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Beyond “to divide or not to divide”: Kinetics matters in hematopoietic stem cells

  • Carys Johnson
    Affiliations
    Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
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  • Serena Belluschi
    Affiliations
    Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
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  • Elisa Laurenti
    Correspondence
    Offprint requests to: Elisa Laurenti, Department of Haematology and Wellcome MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre, Cambridge CB2 0AW, UK
    Affiliations
    Department of Haematology and Wellcome and MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK
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Open ArchivePublished:November 11, 2020DOI:https://doi.org/10.1016/j.exphem.2020.11.003

      Highlights

      • Distinct quiescent states exist in the HSC pool to maintain lifelong function.
      • The CDK4/CDK6/cyclin D complex is key to maintain the balance between HSC quiescence and division.
      • The kinetics of quiescence exit and early G1 influence HSC differentiative output ex vivo.
      • Modulating the quiescence to division kinetics has key implications for HSC expansion and gene therapy.
      Lifelong blood production is ensured by a population of rare and largely quiescent, long-lived hematopoietic stem cells (HSCs). The advent of single-cell technologies has recently highlighted underlying molecular and functional heterogeneity within the HSC pool. Despite heterogenous HSC behaviors, quiescence remains as the most uncontroversial and unifying property of HSCs. Nonetheless, a multifaceted and complex continuum of states has recently been identified within what was previously described as just “quiescent.” Here we review such evidence and discuss how it challenges preconceived ideas on the contribution of cell cycle kinetics to HSC function. Specifically, we detail how both the frequency and kinetics of HSC division, largely determined by a network of molecular regulators linked to early G1, influence long-term HSC functionin vivo. In addition, we present data that indicate lengthening the duration of G1 by inhibiting CDK6 decreases lymphoid differentiation of a subset of lymphoid-primed human HSCs, thus linking cell cycle kinetics to cell fate decisions in HSCs. Finally, we reflect on how these new insights can be helpful to fully harness HSC potential in clinical applications that require ex vivo culture.
      A trillion blood cells are produced daily to maintain blood function at steady state. This immense regenerative output is achieved by the concerted action of rare and infrequently dividing hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Decades of work, primarily in mouse models, have built complex roadmaps of the first steps of hematopoiesis by dissecting the cascade of cell divisions occurring from the most potent HSCs to multilineage and, in turn, unilineage progenitors. Recently, single-cell RNA-seq and clonal tracking have complemented these by defining differentiation trajectories at single-cell resolution [
      • Laurenti E
      • Göttgens B
      From haematopoietic stem cells to complex differentiation landscapes.
      ]. All evidence to date agrees that unilineage progenitor cell types typically divide continuously, re-entering G1 after mitosis. In contrast, HSCs and MPPs do not; they divide occasionally, spending the majority of their cellular life outside of the cell cycle, in a reversible state termed quiescence or G0. As HSCs and MPPs largely differ at single-cell resolution in their self-renewal capacity and lineage preferences [
      • Laurenti E
      • Göttgens B
      From haematopoietic stem cells to complex differentiation landscapes.
      ], this places quiescence as the only fundamental common property of all HSC/MPPs.
      Quiescence can be conceptualized as a poised, restrained, and actively maintained molecular state [
      • Velthoven CTJ van
      • Rando TA
      Stem cell quiescence: dynamism, restraint, and cellular idling.
      ]. Classic hallmarks of quiescence are shared by many adult stem cell types and include low protein synthesis and reliance on glycolytic metabolism. Consistently, in vivo, the maintenance of HSCs in quiescence relies on a specific metabolic context and organelle biology. Initially thought to have low mitochondrial mass, quiescent HSCs are now known to contain relatively high numbers of inactive mitochondria [
      • de Almeida MJ
      • Luchsinger LL
      • Corrigan DJ
      • Williams LJ
      • Snoeck HW
      Dye-independent methods reveal elevated mitochondrial mass in hematopoietic stem cells.
      ] with an abundance of large inactive lysosomes [
      • Liang R
      • Arif T
      • Kalmykova S
      • et al.
      Restraining lysosomal activity preserves hematopoietic stem cell quiescence and potency.
      ]. Long-term maintenance of HSC potency is reliant on restrained glycolytic metabolism [
      • Liang R
      • Arif T
      • Kalmykova S
      • et al.
      Restraining lysosomal activity preserves hematopoietic stem cell quiescence and potency.
      ,
      • Suda T
      • Takubo K
      • Semenza GL
      Metabolic regulation of hematopoietic stem cells in the hypoxic niche.
      ] and a balanced metabolic state maintained by autophagic recycling [
      • Warr MR
      • Binnewies M
      • Flach J
      • et al.
      FOXO3A directs a protective autophagy program in haematopoietic stem cells.
      ,
      • Ho TT
      • Warr MR
      • Adelman ER
      • et al.
      Autophagy maintains the metabolism and function of young and old stem cells.
      ]. Protein synthesis rates are low in quiescent HSCs [
      • Signer RAJ
      • Magee JA
      • Salic A
      • Morrison SJ
      Haematopoietic stem cells require a highly regulated protein synthesis rate.
      ,
      • Signer RAJ
      • Qi L
      • Zhao Z
      • et al.
      The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs.
      ], yet there are high thresholds of protein quality control ensured by high basal expression of endoplasmic reticulum-associated degradation (ERAD) [
      • Liu L
      • Inoki A
      • Fan K
      • et al.
      ER associated degradation preserves hematopoietic stem cell quiescence and self-renewal by restricting mTOR activity.
      ,
      • Xu L
      • Liu X
      • Peng F
      • Zhang W
      • Zheng L
      • Ding Y
      • et al.
      Protein quality control through endoplasmic reticulum-associated degradation maintains haematopoietic stem cell identity and niche interactions.
      ] and integrated stress response pathways [
      • van Galen P
      • Kreso A
      • Mbong N
      • et al.
      The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.
      ,
      • Galen P van
      • Mbong N
      • Kreso A
      • et al.
      Integrated stress response activity marks stem cells in normal hematopoiesis and leukemia.
      ]. Similarly, cellular responses to DNA damage are distinct in HSCs compared with progenitors [
      • Milyavsky M
      • Gan OI
      • Trottier M
      • et al.
      A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal.
      ,
      • Mohrin M
      • Bourke E
      • Alexander D
      • et al.
      Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.
      ]. A consequence of the longevity of HSCs is the accumulation of genotoxic, proteotoxic, and oxidative stress, which, in the absence of cell division, cannot be distributed to progeny. Quiescent HSCs are thus wired to maintain the highest standards of genomic, proteostatic, and organelle integrity, resulting in preferential culling of damaged HSCs to mitigate the risk of leukemic transformation and reduced HSC pool fitness.
      Maintenance of HSC quiescence, exit from quiescence, division, and subsequent return to quiescence must be tightly controlled. This balance has emerged to be intimately linked to HSC function. In the past decade, single-cell resolution and novel tools have revealed that the overall picture is complex and cannot be oversimplified to “HSCs are either more quiescent or more proliferative” in the particular instance studied. Here we focus on a few important findings that have emerged from these studies. We provide definitions and a framework in which to interpret the complex relationship among quiescence, cell cycle kinetics, and HSC fate choices—a key step toward new translational opportunities for HSC expansion, transplantation, and gene therapy.

