Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Distinct roles of the preparatory and payoff phases of glycolysis in hematopoietic stem cells.

In physiological conditions, most adult hematopoietic stem cells (HSCs) maintain a quiescent state. Glycolysis is a metabolic process that can be divided into preparatory and payoff phases. Although the payoff phase maintains HSC function and properties, the role of the preparatory phase remains unknown. In this study, we aimed to investigate whether the preparatory or payoff phases of glycolysis were required for maintenance of quiescent and proliferative HSCs. We used glucose-6-phosphate isomerase (Gpi1) as a representative gene for the preparatory phase and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a representative gene for the payoff phase of glycolysis. First, we identified that stem cell function and survival were impaired in Gapdh-edited proliferative HSCs. Contrastingly, cell survival was maintained in quiescent Gapdh- and Gpi1-edited HSCs. Gapdh- and Gpi1-defective quiescent HSCs maintained adenosine-triphosphate (ATP) levels by increasing mitochondrial oxidative phosphorylation (OXPHOS), whereas ATP levels were decreased in Gapdh-edited proliferative HSCs. Interestingly, Gpi1-edited proliferative HSCs maintained ATP levels independent of increased OXPHOS. Oxythiamine, a transketolase inhibitor, impaired proliferation of Gpi1-edited HSCs, suggesting that the nonoxidative pentose phosphate pathway (PPP) is an alternative means to maintain glycolytic flux in Gpi1-defective HSCs. Our findings suggest that OXPHOS compensated for glycolytic deficiencies in quiescent HSCs, and that in proliferative HSCs, nonoxidative PPP compensated for defects in the preparatory phase of glycolysis but not for defects in the payoff phase. These findings provide new insights into regulation of HSC metabolism, which could have implications for development of novel therapies for hematologic disorders.

Quantitative Analysis of Sympathetic and Nociceptive Innervation Across Bone Marrow Regions in Mice.

Bone marrow (BM) innervation regulates the mobilization of hematopoietic stem and progenitor cells (HSPCs) from BM and stress hematopoiesis either by acting directly on HSPCs or by altering the niche function of mesenchymal and endothelial cells. However, the spatial distribution of BM innervation across bone regions is yet to be fully elucidated. Thus, we aimed to characterize the distribution of sympathetic and nociceptive nerves in each bone and BM region using three-dimensional quantitative microscopy. We discovered that sympathetic and nociceptive nerves were the major fibers throughout the BM. Compared with other femoral regions, central parts of the femoral BM were more densely innervated by both sympathetic and nociceptive nerves. Each region of the sternum was similarly innervated by sympathetic and nociceptive nerves. Further, the majority of sympathetic and nociceptive nerves in the BM ran parallel with arteries and arterioles, whereas the degree varied according to the bone type or BM region. In conclusion, this study provides spatial, topological, and functional information on BM innervation in a quantitative manner and illustrates that sympathetic and nociceptive nerves are two major components in BM innervation, mostly associated with arteries and arterioles.

A culture platform to study quiescent hematopoietic stem cells following genome editing.

Other than genetically engineered mice, few reliable platforms are available for the study of hematopoietic stem cell (HSC) quiescence. Here we present a platform to analyze HSC cell cycle quiescence by combining culture conditions that maintain quiescence with a CRISPR-Cas9 genome editing system optimized for HSCs. We demonstrate that preculture of HSCs enhances editing efficiency by facilitating nuclear transport of ribonucleoprotein complexes. For post-editing culture, mouse and human HSCs edited based on non-homologous end joining and cultured under low-cytokine, low-oxygen, and high-albumin conditions retain their phenotypes and quiescence better than those cultured under the proliferative conditions. Using this approach, HSCs regain quiescence even after editing by homology-directed repair. Our results show that low-cytokine culture conditions for gene-edited HSCs are a useful approach for investigating HSC quiescence ex vivo.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Identification and local manipulation of bone marrow vasculature during intravital imaging.

Physiological regulation of blood flow in bone marrow is important to maintain oxygen and glucose supplies but also the physiological hypoxic state of the hematopoietic stem cell (HSC) niche. However, regulatory mechanisms underlying microcirculation in the bone marrow (BM) niche remain unclear. Here, we identify vessels functioning in control of blood flow in bone marrow and assess their contractility. To evaluate contractile potential of Alexa Fluor 633 (AF633; an arterial marker)-positive vessels, we performed immunohistochemistry for α-smooth muscle actin (α-SMA) and found it expressed around AF633+ vessels in the femoral and calvarial marrow. To validate AF633+ vessel contractility, we developed a simple system to locally administer vasoactive agents that penetrate BM through transcalvarial vessels. After exposure of the calvarial surface to FITC-dextran (70 kDa), FITC intensity in calvarial bone marrow gradually increased. When we evaluated the effect of transcalvarial administration (TCA) of norepinephrine (NE) on vascular tone of AF633+ arteries and behavior of transplanted blood cells, NE administration decreased artery diameter and transendothelial migration of transplanted cells, suggesting that adrenergic signaling regulates the HSC niche microcirculation and blood cell migration into the BM via effects on BMarteries. We conclude that TCA is a useful tool for bone marrow research.

