopus (25) Google Scholar, 26Ergen A.V. Boles N.C. Goodell M.A. Rantes/Ccl5 influences hematopoietic stem cell subtypes and causes myeloid skewing.Blood. 2012; 119: 2500-2509Crossref PubMed Scopus (175) Google Scholar]. It is well established that the function of HSCs changes during early development. For instance, fetal liver HSCs proliferate actively [27Morrison S.J. Hemmati H.D. Wandycz A.M. Weissman I.L. The purification and characterization of fetal liver hematopoietic stem cells.Proc Natl Acad Sci USA. 1995; 92: 10302-10306Crossref PubMed Scopus (464) Google Scholar], whereas adult HSCs acquire a more quiescent nature a few weeks after birth [28Bowie M.B. Kent D.G. Dykstra B. et al.Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties.Proc Natl Acad Sci USA. 2007; 104: 5878-5882Crossref PubMed Scopus (177) Google Scholar]. The dramatically different properties of fetal and adult HSCs can be attributed to a different transcriptional makeup and thus also to differing epigenomic profiles [28Bowie M.B. Kent D.G. Dykstra B. et al.Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties.Proc Natl Acad Sci USA. 2007; 104: 5878-5882Crossref PubMed Scopus (177) Google Scholar, 29Beerman I. Bock C. Garrison B.S. et al.Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging.Cell Stem Cell. 2013; 12: 413-425Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 30Taiwo O. Wilson G.A. Emmett W. et al.DNA methylation analysis of murine hematopoietic side population cells during aging.Epigenetics. 2013; 8: 1114-1122Crossref PubMed Scopus (32) Google Scholar, 31Sun D. Luo M. Jeong M. et al.Epigenomic profiling of young and aged HSCs reveals concerted changes during aging that reinforce self-renewal.Cell Stem Cell. 2014; 14: 673-688Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 32Farlik M. Halbritter F. Muller F. et al.DNA methylation dynamics of human hematopoietic stem cell differentiation.Cell Stem Cell. 2016; 19: 808-822Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar]. The differences in proliferation rates aside, most if not all adult HSCs will inevitably enter the cell cycle and might do so many times during the lifespan of an organism to be able to provide an adequate supply of mature effector cells [33Säwén P. Lang S. Mandal P. Rossi D.J. Soneji S. Bryder D. Mitotic history reveals distinct stem cell populations and their contributions to hematopoiesis.Cell Rep. 2016; 14: 2809-2818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar]. Upon a cell division, the epigenetic features of the parental cell must be reinstated in its daughter cell/s, which could potentially lead to "slipping" of the epigenome and thus gradually give rise to an aged epigenome. We find it particularly noteworthy that changes in the epigenome of HSCs have been reported to occur as a consequence of proliferative stress, with many changes overlapping those seen in chronologically aged HSCs [29Beerman I. Bock C. Garrison B.S. et al.Proliferation-dependent alterations of the DNA methylation landscape underlie hematopoietic stem cell aging.Cell Stem Cell. 2013; 12: 413-425Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar]. Because of this, it is tempting to view HSC aging as a slow and progressive epigenetic drift that starts from fetal to young adult HSCs and continues into the later stages of life. A main finding from our work was the observation that the clonal output from aged HSCs in general was dramatically lower than for young HSCs [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. This also held true for most aged HSC clones with myeloid differentiation potential despite their dramatic amplification with age. We interpret this as evidence for the view that the numerical increase in HSCs with age is an adaptation that might be necessary to maintain appropriate mature blood cell output [34Harrison D.E. Astle C.M. Stone M. Numbers and functions of transplantable primitive immunohematopoietic stem cells. Effects of age.J Immunol. 1989; 142: 3833-3840PubMed Google Scholar]. Perhaps our most dramatic finding was the drastic (fifteenfold) decrease in frequency of T-cell-competent HSC clones with age. Why then would the T-cell potential of HSCs be nearly abolished with age? One obvious reason for a decreased production of naive T cells with age is the involution of the thymus commencing with age, a process under strong influence of sex steroids [35Hince M. Sakkal S. Vlahos K. Dudakov J. Boyd R. Chidgey A. The role of sex steroids and gonadectomy in the control of thymic involution.Cell Immunol. 