      Quiescence is a collection of actively maintained cellular states

      In hierarchically organized stem cell systems such as blood, muscle [
      • Rodgers JT
      • King KY
      • Brett JO
      • et al.
      mTORC1 controls the adaptive transition of quiescent stem cells from G0 to GAlert.
      ], and the subventricular zone of the brain [
      • Llorens-Bobadilla E
      • Zhao S
      • Baser A
      • Saiz-Castro G
      • Zwadlo K
      • Martin-Villalba A
      Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury.
      ], there is now ample evidence that quiescence is not a uniform state and that varying depths of quiescence allow cells to differently and appropriately contribute to tissue maintenance and regeneration. Across all these tissues, the most immature stem cells reside in a deeper quiescent state than their progeny. Depth of quiescence is measured primarily via the time a cell takes to exit this state upon receiving a mitogenic stimulus (quiescence exit: from G0 to end of early G1), often approximated to the time of first division ex vivo. Quiescence is an integral state existing in a wide range of organisms; there is much to be learnt from seminal work in bacteria, yeast, and cell lines that applies to adult stem cells, including HSCs.
      First, protein levels of the CDK4/CDK6 complex are a faithful reporter of quiescence depth. CDK4/CDK6 activation is the key trigger for cells to enter G1 whether they have just divided (from M to G1) or have been quiescent for a given period (from G0 to G1). CDK4/CDK6 activity sustains retinoblastoma protein (Rb) hyperphosphorylation in early G1, which is necessary for a cell to pass the restriction point (R point) and commit to division [
      • Bertoli C
      • Skotheim JM
      • de Bruin RAM
      Control of cell cycle transcription during G1 and S phases.
      ]. In human and mouse HSCs, CDK6 levels govern the degree to which a cell is primed to respond to a mitogenic stimulus. Quiescent long-term HSCs (LT-HSCs, capable of forming blood over serial transplantation) possess undetectable levels of CDK6 protein, whereas quiescent short-term HSCs (ST-HSCs, with transient regenerative potential upon transplantation) express higher CDK6 protein levels to allow faster cell cycle entry following mitogenic stimulation [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Laurenti E
      • Frelin C
      • Xie S
      • et al.
      CDK6 levels regulate quiescence exit in human hematopoietic stem cells.
      ] (Figure 1). Recent work has indicated that reaching the R point is best described as a probabilistic process dependent on levels of CDK4/CDK6 activity in single cells [
      • Chung M
      • Liu C
      • Yang HW
      • Köberlin MS
      • Cappell SD
      • Meyer T
      Transient hysteresis in CDK4/6 activity underlies passage of the restriction point in G1.
      ]. This implies that for a specific level of mitogenic stimulus, cells with high levels of CDK4/CDK6, such as ST-HSCs, have a higher probability of commitment to division than cells with low levels (such as LT-HSCs). It is thus tempting to speculate that LT-HSCs may also require a stronger/longer mitogenic stimulus to divide. LT-HSCs may also be more likely to return to quiescence, as inhibition of CDK4/CDK6 is required for cells to return to G0 after mitosis [
      • Yang HW
      • Cappell SD
      • Jaimovich A
      • et al.
      Stress-mediated exit to quiescence restricted by increasing persistence in CDK4/6 activation.
      ]. In any case, maintenance of distinct depths of quiescence in LT-HSCs and ST-HSCs and the associated division kinetics are necessary to control HSC pool size as well as effective tissue regeneration [
      • Laurenti E
      • Frelin C
      • Xie S
      • et al.
      CDK6 levels regulate quiescence exit in human hematopoietic stem cells.
      ].
      Figure 1
      Figure 1HSC quiescence is a continuum of molecular states associated with specific functional features. The cell cycle has four distinct phases indicated by the inner circle, mitosis (M), gap 1 (G1), the DNA synthesis phase (S), and gap 2 (G2). Quiescence (G0) is a collection of stable states (schematized by color shading) in which retinoblastoma protein (Rb) is unphosphorylated and levels of CDK proteins are low. Dormant HSCs divide very infrequently, reside in a particularly deep state of quiescence and are largely resistant to activation. Dormant HSCs, LT-HSCs and ST-HSCs reside in sequentially shallower states of quiescence, each becoming more primed for cell cycle re-entry. Quiescence exit (indicated by arrow, with arrow thickness indicating strength of activation signal required for cell cycle re-entry) in HSCs is predominantly mediated by levels of CDK6, which form a complex with cyclin D to allow cell cycle re-entry. During early G1, Rb is progressively re-phosphorylated until hyperphosphorylation at the “restriction point” (denoted as R) whereby cell lose dependency on extracellular mitogens for cell cycle progression and commit to complete the cell cycle. Text indicates molecular and functional characteristics associated with different depths of quiescence.
      Second, distinct stressors can induce metabolically different states of quiescence in yeast and bacteria [
      • Coller HA
      • Sang L
      • Roberts JM
      A new description of cellular quiescence.
      ,
      • Klosinska MM
      • Crutchfield CA
      • Bradley PH
      • Rabinowitz JD
      • Broach JR
      Yeast cells can access distinct quiescent states.
      ]. It is therefore unsurprising that alternative states of quiescence are also rendered accessible by injury in mammals. Neural stem cells exist in graded states of activation with characteristic metabolic profiles and acquire a primed state of quiescence after ischemia [
      • Llorens-Bobadilla E
      • Zhao S
      • Baser A
      • Saiz-Castro G
      • Zwadlo K
      • Martin-Villalba A
      Single-cell transcriptomics reveals a population of dormant neural stem cells that become activated upon brain injury.
      ]. Similarly, quiescent muscle stem cells sensing an injury in a distant muscle can reversibly switch into a cell cycle-primed “Galert” phase, marked by mTORC1 activation, in which cells are quiescent but possess enhanced tissue regeneration properties [
      • Rodgers JT
      • King KY
      • Brett JO
      • et al.
      mTORC1 controls the adaptive transition of quiescent stem cells from G0 to GAlert.
      ]. Mouse HSCs can access an mTORC1-high “Galert” state following injury, priming them to divide faster upon successive challenges for example with interferon-γ [
      • Rodgers JT
      • King KY
      • Brett JO
      • et al.
      mTORC1 controls the adaptive transition of quiescent stem cells from G0 to GAlert.
      ]. The “Galert” phase is distinct from the primed quiescent state of ST-HSCs, which actually exhibits minimal levels of mTORC1 activity [
      • Laurenti E
      • Frelin C
      • Xie S
      • et al.
      CDK6 levels regulate quiescence exit in human hematopoietic stem cells.
      ]. The molecular mechanisms as well as the range of stimuli that push HSCs into “Galert” remain to be studied. Nevertheless, these data demonstrate that specific quiescent states may provide at least transient cellular memory for stem cells to respond more efficiently to subsequent injury signals. Given recent evidence of long-term epigenetic memory of mouse HSCs to inflammatory signals [
      • de Laval B
      • Maurizio J
      • Kandalla PK
      • et al.
      C/EBPβ-Dependent epigenetic memory induces trained immunity in hematopoietic stem cells.
      ,
      • Mann M
      • Mehta A
      • de Boer CG
      • et al.
      Heterogeneous responses of hematopoietic stem cells to inflammatory stimuli are altered with age.
      ], it will be interesting to study if and how alternative quiescent states play a role in such responses.
      Finally, a molecular checkpoint that separates the end of G0 from the beginning of G1 has not been identified to date in any organism. Such a checkpoint may very well not exist, and the transition from quiescence to early G1 may instead occur over a continuum of molecular states, with increasing activation of G1 genes and gradual establishment of a metabolic activity distinct from that of quiescence. Consistently, in the mouse, there is a continuum of transcriptional activation from dormant HSCs to quiescent cell cycle-primed HSCs to cell cycle-active HSCs [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ]. Whether this activation continuum corresponds to that of the lineage bias/restriction observed in the HSC/MPP compartment at the single-cell level [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ,
      • Velten L
      • Haas SF
      • Raffel S
      • et al.
      Human haematopoietic stem cell lineage commitment is a continuous process.
      ] remains to be explored.

