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.

A complex proinflammatory cascade mediates the activation of HSCs upon LPS exposure in vivo.

Infections are a key source of stress to the hematopoietic system. While infections consume short-lived innate immune cells, their recovery depends on quiescent hematopoietic stem cells (HSCs) with long-term self-renewal capacity. Both chronic inflammatory stress and bacterial infections compromise competitive HSC capacity and cause bone marrow (BM) failure. However, our understanding of how HSCs act during acute and contained infections remains incomplete. Here, we used advanced chimeric and genetic mouse models in combination with pharmacological interventions to dissect the complex nature of the acute systemic response of HSCs to lipopolysaccharide (LPS), a well-established model for inducing inflammatory stress. Acute LPS challenge transiently induced proliferation of quiescent HSCs in vivo. This response was not only mediated via direct LPS-TLR4 conjugation on HSCs but also involved indirect TLR4 signaling in CD115+ monocytic cells, inducing a complex proinflammatory cytokine cascade in BM. Downstream of LPS-TLR4 signaling, the combined action of proinflammatory cytokines such as interferon (IFN)α, IFNγ, tumor necrosis factor-α, interleukin (IL)-1α, IL-1ß, and many others is required to mediate full HSC activation in vivo. Together, our study reveals detailed mechanistic insights into the interplay of proinflammatory cytokine-induced molecular pathways and cell types that jointly orchestrate the complex process of emergency hematopoiesis and HSC activation upon LPS exposure in vivo.

Extracellular matrix protein Matrilin-4 regulates stress-induced HSC proliferation via CXCR4.

During homeostasis, hematopoietic stem cells (HSCs) are mostly kept in quiescence with only minor contribution to steady-state hematopoiesis. However, in stress situations such as infection, chemotherapy, or transplantation, HSCs are forced to proliferate and rapidly regenerate compromised hematopoietic cells. Little is known about the processes regulating this stress-induced proliferation and expansion of HSCs and progenitors. In this study, we identified the extracellular matrix (ECM) adaptor protein Matrilin-4 (Matn4) as an important negative regulator of the HSC stress response. Matn4 is highly expressed in long-term HSCs; however, it is not required for HSC maintenance under homeostasis. In contrast, Matn4 is strongly down-regulated in HSCs in response to proliferative stress, and Matn4 deficiency results in increased proliferation and expansion of HSCs and progenitors after myelosuppressive chemotherapy, inflammatory stress, and transplantation. This enhanced proliferation is mediated by a transient down-regulation of CXCR4 in Matn4(-/-) HSCs upon stress, allowing for a more efficient expansion of HSCs. Thus, we have uncovered a novel link between the ECM protein Matn4 and cytokine receptor CXCR4 involved in the regulation of HSC proliferation and expansion under acute stress.

Targeting leukemic stem cells by breaking their dormancy.

Transient or long-term quiescence, the latter referred to as dormancy are fundamental features of at least some adult stem cells. The status of dormancy is likely a critical mechanism for the observed resistance of normal HSCs and leukemic stem cells (LSCs) to anti-proliferative chemotherapy. Recent studies have revealed cytokines such as Interferon-alpha (IFNα) and G-CSF as well as arsenic trioxide (As(2)O(3)) to be efficient agents for promoting cycling of dormant HSCs and LSCs. Most interestingly, such cell cycle activated stem cells become exquisitely sensitive to killing by different chemotherapeutic agents, suggesting that dormant LSCs in patients may be targeted by a sequential two-step protocol involving an initial activation by IFNα, G-CSF or As(2)O(3), followed by targeted chemotherapy.

Skin epidermis lacking the c-Myc gene is resistant to Ras-driven tumorigenesis but can reacquire sensitivity upon additional loss of the p21Cip1 gene.

The target gene(s) required for Myc-mediated tumorigenesis are still elusive. Here we show that while endogenous c-Myc is surprisingly dispensable for skin homeostasis and TPA-induced hyperplasia, c-Myc-deficient epidermis is resistant to Ras-mediated DMBA/TPAinduced tumorigenesis. This is mechanistically linked to p21(Cip1), which is induced in tumors by the activated Ras-ERK pathway but repressed by c-Myc. Acute elimination of c-Myc in established tumors leads to the up-regulation of p21(Cip1), and epidermis lacking both p21(Cip1) and c-Myc reacquires normal sensitivity to DMBA/TPA-induced tumorigenesis. This identifies c-Myc-mediated repression of p21(Cip1) as a key step for Ras-driven epidermal tumorigenesis.