2008; 252: 122-138Crossref PubMed Scopus (89) Google Scholar]. However, this relates perhaps less to available functional data on HSC aging, which has mostly been obtained from transplantation experiments in which aged HSCs are transferred into young mice with intact thymi. However, in addition to a role in thymic involution, increased levels of sex steroids also have been reported to affect BM lymphoid-competent cells negatively [36Dudakov J. Goldberg G. Reiseger J. Chidgey A. Boyd R. Withdrawal of sex steroids reverses age- and chemotherapy-related defects in bone marrow lymphopoiesis.J Immunol. 2009; 182: 6247-6260Crossref PubMed Scopus (38) Google Scholar]. Although perhaps a direct argument in favor of the disposable soma theory of aging, the fact that these changes might be intrinsic determinants at the level of aged HSCs [10Wahlestedt M. Norddahl G.L. Sten G. et al.An epigenetic component of hematopoietic stem cell aging amenable to reprogramming into a young state.Blood. 2013; 121: 4257-4264Crossref PubMed Scopus (72) Google Scholar, 18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar, 37Rossi D.J. Bryder D. Zahn J.M. et al.Cell intrinsic alterations underlie hematopoietic stem cell aging.Proc Natl Acad Sci USA. 2005; 102: 9194-9199Crossref PubMed Scopus (819) Google Scholar] should regardless have epigenetic underpinnings. In its simplest form, such an epigenetic drift of aged HSCs could lead to a decreased/defective generation of progenitors destined to home to the thymus. Supporting this are the recent demonstrations of age-associated changes in an early multipotent progenitor fraction, referred to as lymphoid-primed multipotent progenitors (LMPPs) or granulocyte-monocyte-lymphoid progenitors (GMLPs) [38Young K. Borikar S. Bell R. Kuffler L. Philip V. Trowbridge J. Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.J Exp Med. 2016; 213: 2259-2267Crossref PubMed Scopus (52) Google Scholar]. From this work, it has been concluded that aged LMPPs/GMLPs proliferate with faster kinetics [33Säwén P. Lang S. Mandal P. Rossi D.J. Soneji S. Bryder D. Mitotic history reveals distinct stem cell populations and their contributions to hematopoiesis.Cell Rep. 2016; 14: 2809-2818Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 38Young K. Borikar S. Bell R. Kuffler L. Philip V. Trowbridge J. Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.J Exp Med. 2016; 213: 2259-2267Crossref PubMed Scopus (52) Google Scholar] and associate with reduced lymphoid differentiation potential and decreased expression of lymphoid-associated transcripts [38Young K. Borikar S. Bell R. Kuffler L. Philip V. Trowbridge J. Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.J Exp Med. 2016; 213: 2259-2267Crossref PubMed Scopus (52) Google Scholar]. Although such changes could occur separately from changes in the upstream HSCs, with recent demonstrations that LMPPs can be quite long-lived at least during steady-state [39Busch K. Klapproth K. Barile M. et al.Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.Nature. 2015; 518: 542-546Crossref PubMed Scopus (447) Google Scholar], most progenitors eventually appear to be derived from HSCs [40Sawai C. Babovic S. Upadhaya S. et al.Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals.Immunity. 2016; 45: 597-609Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar], although evidence exists that this can be altered in extreme circumstances [41Martins V. Busch K. Juraeva D. et al.Cell competition is a tumour suppressor mechanism in the thymus.Nature. 2014; 509: 465-470Crossref PubMed Scopus (164) Google Scholar]. A central question to us revolves on whether HSC aging is the result of an altered epigenome and, by extension, whether HSC aging might be a reversible process. To begin to unravel this, we undertook an approach in which we reprogrammed hematopoietic progenitor cells, which were derived from barcoded HSCs, into induced pluripotent stem cells (iPSCs) [18Wahlestedt M. Erlandsson E. Kristiansen T. et al.Clonal reversal of ageing-associated stem cell lineage bias via a pluripotent intermediate.Nat Commun. 