These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research.

This is a repository copy of Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms.

SummaryHuman erythro-megakaryopoiesis does not occur in humanized mouse models, preventing the in vivo analysis of human hematopoietic stem cell (HSC) differentiation into these lineages in a surrogate host. Here we show that stably engrafted KIT-deficient NOD/SCID Il2rg−/−KitW41/W41 (NSGW41) mice support much improved human erythropoiesis and platelet formation compared with irradiated NSG recipients. Considerable numbers of human erythroblasts and mature thrombocytes are present in the bone marrow and blood, respectively. Morphology, composition, and enucleation capacity of de novo generated human erythroblasts in NSGW41 mice are comparable with those in human bone marrow. Overexpression of human erythropoietin showed no further improvement in human erythrocyte output, but depletion of macrophages led to the appearance of human erythrocytes in the blood. Human erythropoiesis up to normoblasts and platelet formation is fully supported in NSGW41 mice, allowing the analysis of human HSC differentiation into these lineages, the exploration of certain pathophysiologies, and the evaluation of gene therapeutic approaches.

Initial pathway alternations required for pathogenesis of human acute myeloid leukemia (AML) are poorly understood. Here we reveal that removal of glycogen synthase kinase-3α (GSK-3α) and GSK-3β dependency leads to aggressive AML. Although GSK-3α deletion alone has no effect, GSK-3β deletion in hematopoietic stem cells (HSCs) resulted in a pre-neoplastic state consistent with human myelodysplastic syndromes (MDSs). Transcriptome and functional studies reveal that each GSK-3β and GSK-3α uniquely contributes to AML by affecting Wnt/Akt/mTOR signaling and metabolism, respectively. The molecular signature of HSCs deleted for GSK-3β provided a prognostic tool for disease progression and survival of MDS patients. Our study reveals that GSK-3α- and GSK-3β-regulated pathways can be responsible for stepwise transition to MDS and subsequent AML, thereby providing potential therapeutic targets of disease evolution.

The bone marrow (BM) microenvironment, also called the BM niche, is essential for the maintenance of fully functional blood cell formation (hematopoiesis) throughout life. Under physiologic conditions the niche protects hematopoietic stem cells (HSCs) from sustained or overstimulation. Acute or chronic stress deregulates hematopoiesis and some of these alterations occur indirectly via the niche. Effects on niche cells include skewing of its cellular composition, specific localization and molecular signals that differentially regulate the function of HSCs and their progeny. Importantly, while acute insults display only transient effects, repeated or chronic insults lead to sustained alterations of the niche, resulting in HSC deregulation. We here describe how changes in BM niche composition (ecosystem) and structure (remodeling) modulate activation of HSCs in situ. Current knowledge has revealed that upon chronic stimulation, BM remodeling is more extensive and otherwise quiescent HSCs may be lost due to diminished cellular maintenance processes, such as autophagy, ER stress response, and DNA repair. Features of aging in the BM ecology may be the consequence of intermittent stress responses, ultimately resulting in the degeneration of the supportive stem cell microenvironment. Both chronic stress and aging impair the functionality of HSCs and increase the overall susceptibility to development of diseases, including malignant transformation. To understand functional degeneration, an important prerequisite is to define distinguishing features of unperturbed niche homeostasis in different settings. A unique setting in this respect is xenotransplantation, in which human cells depend on niche factors produced by other species, some of which we will review. These insights should help to assess deviations from the steady state to actively protect and improve recovery of the niche ecosystem in situ to optimally sustain healthy hematopoiesis in experimental and clinical settings.

Granzyme B is expressed by hematopoietic stem cells (HSCs) and stromal cells in response to bacterial products or chemotherapy agents and limits HSC reconstitution potential.

We comment here on the suitability of available mouse models for type 1 diabetes research including research on therapeutic pancreatic islet transplantation. The major emphasis will be laid on models that require minimal invasive procedures. Most biological processes are too complex for a complete recapitulation in a test tube. The study of innate or even adaptive immune responses involves a number of different cell types and organs making in vitro studies unreliable but also providing extreme challenges for the use of surrogate model organisms. Studying these processes directly in humans is impossible due to ethical and technical constraints. To resolve this problem small animal models such as mice or rats are frequently used to study mechanisms of complex diseases. This has brought much insight into hematopoiesis and immune cell function including type 1 diabetes (T1D); however, 65 million years of evolution introduced striking differences between mice and humans 1. In fact, none of the many suggested therapies arising from studies using mice 2 3 that have promised prevention or even reversion of T1D made it into the clinic yet 4 5 6. The reason for this are major species-specific differences between rodents and humans regarding the immune system and beta cells.