Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Wnt/ß-Catenin-Signaling Modulates Megakaryopoiesis at the Megakaryocyte-Erythrocyte Progenitor Stage in the Hematopoietic System.

The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/ß-catenin for MK and RBC differentiation, we activated ß-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1BM-GOF). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1BM-GOFwt/fl, Ctnnb1BM-GOFfl/fl) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1BM-GOF BM into lethally irradiated wildtype recipients (BMT-Ctnnb1BM-GOF) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1BM-GOF mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1BM-GOF BM cells. In conclusion, ß-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.

Isolation of murine bone marrow hematopoietic stem and progenitor cell populations via flow cytometry.

Flow cytometry is a powerful technology that allows not only multiparameter quantitative data analysis at single cell resolution but also simultaneous cell separation of different populations of interest at high speed. It has been rapidly employed in biological research and clinical diagnostics. This technology has enabled the thorough understanding of murine hematopoiesis, especially the physiology of surface marker-defined hematopoietic stem and progenitor cell populations. The isolation of these populations has been well established over the last three decades with a large consensus among leading laboratories. Here, we describe a detailed methodology protocol of two different state-of-the-art approaches to isolate bone marrow cells and purify hematopoietic stem and progenitor cells via flow cytometry. Different gating schemes are introduced to identify well-defined populations of murine hematopoietic stem and multipotent progenitor cells.

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.

Differential Alternative Polyadenylation Landscapes Mediate Hematopoietic Stem Cell Activation and Regulate Glutamine Metabolism.

Alternative polyadenylation (APA) is emerging as an important regulatory mechanism of RNA and protein isoform expression by controlling 3' untranslated region (3'-UTR) composition. The relevance of APA in stem cell hierarchies remains elusive. Here, we first demonstrate the requirement of the APA regulator Pabpn1 for hematopoietic stem cell (HSC) function. We then determine the genome-wide APA landscape (APAome) of HSCs and progenitors by performing low-input 3' sequencing paired with bioinformatic pipelines. This reveals transcriptome-wide dynamic APA patterns and an overall shortening of 3'-UTRs during differentiation and upon homeostatic or stress-induced transition from quiescence to proliferation. Specifically, we show that APA regulates activation-induced Glutaminase (Gls) isoform switching by Nudt21. This adaptation of the glutamine metabolism by increasing the GAC:KGA isoform ratio fuels versatile metabolic pathways necessary for HSC self-renewal and proper stress response. Our study establishes APA as a critical regulatory layer orchestrating HSC self-renewal, behavior, and commitment.

Single-cell analyses to reveal hematopoietic stem cell fate decisions.

Hematopoietic stem cells (HSCs) are the best studied adult stem cells with enormous clinical value. Most of our knowledge about their biology relies on assays at the single HSC level. However, only the recent advances in developing new single cell technologies allowed the elucidation of the complex regulation of HSC fate decision control. This Review will focus on current attempts to investigate individual HSCs at molecular and functional levels. The advantages of these technologies leading to groundbreaking insights into hematopoiesis will be highlighted, and the challenges facing these technologies will be discussed. The importance of combining molecular and functional assays to enlighten regulatory networks of HSC fate decision control, ideally at high temporal resolution, becomes apparent for future studies.

Comparison of the tumor cell secretome and patient sera for an accurate serum-based diagnosis of pancreatic ductal adenocarcinoma.

Pancreatic cancer is the currently most lethal malignancy. Toward an accurate diagnosis of the disease in body liquids, we studied the protein composition of the secretomes of 16 primary and established cell lines of pancreatic ductal adenocarcinoma (PDAC). Compared to the secretome of non-tumorous cells, 112 proteins exhibited significantly different abundances. Functionally, the proteins were associated with PDAC features, such as decreased apoptosis, better cell survival and immune cell regulation. The result was compared to profiles obtained from 164 serum samples from two independent cohorts - a training and a test set - of patients with PDAC or chronic pancreatitis and healthy donors. Eight of the 112 secretome proteins exhibited similar variations in their abundance in the serum profile specific for PDAC patients, which was composed of altogether 189 proteins. The 8 markers shared by secretome and serum yielded a 95.1% accuracy of distinguishing PDAC from healthy in a Receiver Operating Characteristic curve analysis, while any number of serum-only markers produced substantially less accurate results. Utility of the identified markers was confirmed by classical enzyme linked immunosorbent assays (ELISAs). The study highlights the value of cell secretome analysis as a means of defining reliable serum biomarkers.