2017; 8: 1-8Crossref PubMed Scopus (24) Google Scholar]. After barcode determination of the iPSCs, we selected iPSCs derived from aged HSCs that were characterized by pronounced lineage skewing (i.e., that had produced no lymphoid offspring). We next injected blastocysts and morulas with the barcoded iPSCs, followed by implantation of blastocysts/morulas into pseudopregnant mothers. This allowed us to study iPSC-derived hematopoiesis in the resulting chimeric mice. Our underlying reason for this rather complicated experimental paradigm was not only to utilize the well-known epigenetic reset that associates with iPSC reprogramming [42Apostolou E. Hochedlinger K. Chromatin dynamics during cellular reprogramming.Nature. 2013; 502: 462-471Crossref PubMed Scopus (284) Google Scholar] but also to approach such HSC clones that are aged by functional criteria in an appropriate developmental context. Our work revealed that all tested iPSC clones gave rise to HSCs with an ability to generate B, T, and myeloid cells in a similar manner as young HSCs. Further, we found no evidence of a numerical expansion of the HSCs, another hallmark of HSC aging [15Dykstra B. Olthof S. Schreuder J. Ritsema M. de Haan G. Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.J Exp Med. 2011; 208: 2691-2703Crossref PubMed Scopus (301) Google Scholar, 37Rossi D.J. Bryder D. Zahn J.M. et al.Cell intrinsic alterations underlie hematopoietic stem cell aging.Proc Natl Acad Sci USA. 2005; 102: 9194-9199Crossref PubMed Scopus (819) Google Scholar], nor any molecular evidence for the retention of an aging "memory" [43Kim K. Doi A. Wen B. et al.Epigenetic memory in induced pluripotent stem cells.Nature. 2010; 467: 285-290Crossref PubMed Scopus (1714) Google Scholar, 44Mertens J. Paquola A.C. Ku M. et al.Directly reprogrammed human neurons retain aging-associated transcriptomic signatures and reveal age-related nucleocytoplasmic defects.Cell Stem Cell. 2015; 17: 705-718Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar]. Therefore, by all evaluated criteria, the reprogramming process led to the derivation of HSCs that were indistinguishable from chronologically young HSCs. It therefore appears that a driving component of HSC aging must be reversible epigenetic and/or transcriptional alterations (Fig. 1). Importantly, these findings have little compatibility with the view that defined DNA lesions would be the primary driver of all features of HSC aging because such damage should persist during the iPSC induction process and subsequent hematopoietic differentiation. Therefore, whereas dormant aged HSCs have been found to acquire DNA damage [45Beerman I. Seita J. Inlay M.A. Weissman I.L. Rossi D.J. Quiescent hematopoietic stem cells accumulate DNA damage during aging that is repaired upon entry into cell cycle.Cell Stem Cell. 2014; 15: 37-50Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 46Rossi D.J. Bryder D. Seita J. Nussenzweig A. Hoeijmakers J. Weissman I.L. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age.Nature. 2007; 447: 725-729Crossref PubMed Scopus (859) Google Scholar, 47Rübe C.E. Fricke A. Widmann T.A. et al.Accumulation of DNA damage in hematopoietic stem and progenitor cells during human aging.PLoS ONE. 2011; 6 (e17487)Crossref PubMed Scopus (222) Google Scholar, 48Wang J. Morita Y. Han B. Niemann S. Loffler B. Rudolph K. 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However, because the aged HSC compartment contains at least a fraction of HSCs with a blood-forming potential highly similar to that of young HSCs, aging does not affect all HSC clones with the same kinetics. This likely reflects that the acquisition of age-associated epigenetic alterations depends, at least to some extent, on opportunistic and stochastic processes. Although it appears feasible that "youthful" HSC clones found in chronologically aged hosts have remained dormant through the majority of adulthood and recruited into cell cycle late in life, direct evidence for this is at present rather limited. Regardless, it has to be argued that the functionality of an aged HSC clone must be demonstrated to claim that rejuvenation has occurred. This is because any intervening strategy might well enhance the function and/or numbers of cells functionally unaffected by age, but leave functionally compromised aged HSC clones untouched. The use of iPSCs is thought to stand at the frontlines of future cell replacement therapies and personalized medicine, which might be particularly relevant for the aging population. However, whereas the findings that somatic cell