Towards reliable quantification of cell state velocities

A few years ago, it was proposed to use the simultaneous quantification of unspliced and spliced messenger RNA (mRNA) to add a temporal dimension to high-throughput snapshots of single cell RNA sequencing data. This concept can yield additional insight into the transcriptional dynamics of the biological systems under study. However, current methods for inferring cell state velocities from such data (known as RNA velocities) are afflicted by several theoretical and computational problems, hindering realistic and reliable velocity estimation. We discuss these issues and propose new solutions for addressing some of the current challenges in consistency of data processing, velocity inference and visualisation. We translate our computational conclusion in two velocity analysis tools: one detailed method κ-velo and one heuristic method eco-velo, each of which uses a different set of assumptions about the data

IFNα-mediated remodeling of endothelial cells in the bone marrow niche

In the bone marrow, endothelial cells are a major component of the hematopoietic stem cell vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha. In the bone marrow, interferon alpha induces rapid cell cycle entry of hematopoietic stem cells in vivo. However, the effect of interferon alpha on bone marrow endothelial cells has not been described. Here, we demonstrate that acute interferon alpha treatment leads to rapid stimulation of bone marrow endothelial cells in vivo, resulting in increased bone marrow vascularity and vascular leakage. We find that activation of bone marrow endothelial cells involves the expression of key inflammatory and endothelial cell-stimulatory markers. This interferon alpha-mediated activation of bone marrow endothelial cells is dependent in part on vascular endothelial growth factor signaling in bone marrow hematopoietic cell types, including hematopoietic stem cells. Thus, this implies a role for hematopoietic stem cells in remodeling of the bone marrow niche in vivo following inflammatory stress. These data increase our current understanding of the relationship between hematopoietic stem cells and the bone marrow niche under inflammatory stress and also clarify the response of bone marrow niche endothelial cells to acute interferon alpha treatment in vivo

Antigen presentation safeguards the integrity of the hematopoietic stem cell pool

Hematopoietic stem and progenitor cells (HSPCs) are responsible for the production of blood and immune cells. Throughout life, HSPCs acquire oncogenic aberrations that can cause hematological cancers. Although molecular programs maintaining stem cell integrity have been identified, safety mechanisms eliminating malignant HSPCs from the stem cell pool remain poorly characterized. Here, we show that HSPCs constitutively present antigens via major histocompatibility complex class II. The presentation of immunogenic antigens, as occurring during malignant transformation, triggers bidirectional interactions between HSPCs and antigen-specific CD4(+4) T cells, causing stem cell proliferation, differentiation, and specific exhaustion of aberrant HSPCs. This immunosurveillance mechanism effectively eliminates transformed HSPCs from the hematopoietic system, thereby preventing leukemia onset. Together, our data reveal a bidirectional interaction between HSPCs and CD4(+4) T cells, demonstrating that HSPCs are not only passive receivers of immunological signals but also actively engage in adaptive immune responses to safeguard the integrity of the stem cell pool