      All HSC/MPPs are quiescent but very few are truly dormant

      All HSC/MPPs reside predominantly in G0 and divide much more infrequently than progenitors (approximately once every 30 days compared with once every 1–2 days for the latter). Dormant HSCs are defined as the subset of HSCs that reside in the deepest state of quiescence, divide the most infrequently (Figure 1), and have long been exclusively defined functionally, using inducible label retention assays in mice. These studies have allowed careful analysis of the heterogeneity in division frequencies observed in HSCs and MPPs in vivo under homeostatic and stress conditions. Dormancy correlates with the longest duration of quiescent exit and the highest degree of long-term repopulation capacity [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Laurenti E
      • Frelin C
      • Xie S
      • et al.
      CDK6 levels regulate quiescence exit in human hematopoietic stem cells.
      ,
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ,
      • Foudi A
      • Hochedlinger K
      • Van Buren D
      • et al.
      Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ,
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ,
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ]. In fact, the correlation is so strong that both long quiescence exit duration and extremely infrequent divisions can be used as surrogate markers for the long-term repopulation capacity of an HSC. The dormant HSC subset in mouse cycles less than once every 120 days [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ], has superior serial repopulation capacity to all other HSC/MPP subsets, and is recruited into cycling only upon transplantation or severe stress [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ]. Only recently, reporter mice allowing prospective identification of dormant HSCs have been developed. These respectively trace dormant HSCs based on either Gprc5c expression [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ], p27 activity [
      • Fukushima T
      • Tanaka Y
      • Hamey FK
      • et al.
      Discrimination of dormant and active hematopoietic stem cells by g0 marker reveals dormancy regulation by cytoplasmic calcium.
      ], or retinoic acid signalling [
      • Lauridsen FKB
      • Jensen TL
      • Rapin N
      • et al.
      Differences in cell cycle status underlie transcriptional heterogeneity in the HSC compartment.
      ].
      Self-renewal capacity has been suggested to decline upon each successive division, with dormant HSCs becoming less likely to return to quiescence [
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ]. The number of symmetric divisions sufficient to exhaust self-renewal capacity is still under debate [
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ,
      • Morcos MNF
      • Zerjatke T
      • Glauche I
      • et al.
      Continuous mitotic activity of primitive hematopoietic stem cells in adult mice.
      ]. Extrinsic stresses such as those induced by 5-FU [
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ], lipopolysaccharide (LPS) [
      • Takizawa H
      • Regoes RR
      • Boddupalli CS
      • Bonhoeffer S
      • Manz MG
      Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation.
      ] or polyinosinic:polycytidylic acid (pI–pC) [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ] to mimic bacterial and viral infections, force dormant HSCs to re-enter the cell cycle and become activated. Importantly, injury-activated HSCs are later able to return to dormancy, and restored proportions of dormant HSCs can be detected within days of the initial stress signal [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ,
      • Wilson A
      • Laurenti E
      • Oser G
      • et al.
      Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.
      ]. However, a recent study revealed that HSCs permanently accumulate dysfunctional mitochondria upon return to quiescence [
      • Hinge A
      • He J
      • Bartram J
      • et al.
      Asymmetrically segregated mitochondria provide cellular memory of hematopoietic stem cell replicative history and drive HSC attrition.
      ], providing mechanistic insights into how HSC divisional histories contribute to functional decline.
      Altogether, label retention studies indicate that the contribution of dormant HSCs to daily or steady-state hematopoiesis is minimal, complementing insights obtained from clonal tracking experiments. This reinforces the concept that diversity of quiescent states within the HSC/MPP compartment is key to the resilience of blood production, with distinct dynamics underlying both a rapid stress response and protection from untoward HSC/MPP exhaustion. However, if and how the balance of quiescent states is altered with ageing or disease remains poorly characterised. Interestingly, in the ageing mouse brain, changes in the niche enforce increased neuronal stem cell quiescence [
      • Kalamakis G
      • Brüne D
      • Ravichandran S
      • et al.
      Quiescence modulates stem cell maintenance and regenerative capacity in the aging brain.
      ]. Future studies will have to consider the further complexity that arises in human HSCs with age, due to niche-driven effects, life histories of infection/inflammation [
      • Bogeska R
      • Kaschutnig P
      • Fawaz M
      • et al.
      Hematopoietic stem cells fail to regenerate following inflammatory challenge.
      ,
      • Chavez JS
      • Rabe JL
      • Loeffler D
      • et al.
      PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.
      ] as well as age-related clonal hematopoiesis [
      • Cabezas-Wallscheid N
      • Buettner F
      • Sommerkamp P
      • et al.
      Vitamin A–retinoic acid signaling regulates hematopoietic stem cell dormancy.
      ].