reprogramming can erase the marks of functionally compromised aged cells might have broad implications, we wish to emphasize that our work investigated whether rejuvenation could be achieved for a very limited number of aged HSCs (five individual clones). Furthermore, our approach did not make it possible to reveal aged HSC clones too dysfunctional to be rejuvenated. Therefore, future studies should also investigate the frequency of HSC clones that cannot be rejuvenated by epigenetic reversal and why. A few recent studies have found that old age can coincide with mutations associated with clonal hematopoiesis (summarized by Steensma et al [58Steensma D. Bejar R. Jaiswal S. et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (1091) Google Scholar].). Intriguingly, among the most commonly mutated genes are ASXL1, DNMT3A, and TET2, which not only are key epigenetic regulators, but also have a prominent connection to hematological disease [58Steensma D. Bejar R. Jaiswal S. et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (1091) Google Scholar]. Indeed, carriers of these mutations associated with an increased risk of developing hematological disease later in life. Although supportive of the theory that DNA damage might underlie hematopoietic cell/HSC aging, the fact that only a minor fraction of the studied population do acquire such mutations [58Steensma D. Bejar R. Jaiswal S. et al.Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes.Blood. 2015; 126: 9-16Crossref PubMed Scopus (1091) Google Scholar] perhaps argues against this as a major determinant of HSC aging. In addition, whereas it appears intuitive that the acquisition of such defined mutations is associated with a preleukemic state, its impact on normal hematopoietic aging and information on the stages of hematopoiesis at which the mutations initially arise remain unknown. Because the physiological processes of aging coincide with an increased incidence of many diseases and worsened organismal function in general, the successful development of strategies that increase the health span and an overall healthier aging process should be of great benefit. Toward this goal, one important step is to achieve cellular rejuvenation directly in vivo. Although much effort has been devoted to increasing the lifespan of model organisms, it remains for the most part more unclear whether these strategies target cells functionally affected by age. Recently, one study observed that brief in vivo activation of the reprogramming factors used in our studies (Oct4, Sox2, Klf4, and c-Myc) improved several aspects of aging both in chronologically aged mice and in a model of premature aging [57Ocampo A. Reddy P. Martinez-Redondo P. et al.In vivo amelioration of age-associated hallmarks by partial reprogramming.Cell. 2016; 167 (e12): 1719-1733Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar]. Although providing additional support to an altered epigenome as a major determinant of the aging process, combining the clonal/barcoding aspects with the in vivo "reprogramming" regime of Ocampo et al [57Ocampo A. Reddy P. Martinez-Redondo P. et al.In vivo amelioration of age-associated hallmarks by partial reprogramming.Cell. 2016; 167 (e12): 1719-1733Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar] might also reveal whether cells strongly functionally compromised with age can be rejuvenated directly in vivo. Future studies that combine epigenomics, transcriptomics, and mutational analyses with the functional readout of individual aged HSCs will be necessary to dissect and understand the process of hematopoietic aging fully. This includes the development of strategies that permit the functional evaluation of HSC activity in more native settings, as opposed to the transplantation-based approaches that currently prevail. Although such approaches to a large extent have been hindered by technical limitations, the times are exciting with recent and rapid developments in sequencing-based approaches, genome editing, and iPSC technologies. Therefore, we anticipate advances at an accelerated pace in this relatively young field of research, with the final goal of making epigenetically based therapies available to achieve healthier late stages of life. D.B. is supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, ERC Consolidator Grant 615068, and the Tobias Foundation.
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