Forkhead box O and the control of cellular oxidative stress

FOXO Forkhead transcription factors are a subfamily of the large superfamily of Forkhead transcription factors, which consists of over 100 different members, expressed in species ranging from yeast to humans. Members of this family of transcription factors have been demonstrated to play important roles in cell proliferation and differentiation, both during development and in the adult organism. In addition to these roles in normal development, several Forkhead transcription factors have also been suggested to be involved in neoplasia. FOXO transcription factors, for example, have been shown to be part of chromosomal translocations associated with several forms of human cancer. In recent years, a lot of research is performed to investigate the regulation and the role of the FOXO Forkhead transcription factors. Members of this subfamily have been identified in several organisms including in Caenorhabditis elegans, Drosophila Melanogaster, mice, and humans. FOXOs are mainly regulated by the phosphoinositide 3-kinase (PI-3K)/protein kinase B (PKB) pathway. Upon growth factor stimulation FOXOs are directly phosphorylated by PKB, resulting in nuclear export of FOXO and inhibition of the transcriptional activity. Several other kinases, which regulate phosphorylation of FOXO, have been identified as well. Also other processes like acetylation and ubiquitination may play a role in the regulation of FOXO activity. FOXO transcription factors have been implicated in to play a role in the regulation of a multitude of biological processes including cell cycle, apoptosis, DNA repair, differentiation, metabolism, and protection from oxidative stress. These data indicate a role for FOXOs as a meeting point within the cell, integrating several signalling pathways and regulating cell fate. In C. elegans, the FOXO orthologue DAF-16 is involved in the regulation of longevity, dauer formation and stress resistance. Overexpression of DAF-16 in the worm will lead to an increased lifespan, correlated with increased resistance to oxidative stress. Recent data in mammalian systems show that FOXOs are involved in inducing a cell cycle arrest and quiescence. On the other side, FOXOs can protect cells from oxidative stress by regulating the expression of MnSOD and catalase, two anti-oxidant enzymes important in the defense to oxygen radicals. Thus, these data indicate, that, like in C. elegans, also in mammalian systems FOXOs can regulate both quiescence and stress resistance. In this thesis we tried to further understand the role of mammalian FOXOs in the control of cellular oxidative stress. We focus on the possibility that FOXOs not only repress cellular oxidative stress by increasing anti-oxidants expression, but that cellular oxidative stress itself also signals to FOXOs, creating a feedback mechanism. FOXO transcription factors can directly bind to regions within promoters of their target genes, thereby regulating the transcription of these genes. However, in recent years, a variety of cofactors for FOXO-induced effects on transcription have been described, for example p300 and several nuclear receptors. A yeast-two-hybrid screen for new interactors for -catenin revealed FOXOs as potential binding partners. -catenin is a multifunctional protein that regulates gene transcription within the Wnt signalling pathway by direct binding to members of the Lef-1/TCF family of transcription factors. The results described in Chapter 2 show that the binding of FOXO to -catenin also occurs in cells, and that this binding enhances the transcriptional activity of FOXO. The binding between FOXO and -catenin is induced under conditions of increased cellular oxidative stress, further increasing FOXO transcriptional activity under these conditions. In addition to the biochemical data, our genetic analysis in C. elegans demonstrate that this interaction is evolutionary conserved. The -catenin homologue BAR-1 is required for DAF-16 dependent dauer formation, lifespan regulation, oxidative stress resistance and the expression of the DAF-16 target gene SOD-3 following oxidative stress. These data implicate -catenin binding to FOXO as part of the mechanism by which cellular oxidative stress signals to FOXO. The binding of -catenin to FOXOs not only influences the function of FOXOs; this interaction also results in the inhibition of -catenin/TCF transcriptional activity (Chapter 3). The -catenin/TCF complex is activated upon Wnt signalling and it regulates transcription of several downstream target genes. -catenin levels in a cell are tightly controlled by the APC/GSK3/Axin complex. In the absence of a Wnt signal, this complex targets -catenin for degradation by inducing phosphorylation of -catenin. The inhibition of -catenin/TCF transcriptional activity is probably due to competition between TCF and FOXO for binding to -catenin. The FOXO mediated inhibition also correlated with the ability of FOXOs to shuttle -catenin out of the nucleus. Mutants of FOXO that are predominantly localized within the nucleus are impaired in their ability to inhibit -catenin/TCF transcriptional activity and do not increase cytosolic levels of -catenin, whereas mutants of FOXO that do inhibit -catenin/TCF activity are predominantly localized in the cytosol and also increase cytosolic -catenin levels. Finally, the increased binding of FOXO to -catenin upon cellular oxidative stress also results in a further enhancement of the ability of FOXOs to inhibit -catenin/TCF activity. These results further demonstrate a cross talk between FOXO and TCF signalling in which -catenin is the central player. Phosphorylation of FOXOs is regulated by several kinases, including PKB. Activation of the Ras/Ral pathway also results in phosphorylation of FOXO, as was described for FOXO4. Activation of Ral results in phosphorylation of FOXO4 on two residues in the C-terminal part of the protein, Thr447 and Thr451. In Chapter 4 we show that phosphorylation of these sites by the Ral pathway is increased under conditions of cellular oxidative stress. Upon treatment of cells with H2O2, Ral is activated and this results in JNK dependent phosphorylation of Thr447 and Thr451. Oxidative stress induces nuclear localization of FOXO4 and an increase in the transcriptional activity of the protein. In addition, we show that this signalling pathway is also employed by TNFa to activate FOXO4 transcriptional activity. Taken together, these data provide evidence for a feedback mechanism from oxidative stress to FOXO transcription factors, involving the small GTPase Ral and the stress kinase JNK. In the Addendum of Chapter 4 we compare different forms of stress and the ability of FOXOs to reduce JNK activation in cells in response to these stresses. Inhibition of JNK activation by FOXOs in response to oxidative stress is dependent on MnSOD, since FOXOs can no longer inhibit JNK activity after H2O2 treatment in SOD-/- MEFs. However, FOXOs can still reduce JNK activation in response to UV treatment in these cells. Experiments in TKO MEFs, which lack all members of the RB family and thus are incapable of going into cell cycle arrest, indicate an involvement of a cell cycle arrest in stress induced by H2O2, whereas UV induced stress is likely to be independent of the ability to go into cell cycle arrest. In conclusion, this thesis describes the role of FOXOs in oxidative stress signalling in more detail. We show that FOXOs are part of a feedback mechanism, in which an increase in ROS levels in a cell will signal towards FOXO, leading to Ral-mediated, JNK-dependent phosphorylation and activation of FOXO. This results in activation of antioxidant mechanisms in a cell, and thus protection of the cell. Furthermore, we have identified a new binding partner for FOXOs, -catenin. Interaction between these two proteins is increased under conditions of oxidative stress and affects both FOXO and -catenin/TCF signalling in a cell. These findings indicate an important role for FOXO in the defense against oxidative stress. Together with its role in cell cycle regulation this further indicates a link between the protection against oxidative stress and the regulation of the cell cycle and quiescence via the regulation of one subfamily of transcription factors, FOXOs

Inhibition of cell proliferation by lithium is associated with interference in cdc2 activation