Potential involvement of semaphorin 3A in maintaining intervertebral disc tissue homeostasis.

Intervertebral discs (IVDs) are avascular; however, ingrowth of blood vessels into their outer regions has been noted during the progression of degeneration. The mechanisms underlying vascularization in IVD degeneration are not completely understood. Semaphorin 3A (Sema3A), originally characterized as a chemorepulsive factor for growing axons in the developing nervous system, inhibits angiogenesis. This study aimed to elucidate the potential involvement of Sema3A in maintaining tissue homeostasis within the avascular IVD. We demonstrated that the mRNA expression of Sema3A was higher in rat annulus fibrosus (AF) than in nucleus pulposus (NP) and that its expression level decreased with age. Both mRNA and protein expression level of Sema3A was also markedly suppressed in AF tissues of a rat IVD degeneration model. Both real-time RT-PCR and Western blot clearly indicated that Sema3A expression significantly reduced by treating inflammatory cytokines in rat AF cells. In a gain- and loss-of-function study, we observed that Sema3A reduced the catabolic shift in rat AF cells. In addition, our results indicated that Sema3A potentially inhibited the IL-6/JAK/STAT pathway. Finally, BrdU assay and tube formation assay revealed that treatment of recombinant Sema3A significantly blocks both proliferation and tube formation of HUVEC. Our results indicate that Sema3A may help maintain IVD tissue homeostasis. Thus, although further studies are needed, Sema3A may be a potential molecular target for suppressing IVD degeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Quiescence of adult oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1.

The adult mammalian central nervous system (CNS) contains a population of slowly dividing oligodendrocyte precursor cells (OPCs), i.e., adult OPCs, which supply new oligodendrocytes throughout the life of animal. While adult OPCs develop from rapidly dividing perinatal OPCs, the mechanisms underlying their quiescence remain unknown. Here, we show that perinatal rodent OPCs cultured with thyroid hormone (TH) under hypoxia become quiescent and acquire adult OPCs-like characteristics. The cyclin-dependent kinase inhibitor p15/INK4b plays crucial roles in the TH-dependent cell cycle deceleration in OPCs under hypoxia. Klf9 is a direct target of TH-dependent signaling. Under hypoxic conditions, hypoxia-inducible factors mediates runt-related transcription factor 1 activity to induce G1 arrest in OPCs through enhancing TH-dependent p15/INK4b expression. As adult OPCs display phenotypes of adult somatic stem cells in the CNS, the current results shed light on environmental requirements for the quiescence of adult somatic stem cells during their development from actively proliferating stem/progenitor cells.

Overexpression of cyclin dependent kinase inhibitor P27/Kip1 increases oligodendrocyte differentiation from induced pluripotent stem cells.

Cell transplantation therapy with oligodendrocyte precursor cells (OPCs) is a promising and effective treatment for diseases involving demyelination in the central nervous system (CNS). In previous studies, we succeeded in producing O4(+) oligodendrocytes (OLs) from mouse- and human-induced pluripotent stem cells (iPSCs) in vitro; however, the efficiency of differentiation into OLs was lower for iPSCs than that for embryonic stem cells (ESCs). To clarify the cause of this difference, we compared the expression of proteins that contribute to OL differentiation in mouse iPSC-derived cells and in mouse ESC-derived cells. The results showed that the expression levels of cyclin dependent kinase inhibitor P27/Kip1, mitogen-activated protein kinase (MAPK) JNK3, and transcription factor Mash1 were lower in iPSC-derived cells. In contrast, the expression levels of MAPK P38α, P38γ, and thyroid hormone receptor ß1 were higher in iPSC-derived cells. We attempted to compensate for the expression changes in P27/Kip1 protein and Mash1 protein in iPSC-derived cells through retrovirus vector-mediated gene expression. Although the overexpression of Mash1 had no effect, the overexpression of P27/Kip1 increased the differentiation efficiency of iPSC-derived cells into O4(+) OLs.