      The importance of the early G1 regulatory network for HSC function

      Many studies have investigated whether manipulating the levels of cell cycle regulators can influence HSC self-renewal and/or differentiation (reviewed in [
      • Matsumoto A
      • Nakayama KI.
      Role of key regulators of the cell cycle in maintenance of hematopoietic stem cells.
      ,
      • Nakamura-Ishizu A
      • Takizawa H
      • Suda T
      The analysis, roles and regulation of quiescence in hematopoietic stem cells.
      ,
      • Pietras EM
      • Warr MR
      • Passegué E
      Cell cycle regulation in hematopoietic stem cells.
      ]). It is no surprise that complex control of the molecular network promoting Rb hyperphosphorylation, and hence commitment to division, is absolutely fundamental to regulate how often and how fast HSCs exit from quiescence (as discussed above), in addition to the kinetics of successive divisions. This network includes Rb, CDK4/CDK6, their cyclin Ds partners, as well as their specific CDK inhibitors (p16, p18, p27, p57; Figure 2).
      Figure 2
      Figure 2Quiescence exit and cell cycle re-entry in HSCs is regulated by CDK6/CCND-mediated Rb phosphorylation. After dephosphorylation in M, G0, and early G1, unphosphorylated Rb is physically associated with E2F factors blocking the transactivation domain and inhibiting cell cycle progression. Upon cell cycle re-entry from quiescence, Rb is sequentially phosphorylated by kinase complexes of cyclin D (CCND) with CDK6 and later cyclin E (CCNE) with CDK2. Hyperphosphorylation of Rb causes a conformational change permitting E2F release and transcription of CDK2, cyclin E (CCNE), cyclin A (CCNA), MYC, and other genes involved in cell cycle progression and nucleotide biosynthesis. At this “restriction point” (denoted as R), cells lose dependency on extracellular mitogenic signals and commit to entering G1/S transition and to completing the cell cycle. Edge and fill colors for each protein respectively indicate the cell cycle progression/frequency of division and long-term repopulation phenotypes observed when the corresponding genes were knocked out, as reported for p16
      [
      • Janzen V
      • Forkert R
      • Fleming HE
      • et al.
      Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.
      ]
      , p18
      [
      • Yuan Y
      • Shen H
      • Franklin DS
      • Scadden DT
      • Cheng T
      In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C.
      ]
      , p27
      [
      • Cheng T
      • Rodrigues N
      • Dombkowski D
      • Stier S
      • Scadden DT
      Stem cell repopulation efficiency but not pool size is governed by p27(kip1).
      ]
      , p57
      [
      • Matsumoto A
      • Takeishi S
      • Kanie T
      • et al.
      p57 is required for quiescence and maintenance of adult hematopoietic stem cells.
      ]
      , p27/p57
      [
      • Zou P
      • Yoshihara H
      • Hosokawa K
      • et al.
      p57Kip2 and p27Kip1 cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70.
      ]
      , CDK6
      [
      • Scheicher R
      • Hoelbl-Kovacic A
      • Bellutti F
      • et al.
      CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.
      ]
      , CycD triple KO
      [
      • Kozar K
      • Ciemerych MA
      • Rebel VI
      • et al.
      Mouse development and cell proliferation in the absence of D-cyclins.
      ]
      , and Rb/p107/p130
      [
      • Viatour P
      • Somervaille TC
      • Venkatasubrahmanyam S
      • et al.
      Hematopoietic stem cell quiescence is maintained by compound contributions of the retinoblastoma gene family.
      ]
      . KO=knockout; Cyc=cyclin.
      Given the importance of maintaining the lifelong capacity for HSC division, it is expected that there is a high degree of redundancy between members of the CDK4/CDK6-Rb network, with phenotypes observed only when several homologues are genetically deleted in mice [
      • Zou P
      • Yoshihara H
      • Hosokawa K
      • et al.
      p57Kip2 and p27Kip1 cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70.
      ,
      • Kozar K
      • Ciemerych MA
      • Rebel VI
      • et al.
      Mouse development and cell proliferation in the absence of D-cyclins.
      ,
      • Viatour P
      • Somervaille TC
      • Venkatasubrahmanyam S
      • et al.
      Hematopoietic stem cell quiescence is maintained by compound contributions of the retinoblastoma gene family.
      ]. Constitutive deletion of Cdk6 has minor effects on hematopoiesis, likely because of compensatory effects from Cdk4. However, Cdk6 is strictly necessary for HSC exit from quiescence and activation under stress conditions. Cdk6 action during this transition cannot be compensated by Cdk4, as demonstrated with constitutive [
      • Scheicher R
      • Hoelbl-Kovacic A
      • Bellutti F
      • et al.
      CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.
      ] and conditional Cdk6 knockout models [
      • Maurer B
      • Brandstoetter T
      • Kollmann S
      • Sexl V
      • Prchal-Murphy M
      Inducible deletion of CDK4 and CDK6—Deciphering CDK4/6 inhibitor effects in the hematopoietic system.
      ].
      In addition, when HSC function fails either in the context of an experimental model or in patients, teasing apart whether cell cycle changes are drivers of such loss or merely consequences of other signalling events is an ongoing challenge in the field. Indeed, many cell cycle regulators possess functions beyond pushing cells towards division [
      • Hydbring P
      • Malumbres M
      • Sicinski P
      Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases.
      ,
      • Uras IZ
      • Sexl V
      • Kollmann K
      CDK6 inhibition: a novel approach in AML management.
      ]. CDK6 can directly phosphorylate EGR1 [
      • Scheicher R
      • Hoelbl-Kovacic A
      • Bellutti F
      • et al.
      CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.
      ], a transcription factor involved in HSC function, but also TSC2, thereby promoting mTORC1 activity and linking cell cycle with cell growth [
      • Romero-Pozuelo J
      • Figlia G
      • Kaya O
      • Martin-Villalba A
      • Teleman AA
      Cdk4 and Cdk6 couple the cell-cycle machinery to cell growth via mTORC1.
      ]. CDK6 can directly block RUNX1 transcriptional activity [
      • Fujimoto T
      • Anderson K
      • Jacobsen SEW
      • Nishikawa S
      • Nerlov C
      Cdk6 blocks myeloid differentiation by interfering with Runx1 DNA binding and Runx1–C/EBPα interaction.
      ] or contact the chromatin modulating NFkB-p65 [
      • Handschick K
      • Beuerlein K
      • Jurida L
      • et al.
      Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-κB-dependent gene expression.
      ], p16, VEGF-A [
      • Kollmann K
      • Heller G
      • Schneckenleithner C
      • et al.
      A kinase-independent function of CDK6 links the cell cycle to tumor angiogenesis.
      ], and p53 antagonist activity [
      • Bellutti F
      • Tigan AS
      • Nebenfuehr S
      • et al.
      CDK6 antagonizes p53-induced responses during tumorigenesis.
      ], broadly affecting HSC function in a cell cycle-independent manner.
      The whole picture is further complicated by the fact that the cell cycle machinery is activated or repressed by a multitude of external stimuli. A very substantial body of work has collectively identified more than 100 genes and dozens of environmental components [
      • Pinho S
      • Frenette PS
      Haematopoietic stem cell activity and interactions with the niche.
      ] that contribute to regulating the delicate balance between HSC quiescence and division. A common interpretation of all these studies is that excessive division in vivo almost inevitably leads to loss of HSC self-renewal. We would argue that this is an oversimplification. This can certainly be driven by exhaustion through excessive division coupled to differentiation (loss of self-renewal, “locked out” of quiescence). Nonetheless, failure to divide and produce differentiated progeny (“locked in” quiescence) eventually leads to the same failure in blood production. Most experimental assays require division to read out HSC function, so caution must be taken when interpreting HSC phenotypes by determining if a particular perturbation affects quiescence, quiescence exit or division.

