Title: Hematopoietic stem cell fate through metabolic control
Abstract: •Specific modes of metabolism play important roles in hematopoietic stem cell (HSC) self-renewal.•Heterogeneity and technical challenges have prevented the elucidation of HSC behavior.•Recent advances have highlighted mitochondrial quality control as a key HSC fate factor.•A deeper understanding of HSC fate via metabolic control has clinical implications. Hematopoietic stem cells maintain a quiescent state in the bone marrow to preserve their self-renewal capacity, but also undergo cell divisions as required. Organelles such as the mitochondria sustain cumulative damage during these cell divisions and this damage may eventually compromise the cells' self-renewal capacity. Hematopoietic stem cell divisions result in either self-renewal or differentiation, with the balance between the two affecting hematopoietic homeostasis directly; however, the heterogeneity of available hematopoietic stem cell-enriched fractions, together with the technical challenges of observing hematopoietic stem cell behavior, has long hindered the analysis of individual hematopoietic stem cells and prevented the elucidation of this process. Recent advances in genetic models, metabolomics analyses, and single-cell approaches have revealed the contributions made to hematopoietic stem cell self-renewal by metabolic cues, mitochondrial biogenesis, and autophagy/mitophagy, which have highlighted mitochondrial quality control as a key factor in the equilibrium of hematopoietic stem cells. A deeper understanding of precisely how specific modes of metabolism control hematopoietic stem cells fate at the single-cell level is therefore not only of great biological interest, but will also have clear clinical implications for the development of therapies for hematological diseases. Hematopoietic stem cells maintain a quiescent state in the bone marrow to preserve their self-renewal capacity, but also undergo cell divisions as required. Organelles such as the mitochondria sustain cumulative damage during these cell divisions and this damage may eventually compromise the cells' self-renewal capacity. Hematopoietic stem cell divisions result in either self-renewal or differentiation, with the balance between the two affecting hematopoietic homeostasis directly; however, the heterogeneity of available hematopoietic stem cell-enriched fractions, together with the technical challenges of observing hematopoietic stem cell behavior, has long hindered the analysis of individual hematopoietic stem cells and prevented the elucidation of this process. Recent advances in genetic models, metabolomics analyses, and single-cell approaches have revealed the contributions made to hematopoietic stem cell self-renewal by metabolic cues, mitochondrial biogenesis, and autophagy/mitophagy, which have highlighted mitochondrial quality control as a key factor in the equilibrium of hematopoietic stem cells. A deeper understanding of precisely how specific modes of metabolism control hematopoietic stem cells fate at the single-cell level is therefore not only of great biological interest, but will also have clear clinical implications for the development of therapies for hematological diseases. 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Because HSC populations are precisely controlled within certain limits in vivo, once hematopoietic recovery is complete, it is believed that HSCs return to a quiescent state (dormancy). This suspension of the cell cycle is thought to make a critical contribution to the maintenance of stem cells' self-renewal capacity and multipotency because deletion of the genes involved in quiescence often leads to HSC exhaustion due to uncontrolled proliferation [14Ito K Carracedo A Weiss D et al.A PML-PPAR-delta pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance.Nat Med. 2012; 18: 1350-1358Crossref PubMed Scopus (216) Google Scholar, 15Wilson A Laurenti E Oser G et al.Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.Cell. 2008; 135: 1118-1129Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 16Cheng T Rodrigues N Shen H et al.Hematopoietic stem cell quiescence maintained by p21cip1/waf1.Science. 2000; 287: 1804-1808Crossref PubMed Scopus (937) Google Scholar, 17Pietras EM Warr MR Passegue E Cell cycle regulation in hematopoietic stem cells.J Cell Biol. 2011; 195: 709-720Crossref PubMed Scopus (174) Google Scholar, 18Trumpp A Essers M Wilson A Awakening dormant haematopoietic stem cells.Nat Rev Immunol. 2010; 10: 201-209Crossref PubMed Scopus (213) Google Scholar, 19Nakamura-Ishizu A Takizawa H Suda T The analysis, roles and regulation of quiescence in hematopoietic stem cells.Development. 2014; 141: 4656-4666Crossref PubMed Scopus (43) Google Scholar]. 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Vitamin a-retinoic acid signaling regulates hematopoietic stem cell dormancy.Cell. 2017; 169 (e19): 807-823Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar] (Figure 1). In this review, we highlight recent advances in our understanding of the intriguing relationship among cellular metabolism, mitochondrial quality control, and HSC fate decisions. 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Their possible division options are: symmetric self-renewal expansion (symmetric division, SD, in which both daughter cells have the same function as the original cell), self-renewal maintenance (asymmetric division, AD), and differentiation (symmetric commitment, SC, in which both daughter cells are differentiated from the original parent cell) and their eventual division pattern is determined by the in vivo repopulation capacity of their daughter cells. In cases in which at least one daughter cell is a long-term HSC (LT-HSC), the original cell must also be an LT-HSC. However, if both daughter cells are non-LT-HSCs, interpreting the resulting data can be complex because a cell's original function can affect its division pattern (Figure 2A). Further, the homogeneity of the cell population is critical to accurate division pattern analysis. Tracking the divisions of individual cells from a heterogeneous population has proved difficult and any contamination of non-HSCs can lead to an overestimate of the rate of SC. As an example, let us consider a 30% pure population (low purity) in which three out of 10 single cells in the HSC fraction must be "real" HSCs and a case in which one of these HSCs undergoes SD (33%), whereas another undergoes AD (33%) and the third undergoes SC (33%). Because committed cells cannot produce HSCs upon their division, their division patterns must be regarded as SC. The resulting division balance of the entire population would therefore be SD 11%, AD 11%, and SC 78% (Figure 2B). HSCs have been identified retrospectively after single-cell transplantation by clonal assays and these assays have demonstrated the heterogeneity of currently available HSC-enriched fractions [32Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 35Osawa M Hanada K Hamada H Nakauchi H Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Google Scholar, 36Benveniste P Cantin C Hyam D Iscove NN Hematopoietic stem cells engraft in mice with absolute efficiency.Nat Immunol. 