Lithium can interfere with embryonal development in a variety of organisms. We investigated the effect of lithium on the proliferation of early embryonal cells. [3H]Thymidine incorporation of non-committed mouse P19 embryonal carcinoma cells was inhibited by lithium treatment. Similar effects were seen in a variety of other cells. This growth inhibition occurred in the G2 phase, since cells accumulated with a 4N DNA content, but the appearance of mitotic cells was blocked. Lithium could also prevent the activation of cdc2, thereby inhibiting cyclin B/cdc2 kinase activity. These data indicate that lithium might disturb embryonal development through interference in embryonal cell cycle regulation

Acetate Does Not Affect Palmitate Oxidation and AMPK Phosphorylation in Human Primary Skeletal Muscle Cells

Our recent in vivo human studies showed that colonic administration of sodium acetate (SA) resulted in increased circulating acetate levels, which was accompanied by increments in whole-body fat oxidation in overweight-obese men. Since skeletal muscle has a major role in whole-body fat oxidation, we aimed to investigate effects of SA on fat oxidation and underlying mechanisms in human primary skeletal muscle cells (HSkMC). We investigated the dose (0-5 mmol/L) and time (1, 4, 20, and 24 h) effect of SA on complete and incomplete endogenous and exogenous oxidation of C-14-labeled palmitate in HSkMC derived from a lean insulin sensitive male donor. Both physiological (0.1 and 0.25 mmol/L) and supraphysiological (0.5, 1 and 5 mmol/L) concentrations of SA neither increased endogenous nor exogenous fat oxidation over time in HSkMC. In addition, no effect of SA was observed on Thr(172)-AMPK alpha phosphorylation. In conclusion, our previously observed in vivo effects of SA on whole-body fat oxidation in men may not be explained via direct effects on HSkMC fat oxidation. Nevertheless, SA-mediated effects on whole-body fat oxidation may be triggered by other mechanisms including gut-derived hormones or may occur in other metabolically active tissues

Negative growth regulation of SK-N-MC cells by bFGF defines a growth factor-sensitive point in G2

Basic fibroblast growth factor (bFGF) has been shown to induce growth inhibition of the neuroepithelioma cell line SK-N-MC. Here we show that this growth inhibition occurs in G2. We show that bFGF is active on these cells during S and early G2 phase. Therefore, this constitutes a rather unusual mechanism of growth inhibition, because it is generally believed that cells become refractory to extracellular signals after passage through the restriction point. We show that bFGF treatment inhibits Tyr-15 dephosphorylation of cdc2 and prevents activation of Cdc25C, similar to what is seen upon activation of the G2 DNA damage checkpoint. Interestingly, both DNA damage- and bFGF-induced effects on cdc2 phosphorylation are reverted by caffeine. To confirm the involvement of similar pathways induced by bFGF and DNA damage, we generated tetracycline-regulatable SK-N-MC clones expressing Cdc25C-S216A. Expression of this Cdc25C mutant can revert the bFGF-induced effects on cdc2 phosphorylation and can rescue cells from the block in G2 imposed by bFGF. Taken together, these data define a growth factor-sensitive point in G2 that most likely involves regulation of Cdc25C phosphorylation

Human haematopoietic stem cell lineage commitment is a continuous process

Blood formation is believed to occur through stepwise progression of haematopoietic stem cells (HSCs) following a tree-like hierarchy of oligo-, bi- and unipotent progenitors. However, this model is based on the analysis of predefined flow-sorted cell populations. Here we integrated flow cytometric, transcriptomic and functional data at single-cell resolution to quantitatively map early differentiation of human HSCs towards lineage commitment. During homeostasis, individual HSCs gradually acquire lineage biases along multiple directions without passing through discrete hierarchically organized progenitor populations. Instead, unilineage-restricted cells emerge directly from a 'continuum of low-primed undifferentiated haematopoietic stem and progenitor cells' (CLOUD-HSPCs). Distinct gene expression modules operate in a combinatorial manner to control stemness, early lineage priming and the subsequent progression into all major branches of haematopoiesis. These data reveal a continuous landscape of human steady-state haematopoiesis downstream of HSCs and provide a basis for the understanding of haematopoietic malignancies

Inflammatory exposure drives long-lived impairment of hematopoietic stem cell self-renewal activity and accelerated aging

Hematopoietic stem cells (HSCs) mediate regeneration of the hematopoietic system following injury, such as following infection or inflammation. These challenges impair HSC function, but whether this functional impairment extends beyond the duration of inflammatory exposure is unknown. Unexpectedly, we observed an irreversible depletion of functional HSCs following challenge with inflammation or bacterial infection, with no evidence of any recovery up to 1 year afterward. HSCs from challenged mice demonstrated multiple cellular and molecular features of accelerated aging and developed clinically relevant blood and bone marrow phenotypes not normally observed in aged laboratory mice but commonly seen in elderly humans. In vivo HSC self-renewal divisions were absent or extremely rare during both challenge and recovery periods. The progressive, irreversible attrition of HSC function demonstrates that temporally discrete inflammatory events elicit a cumulative inhibitory effect on HSCs. This work positions early/mid-life inflammation as a mediator of lifelong defects in tissue maintenance and regeneration