      Is there a causal relationship between G1 length and HSC fate decisions?

      A possible causal relationship between cell cycle and fate decisions has long been pursued in stem cell biology. The position in the cell cycle may control how cells respond to external stimuli [
      • Orford KW
      • Scadden DT
      Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation.
      ], and the duration of a particular cell cycle phase [
      • Mummery CL
      • van den Brink CE
      • de Laat SW
      Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent.
      ] may influence cell fate. The duration of G1 causally influences cell fate decisions in embryonic stem cells, specifically which differentiation route cells take [
      • Canu G
      • Athanasiadis E
      • Grandy RA
      • et al.
      Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation.
      ,
      • Pauklin S
      • Vallier L
      The cell-cycle state of stem cells determines cell fate propensity.
      ,
      • Singh AM
      • Chappell J
      • Trost R
      • et al.
      Cell-cycle control of developmentally regulated transcription factors accounts for heterogeneity in human pluripotent cells.
      ]. Lengthening of G1 is also associated with commitment in neural stem cells [
      • Lange C
      • Huttner WB
      • Calegari F
      Cdk4/cyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors.
      ,
      • Takahashi T
      • Nowakowski RS
      • Caviness VS
      The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.
      ]. Whether similar mechanisms are at play in HSCs is still under debate. In human LT-HSCs, overexpression of CDK6 protein alone leads to a shorter early G1 and confers a competitive advantage over serial transplantation without causing any overt changes in differentiation ability [
      • Laurenti E
      • Frelin C
      • Xie S
      • et al.
      CDK6 levels regulate quiescence exit in human hematopoietic stem cells.
      ]. Overexpression of both CDK4 and cyclin D1 shortened early G1 further and increased myeloid differentiation upon xenotransplantation in vivo, but this was not supported in vitro [
      • Mende N
      • Kuchen EE
      • Lesche M
      • et al.
      CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.
      ]. Given the pleiotropic effects of CDKs and cyclins, it is difficult to conclude from these genetic experiments if the slight bias in lineage differentiation is caused by different cell cycle lengths or by other factors.
      We reasoned that we could gain insights into the causal relationship between G1 length and human HSC differentiation capacity by lengthening their time of first division. We chose to use palbociclib, a dual and highly specific CDK4/CDK6 inhibitor (hereafter referred to as CDK6i), which inhibits the kinase-dependent effects of these proteins without affecting their kinase-independent functions. Our group recently identified a subpopulation of long-term repopulating cells within the phenotypic human LT-HSC (CD49f+ CD90+) compartment [
      • Notta F
      • Doulatov S
      • Laurenti E
      • Poeppl A
      • Jurisica I
      • Dick JE
      Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.
      ] that produce lymphoid and myeloid (My) cells but no erythroid (Ery) or megakaryocytic (Meg) cells in vitro and upon xenotransplantation [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ]. This population, which we termed CLEC9Alo LT-HSCs (purified as CD34hiCD38CD45RACD90+CD49f+CLEC9Alo), was also characterized by shorter quiescence exit and higher CDK6 levels than LT-HSCs or CLEC9Ahi LT-HSCs (CD34loCD38CD45RACD90+CD49f+CLEC9Ahi) [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ]. We thus sorted single CLEC9Alo LT-HSCs and cultured them under conditions that sustain simultaneous differentiation of My, Ery, megakaryocytic (Meg), and lymphoid (NK) cells [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ], in the presence or absence of 200 nmol/L CDK6i for the first 3 days (see Supplementary Methods, online only, available at www.exphem.org). Single CLEC9Ahi LT-HSCs were also cultured in parallel as a control. Time to first division was recorded, and the differentiation output of each cell was determined 3 weeks later by flow cytometry. First, CDK6i lengthened the duration of CLEC9Alo LT-HSCs first division to that of CLEC9Ahi LT-HSCs (Figure 3A), without altering clonogenic efficiency (Figure 3B). Interestingly, upon CDK6i, the proportion of CLEC9Alo LT-HSC derived colonies containing NK cells decreased significantly with CDK6i (p = 0.027; Figure 3C), principally because of a decrease in colonies containing both My and NK cells (Figure 3D). However, colony output of CDK6i CLEC9Alo LT-HSCs was still vastly different from that of CLEC9Ahi LT-HSCs (Figure 3D). These results suggest that the inability of CLEC9Alo LT-HSCs to produce Ery and Meg cells is not caused by their shorter time of quiescence exit. Rather, we propose that restriction to the myelolymphoid lineages is irreversibly determined epigenetically and transcriptionally, in line with CLEC9Alo LT-HSCs possessing transcriptional features intermediate between CLEC9Ahi LT-HSCs and lymphoid-primed multipotent progenitors [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ]. However, when G1 was lengthened, CLEC9Alo LT-HSCs commitment to the myeloid and lymphoid lineages was significantly imbalanced in vitro. More broadly this suggests that changes in cell cycle duration may also influence lineage differentiation in hematopoietic stem and progenitor cells and warrant further investigation.
      Figure 3
      Figure 3Lengthening the time to first division decreases lymphoid differentiation of Ly-ST-HSCs. (A,B) Single cells from the indicated populations were cultured for 3 days in the presence or absence of CDK6i (palbociclib, 200 nmol/L), then without CDK6i for 3 weeks. Mean time to first division (EC50 of nonlinear fit of cumulative first-division kinetics) (A) and clonogenic efficiency of single cells from the indicated populations (B). n = 2 experiments with independent CB samples, 144 total cells plated for CLEC9Ahi LT-HSC, 336 for CLEC9Alo LT-HSC, and 192 for CLEC9Alo LT-HSC CDK6i. (C,D) Percentage of colonies containing differentiated cells of the indicated lineages (C) and of the indicated colony type generated by single cells of the indicated populations (D). n = 2 experiments with independent CB samples, n = 128 colonies from CLEC9Ahi LT-HSC, n = 105 colonies from CLEC9Alo LT-HSC, n = 161 colonies from CLEC9Alo LT-HSC CDK6i). Statistical significance shown was calculated with Fisher's test using the number of colonies obtained from both experiments. (NK: expmt 1 p = 0.048, expmt 2 p = 0.024; My: expmt 1 p = 0.039, expmt 2 p = 0.056; MyNK: expmt 1 p < 0.001, expmt 2 p = 0.003; NK only: expmt 1 = 0.016, expmt 2 p = 0.418). Values are expressed as the mean ± SEM.