2003; 4: 708-713Crossref PubMed Scopus (112) Google Scholar, 37Kiel MJ Yilmaz OH Iwashita T Yilmaz OH Terhorst C Morrison SJ SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.Cell. 2005; 121: 1109-1121Abstract Full Text Full Text PDF PubMed Scopus (1891) Google Scholar, 38Dykstra 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 (301) Google Scholar]. Unfortunately, the reported frequency of HSCs in these fractions is generally lower than 30% and it is worth pointing out that, in the case described earlier (SD: AD: SC = 1: 1: 1), an HSC purity of even ∼40% would be regarded as low because the overestimation of SC would lead to a significant shift in the assessed division balance (to a maximum of 44% SC in n = 27, and 41% SC in n = 50 divisions assessed, respectively. *p < 0.05 by Chi-squared test). However, when we have a high-purity population of real HSCs, we can more accurately determine their division pattern (Figure 2B). To avoid this imprecision, researchers have long sought a reliable marker for individual HSCs that is strongly associated with repopulation capacity and does not fluctuate with changes in the surrounding environment and/or cell cycle. In various attempts to detect purified HSCs, recent studies have utilized combinations of cell surface markers, the reporter Cre-recombinase, and antibody positivity, but so far, these efforts have met with only limited success [8Busch K Klapproth K Barile M et al.Fundamental properties of unperturbed haematopoiesis from stem cells in vivo.Nature. 2015; 518: 542-546Crossref PubMed Scopus (192) Google Scholar, 39Gazit R Mandal PK Ebina W et al.Fgd5 identifies hematopoietic stem cells in the murine bone marrow.J Exp Med. 2014; 211: 1315-1331Crossref PubMed Scopus (41) Google Scholar, 40Sanjuan-Pla A Macaulay IC Jensen CT et al.Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.Nature. 2013; 502: 232-236Crossref PubMed Scopus (156) Google Scholar, 41Acar M Kocherlakota KS Murphy MM et al.Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.Nature. 2015; 526: 126-130Crossref PubMed Scopus (145) Google Scholar, 42Challen GA Boles NC Chambers SM Goodell MA 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 (294) Google Scholar, 43Chen JY Miyanishi M Wang SK et al.Hoxb5 marks long-term haematopoietic stem cells and reveals a homogenous perivascular niche.Nature. 2016; 530: 223-227Crossref PubMed Scopus (71) Google Scholar, 44Carrelha J Meng Y Kettyle LM et al.Hierarchically related lineage-restricted fates of multipotent haematopoietic stem cells.Nature. 2018; 554: 106-111Crossref PubMed Scopus (7) Google Scholar]. Until recently, HSC number and capacity were believed to decrease rather than increase with age and it has proved very challenging to expand the HSC population while maintaining stem-ness. Indeed, although division patterns in hematopoietic stem and progenitor cells (HSPCs) were thought to be controlled by the balance between SC and AD [14Ito K Carracedo A Weiss D et al.A PML-PPAR-delta pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance.Nat Med. 2012; 18: 1350-1358Crossref PubMed Scopus (216) Google Scholar, 32Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 33Suda T Suda J Ogawa M Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors.Proc Natl Acad Sci U S A. 1984; 81: 2520-2524Crossref PubMed Google Scholar], advanced single-cell approaches have recently confirmed that HSCs are capable of symmetric self-renewing division (or SD) [32Yamamoto R Morita Y Ooehara J et al.Clonal analysis unveils self-renewing lineage-restricted progenitors generated directly from hematopoietic stem cells.Cell. 2013; 154: 1112-1126Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 45Ito K Turcotte R Cui J et al.Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.Science. 2016; 354: 1156-1160Crossref PubMed Scopus (42) Google Scholar]. 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Our use of Tie2 positivity as a marker has allowed us to identify a purified population of HSCs and we have demonstrated with our local transplantation protocol that single HSCs from this population exhibit high reconstitution capacity in vivo [45Ito K Turcotte R Cui J et al.Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.Science. 2016; 354: 1156-1160Crossref PubMed Scopus (42) Google Scholar, 47Turcotte R Alt C Runnels JM et al.Image-guided transplantation of single cells in the bone marrow of live animals.Sci Rep. 2017; 7: 3875Crossref PubMed Scopus (2) Google Scholar]. Our tracking technique allowed us to determine the function of the paired daughter cells resulting from single HSC divisions, which in turn enabled us to more accurately visualize division patterns and distinguish self-renewal expansion from self-renewal maintenance. In these studies, we found that only top hierarchical HSCs underwent SD, in which both daughter cells are HSCs and retain Tie2 positivity [45Ito K Turcotte R Cui J et al.Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.Science. 2016; 354: 1156-1160Crossref PubMed Scopus (42) Google Scholar]. Because increasing evidence supports the essential contributions of metabolic control to HSC division patterns, determining the metabolic mode of purified HSCs is of crucial importance [14Ito K Carracedo A Weiss D et al.A PML-PPAR-delta pathway for fatty acid oxidation regulates hematopoietic stem cell maintenance.Nat Med. 2012; 18: 1350-1358Crossref PubMed Scopus (216) Google Scholar, 28Ito K Ito K Metabolism and the control of cell fate decisions and stem cell renewal.Annu Rev Cell Dev Biol. 2016; 32: 399-409Crossref PubMed Scopus (4) Google Scholar]. 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