      Understanding quiescence exit and division ex vivo is essential to improve HSC clinical approaches

      Recent years have seen important milestones towards clinical applications of HSCs that require ex vivo culture of HSCs, such as within HSC gene therapy and protocols for HSC expansion [
      • Cavazzana M
      • Bushman FD
      • Miccio A
      • André-Schmutz I
      • Six E
      Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges.
      ,
      • Wilkinson AC
      • Nakauchi H
      Stabilizing hematopoietic stem cells in vitro.
      ]. One critical challenge remains: prolonged culture ex vivo leads to a net decline in HSC long-term repopulation capacity [
      • Kallinikou K
      • Anjos‐Afonso F
      • Blundell MP
      • et al.
      Engraftment defect of cytokine-cultured adult human mobilized CD34+ cells is related to reduced adhesion to bone marrow niche elements.
      ,
      • Larochelle A
      • Gillette JM
      • Desmond R
      • et al.
      Bone marrow homing and engraftment of human hematopoietic stem and progenitor cells is mediated by a polarized membrane domain.
      ,
      • Glimm H
      • Oh I-H
      • Eaves CJ
      Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not reenter G0.
      ], as a result of divisions shifted to producing daughter cells destined for differentiation but also reduced homing. The causes of such decline are multiple and highly dependent on the culture medium composition and culture duration [
      • Csaszar E
      • Kirouac DC
      • Yu M
      • et al.
      Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.
      ,
      • Sauvageau G
      • Iscove NN
      • Humphries RK
      In vitro and in vivo expansion of hematopoietic stem cells.
      ]. Interestingly though, whereas in vivo self-renewing HSCs return to quiescence after division, ex vivo HSCs fail to do so. We would argue that understanding how quiescence exit and division differ ex vivo from in vivo will be essential to successfully recreate conditions maintaining divisions where one (self-renewal) or both (expansion) daughter cells retain HSC identity. This will be equally beneficial in the context of gene therapy, where significant improvement to gene transfer [
      • Petrillo C
      • Cesana D
      • Piras F
      • et al.
      Cyclosporin A and rapamycin relieve distinct lentiviral restriction blocks in hematopoietic stem and progenitor cells.
      ] and editing rates [
      • Shin JJ
      • Schröder MS
      • Caiado F
      • et al.
      Controlled cycling and quiescence enables efficient HDR in engraftment-enriched adult hematopoietic stem and progenitor cells.
      ] has been achieved by manipulating cell cycle-related parameters.
      Evidence has accumulated both in vivo [
      • Pei W
      • Shang F
      • Wang X
      • et al.
      Resolving fates and single-cell transcriptomes of hematopoietic stem cell clones by PolyloxExpress Barcoding.
      ,
      • Rodriguez-Fraticelli AE
      • Weinreb C
      • Wang SW
      • et al.
      Single-cell lineage tracing unveils a role for TCF15 in haematopoiesis.
      ,
      • Grinenko T
      • Eugster A
      • Thielecke L
      • et al.
      Hematopoietic stem cells can differentiate into restricted myeloid progenitors before cell division in mice.
      ,
      • Dong F
      • Hao S
      • Zhang S
      • et al.
      Differentiation of transplanted haematopoietic stem cells tracked by single-cell transcriptomic analysis.
      ] and ex vivo [
      • Wohrer S
      • Knapp DJHF
      • Copley MR
      • et al.
      Distinct stromal cell factor combinations can separately control hematopoietic stem cell survival, proliferation, and self-renewal.
      ,
      • Kent DG
      • Dykstra BJ
      • Cheyne J
      • Ma E
      • Eaves CJ
      Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells.
      ,
      • Knapp DJHF
      • Hammond CA
      • Miller PH
      • et al.
      Dissociation of survival, proliferation, and state control in human hematopoietic stem cells.
      ] that self-renewal and differentiation can in certain circumstances be uncoupled from division. This implies that there is no theoretical impossibility for HSC maintenance or expansion ex vivo, as long as survival can be guaranteed. Strategies mimicking quiescence-inducing factors normally found in the in vivo niche have shown success in minimising the decline in long-term repopulation ability of cultured HSCs [
      • Kobayashi H
      • Morikawa T
      • Okinaga A
      • et al.
      Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.
      ,
      • Yamazaki S
      • Iwama A
      • Takayanagi S
      • Eto K
      • Ema H
      • Nakauchi H
      TGF-β as a candidate bone marrow niche signal to induce hematopoietic stem cell hibernation.
      ,
      • Nakahara F
      • Borger DK
      • Wei Q
      • et al.
      Engineering a haematopoietic stem cell niche by revitalizing mesenchymal stromal cells.
      ], most likely by favouring self-renewing asymmetric divisions. Recent strategies with small molecule inhibitors [
      • Fares I
      • Chagraoui J
      • Gareau Y
      • et al.
      Pyrimidoindole derivatives are agonists of human hematopoietic stem cell self-renewal.
      ,
      • Wagner JE
      • Brunstein CG
      • Boitano AE
      • et al.
      Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft.
      ], aimed at removing differentiation factors [
      • Csaszar E
      • Kirouac DC
      • Yu M
      • et al.
      Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.
      ,
      • Kobayashi H
      • Morikawa T
      • Okinaga A
      • et al.
      Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.
      ,
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ], limiting accumulation of damaging agents [
      • Luchsinger LL
      • Strikoudis A
      • Danzl NM
      • et al.
      Harnessing hematopoietic stem cell low intracellular calcium improves their maintenance in vitro.
      ], and/or retaining quality control mechanisms associated with quiescence in vivo [
      • Xie SZ
      • Garcia-Prat L
      • Voisin V
      • et al.
      Sphingolipid modulation activates proteostasis programs to govern human hematopoietic stem cell self-renewal.
      ] have managed to obtain fully functional HSCs in numbers similar to or higher than those at the start of the cultures. The most impressive expansion to date has been reported to be around 550-fold over 2 months of mouse HSC culture with polyvinyl alcohol (PVA) [
      • Wilkinson AC
      • Ishida R
      • Kikuchi M
      Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.
      ]. Of note, no such expansion has been obtained with human cells so far, and all protocols above generate mixed cultures in which functional HSCs remain a minority. Nonetheless these descriptive studies represent major steps forward. All aforementioned studies measure HSC expansion by assessing HSC function through transplantation rather than mere phenotypic characterisation, which is absolutely essential for this field to progress. Altogether, these studies indicate that the entire underlying molecular network, stress response regulation, and macromolecular organelle biology associated with quiescence in vivo should be preserved when aiming to maintain or expand self-renewal ex vivo.
      There is much to be learnt from how each of these protocols mechanistically acts on HSC identity and how they affect quiescence exit and division. For instance, we do not know if successful expansion strategies are effectively allowing at least some HSCs to return to quiescence ex vivo and potentially maintain a hierarchy of cells characterised by accelerated division kinetics and progressively decreasing self-renewal as in vivo. There is no doubt that better understanding quiescence exit and return to quiescence (or G1) at single-cell resolution both in vivo and in clinically relevant in vitro settings will have a major impact not only on our understanding of HSC biology but also on HSC transplantation success rates.

      Conclusions

      Quiescence unites but also divides HSCs. Subtle differences in metabolism, quality control mechanisms, and organelle biology during quiescence lead to seemingly small (several hours) yet important differences in division kinetics and distinct division frequency over very long time frames (several months). Together these two parameters control how likely a cell is to divide and contribute to blood formation both at steady state and under stress. Given the extent to which divisional histories contribute to HSC function [
      • Bernitz JM
      • Kim HS
      • MacArthur B
      • Sieburg H
      • Moore K
      Hematopoietic stem cells count and remember self-renewal divisions.
      ,
      • Qiu J
      • Papatsenko D
      • Niu X
      • Schaniel C
      • Moore K
      Divisional history and hematopoietic stem cell function during homeostasis.
      ,
      • Morcos MNF
      • Zerjatke T
      • Glauche I
      • et al.
      Continuous mitotic activity of primitive hematopoietic stem cells in adult mice.
      ,
      • Hinge A
      • He J
      • Bartram J
      • et al.
      Asymmetrically segregated mitochondria provide cellular memory of hematopoietic stem cell replicative history and drive HSC attrition.
      ] and the fact that cellular memories of injury may affect subsequent divisions, we propose that diversity in quiescent states is perhaps the most relevant aspect of HSC heterogeneity. It is already established that the reversible shift from quiescence to division plays a key role in leukemia, particularly influencing drug resistance and relapse [
      • Vetrie D
      • Helgason GV
      • Copland M
      The leukaemia stem cell: similarities, differences and clinical prospects in CML and AML.
      ]. HSC quiescent states, quiescence exit kinetics, and potentially the lineage biases associated with the latter are likely to shift over time, with indications that such shifts may be detrimental in aging and for progression from clonal hemopoiesis to overt malignancy. This is a field that we bet will not become dormant in the near future.

      Conflict of interest disclosure

      GlaxoSmithKline provided research funding to EL and CJ.

      Acknowledgments

      We thank the Cambridge Blood and Stem Cell Biobank, specifically Joanna Baxter and the team of nurses consenting and collecting cord blood samples and the Cambridge NIHR BRC Cell Phenotyping Hub for their flow cytometry services. EL is supported by a Sir Henry Dale fellowship from Wellcome/Royal Society ( 107630/Z/15/Z ). Research in EL's laboratory is supported by Wellcome, BBSRC, EHA, BIRAX, and the Royal Society and by core support grants from Wellcome and MRC to the Wellcome–MRC Cambridge Stem Cell Institute ( 203151/Z/16/Z ). CJ is supported by an MRC iCASE PhD studentship, and SB, by a CRUK Cambridge Cancer Centre PhD fellowship.

      Supplementary Methods

      Human Cord Blood Samples

      Anonymized umbilical cord blood (CB) samples were obtained with informed consent from healthy donors through the Cambridge Blood and Stem Cell Biobank (CBSB) in accordance with regulated procedures approved by the relevant Research and Ethics Committees (07/MRE05/44 research study). CB units received on the same day were pooled independently of sex and processed as a single sample.

      Human Cord Blood CD34+ cell selection

      Mononuclear cells (MNCs) were isolated from CB by density gradient centrifugation of pre-diluted CB (1:1 ratio with PBS) using Pancoll (PAN-biotech). The collected MNC fractions were then depleted of red blood cells (RBCs) by incubation with RBC lysis buffer (15 minutes / 4˚C) (BioLegend). CB CD34+ cells were positively selected by incubation (30 minutes / 4˚C) with CD34+ microbeads (30ul/ 108 cells) (Miltenyi Biotech) and FcR blocking reagent (30ul/108 cells) in PBS + 3% Fetal Bovine Serum (FBS) (90ul/108 cells) before separation using the AutoMACS cell seperator (Myltenyi Biotech). CB CD34+ enriched cells were stored at -150˚C until use.

      Fluorescence activated cell sorting

      To isolate cell populations from CD34+ CB cells, frozen CB CD34+ samples were thawed by drop-wise addition of pre-warmed Iscove's Modified Dulbecco's Medium (IMDM, Life Technologies) + 0.1 mg/ml DNase (Sigma) + 50% Fetal Bovine Serum (FBS, Life Technologies) and counted before re-suspension in an antibody mix for a panel of cell surface markers (2 × 106 cells/ml in PBS + 3% FBS antibody mix) (Suppl. Table 1). Cells were then incubated (20 minutes / dark / room temperature (RT)) and washed with an appropriate volume of PBS + 3% FBS. Cells were sorted using single cell purity and index sorting to allow retrospective correlation between the cell surface marker expression obtained from each single cell with in vitro functional lineage output. CLEC9Alo LT-HSCs were defined as: CD34hiCD38CD45RACD90+CD49f+CLEC9Alo; CLEC9Ahi LT-HSCs were defined as: CD34loCD38CD45RACD90+CD49f+CLEC9Ahi.
      Supplementary Table 1Antibodies and antibody panels used for isolation of cell populations derived from CB CD34+ cells.
      Antibody (clone)DilutionFluorochrome
      CD19 (HIB19)1:300Alexa 700
      CD34 (581)1:100APC-Cy7
      CD38 (HIT2)1:100PE-Cy7
      CD45RA (HI100)*1:100FITC
      CD49f (GoH3)*1:100PE-Cy5
      CD90 (5E10)*1:100APC
      CLEC9A (8F9)1:75PE
      Zombie1:2000Aqua
      Antibodies used for single cell sorting of CLEC9Alo LT-HSCs and CLEC9Ahi LT-HSCs. All the antibodies listed were purchased from BioLegend except those indicated by * which were purchased from BD Biosciences.

      Time to first division and single cell differentiation in vitro assay

      CLEC9Ahi and CLEC9AloLT-HSCs were single cell FACS sorted as in [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ] into 96-well round-bottom plates containing 100 μl/well of StemPro base media supplemented with the following cytokines: SCF 100 ng/ml, Flt3-L 20 ng/ml, TPO 100 ng/ml, IL-6 50 ng/ml, IL-3 10 ng/ml, IL-11 50 ng/ml, GM-CSF 20 ng/ml (all Miltenyi Biotec), in addition to nutrients supplement (Life Technologies), h-LDL 50 ng/ml (Stem Cell Technologies), 1% L-Glutamine (Life Technologies) and 1% Pen/Strep (Life Technologies). 200nM of CDK6i (Palbociclib, PD0332991) was added to wells where appropriate. After cell sorting, plates were centrifuged (400 g x 5 minutes) and incubated (37˚C / 5% CO2). Single cells visualised and counted manually every 8-12 hours for a total duration of 4 days. The time of first division was recorded for each cell and empty wells were excluded from the experiment. After 4 days, plates were centrifuged (400 g x 5 minutes), medium removed and 100μl of MEM (Myeloid (My)-Erythroid (Ery)-Megakaryocytic (Meg)) medium was added in absence of CDK6i in order to support differentiation toward My-Ery-Meg lineages. The composition of complete MEM medium differs from the medium used for counting time to first division and was as follows: StemPro base medium with nutrients supplement (Life Technologies) and supplemented with the following cytokines: SCF 100 ng/ml, Flt3-L 20 ng/ml, TPO 100 ng/ml, IL-6 50 ng/ml, IL-3 10 ng/ml, IL-11 50 ng/ml, GM-CSF 20 ng/ml, IL-2 10 ng/ml, IL-7 20 ng/ml (all Miltenyi Biotec), EPO 3 units/ml (Eprex, Janssen-Cilag), h-LDL 50 ng/ml (Stem Cell Technologies), 1% L-Glutamine (Life Technologies) and 1% Pen/Strep (Life Technologies). The type (lineage determination) and the size of the colonies formed were assessed after 21 days of culture by high-throughput flow-cytometry using the BD LSR II HTC Analyser (Suppl. Table 2). First, all single cell derived colonies were harvested into 96 u-bottom plates. Each colony was then stained using the antibody panel shown in Table 5. Plates were stained by incubation (20 minutes / dark / RT) with 50 μl/well of antibody mix and then washed with 100 μl/well of PBS + 3% FBS.
      Supplementary Table 2Antibodies used to assess the differentiation output in single cell differentiation assay from CLEC9Alo LT-HSCs and CLEC9Ahi LT-HSCs.
      LineageAntibody (clone)DilutionFluorochrome
      MegakaryocyticCD41(HIP8)1:1000FITC
      ErythroidGlyA (HIR2) *1:1000PE
      hHSC markerCD45 (HI30)1:300PE-Cy5
      MonocyticCD14 (M5E2)1:1000PE-Cy7
      NK cellsCD56 (HCD56)1:200APC
      MyeloidCD11b (ICRF44)1:300APC-Cy7
      GranulocyticCD15 (MC-480)1:200BV421
      All antibodies were purchased from Biolegend unless indicated with * which were purchased by BD Biosciences.

      Single cell differentiation assay analysis

      Colony output was determined using the same gating strategy as in [
      • Belluschi S
      • Calderbank EF
      • Ciaurro V
      • et al.
      Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors.
      ]. A single cell was defined as giving rise to a colony if the sum of cells detected in the CD45+ and GlyA+ gates was ≥30 cells. Ery colonies were identified as CD45 GlyA+≥30 cells, Meg colonies as CD45- CD41+≥30 cells, My colonies as [(CD45+ CD14+) + (CD45+ CD15+)] ≥30 cells, NK colonies as CD45+ CD56+≥30 cells. My colonies were further classified as follows: Granulocytes (Gran) colonies were identified as CD45+ CD15+≥30 cells and CD45+ CD14+≤ 30, Monocyte (Mono) colonies as CD45+ CD15+≤30 cells and CD45+ CD14+≥30 and Monocyte/Granuocyte (MonoGran) as CD45+ CD15+≥30 cells and CD45+ CD14+≥30 cells. All high-throughput screening flow cytometry data was recorded in a blinded way, and correlation between the colony phenotype and originating population was only performed at the final stage of analysis. Gating analysis was performed using FlowJo (v9.9) software. All data obtained from gating analysis were exported into an Excel file and where appropriate, correlated with the first time of division. The subsequent lineage output analysis was performed using the R Studio software.

      Statistical analysis

      Statistical significance of lineage output between CLEC9Alo LT-HSCs +/- CDK6i was determined both within each biological repeat and with repeats combined using the number of colonies produced and the Fishers Exact test on GraphPad Prism (v8.4).

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