Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166.

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM) are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single-cell mRNA-seq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM confirmed that the loss of these molecules significantly reduced the ability of OM to augment the osteoblast-mediated hematopoietic-enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various in vitro manipulations of stem cells.

Niche Tet maintains germline stem cells independently of dioxygenase activity.

Ten-eleven translocation (TET) proteins are dioxygenases that convert 5-methylcytosine (5mC) into 5-hydroxylmethylcytosine (5hmC) in DNA and RNA. However, their involvement in adult stem cell regulation remains unclear. Here, we identify a novel enzymatic activity-independent function of Tet in the Drosophila germline stem cell (GSC) niche. Tet activates the expression of Dpp, the fly homologue of BMP, in the ovary stem cell niche, thereby controlling GSC self-renewal. Depletion of Tet disrupts Dpp production, leading to premature GSC loss. Strikingly, both wild-type and enzyme-dead mutant Tet proteins rescue defective BMP signaling and GSC loss when expressed in the niche. Mechanistically, Tet interacts directly with Bap55 and Stat92E, facilitating recruitment of the Polybromo Brahma associated protein (PBAP) complex to the dpp enhancer and activating Dpp expression. Furthermore, human TET3 can effectively substitute for Drosophila Tet in the niche to support BMP signaling and GSC self-renewal. Our findings highlight a conserved novel catalytic activity-independent role of Tet as a scaffold protein in supporting niche signaling for adult stem cell self-renewal.

Semaphorins and the bone marrow microenvironment: New candidates that influence the hematopoietic system.

The bone marrow is a haven for hematopoietic and non-hematopoietic cells, creating complex micro-anatomical regions called niches. These distinct niches all participate in an intricate orchestra of cellular interactions that regulates the hematopoietic stem cell and its progenies. In this review, we provide a detailed description of the three most well-known bone marrow niches and their participation in hematopoiesis. We use pre-clinical data, including different in vitro and in vivo studies to discuss how a group of proteins called Semaphorins could potentially modulate both hematopoietic and non-hematopoietic cells, establishing links between the niches, semaphorins, and hematopoietic regulation. Thus, here we provide a deep dive into the inner functioning of the bone marrow and discuss the overarching implications that semaphorins might have on blood formation.

Diffusible fraction of niche BMP ligand safeguards stem-cell differentiation.

Drosophila male germline stem cells (GSCs) reside at the tip of the testis and surround a cluster of niche cells. Decapentaplegic (Dpp) is one of the well-established ligands and has a major role in maintaining stem cells located in close proximity. However, the existence and the role of the diffusible fraction of Dpp outside of the niche have been unclear. Here, using genetically-encoded nanobodies called Morphotraps, we physically block Dpp diffusion without interfering with niche-stem cell signaling and find that a diffusible fraction of Dpp is required to ensure differentiation of GSC daughter cells, opposite of its role in maintenance of GSC in the niche. Our work provides an example in which a soluble niche ligand induces opposed cellular responses in stem cells versus in differentiating descendants to ensure spatial control of the niche. This may be a common mechanism to regulate tissue homeostasis.

Differential adhesion during development establishes individual neural stem cell niches and shapes adult behaviour in Drosophila.

Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. We first found that CG wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. We then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, we link the loss of these 2 adhesion complexes specifically during development to locomotor hyperactivity in the resulting adults. Altogether, our findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour.

[Imaging techniques for the bone marrow environment].

Hematopoietic stem and progenitor cells in mammals primarily reside in the bone marrow after birth. There, the cellular dynamics and subsequent fate of those cells are regulated by the adjacent microenvironment, known as the niche, to sustain lifelong blood cell production. To analyze and study physiological hematopoiesis and various hematopoietic disorders, it is essential to deeply understand how the niche regulates hematopoiesis and how niche dysregulation occurs. However, the dynamics of hematopoietic stem and progenitor cells and their interactions with the niche are dynamic and complex, and our knowledge of the spatial organization of bone marrow cells and niche factors is still limited. In this review, I provide an overview of classical techniques for spatiotemporal understanding of the cellular communities in bone marrow, as well as recent advances in bone marrow imaging techniques and valuable animal models, and discuss future prospects in this field.

A comprehensive analysis of cell-autonomous and non-cell-autonomous regulation of myeloid leukemic cells: The prospect of developing novel niche-targeting therapies.

Leukemic cells (LCs) arise from the hematopoietic stem/and progenitor cells (HSCs/HSPCs) and utilize cues from the bone marrow microenvironment (BMM) for their regulation in the same way as their normal HSC counterparts. Mesenchymal stromal cells (MSCs), a vital component of the BMM promote leukemogenesis by creating a protective and immune-tolerant microenvironment that can support the survival of LCs, helping them escape chemotherapy, thereby resulting in the relapse of leukemia. Conversely, MSCs also induce apoptosis in the LCs and inhibit their proliferation by interfering with their self-renewal potential. This review discusses the work done so far on cell-autonomous (intrinsic) and MSCs-mediated non-cell-autonomous (extrinsic) regulation of myeloid leukemia with a special focus on the need to investigate the extrinsic regulation of myeloid leukemia to understand the contrasting role of MSCs in leukemogenesis. These mechanisms could be exploited to formulate novel therapeutic strategies that specifically target the leukemic microenvironment.

Live Imaging of the Drosophila Testis Stem Cell Niche.

Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here, we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 18 h allows for tracking of cell-type-specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. By using live imaging in intact niches, we can better uncover the cellular and molecular mechanisms that regulate stem cell function in vivo.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Young donor hematopoietic stem cells revitalize aged or damaged bone marrow niche by transdifferentiating into functional niche cells.

The bone marrow niche maintains hematopoietic stem cell (HSC) homeostasis and declines in function in the physiologically aging population and in patients with hematological malignancies. A fundamental question is now whether and how HSCs are able to renew or repair their niche. Here, we show that disabling HSCs based on disrupting autophagy accelerated niche aging in mice, whereas transplantation of young, but not aged or impaired, donor HSCs normalized niche cell populations and restored niche factors in host mice carrying an artificially harassed niche and in physiologically aged host mice, as well as in leukemia patients. Mechanistically, HSCs, identified using a donor lineage fluorescence-tracing system, transdifferentiate in an autophagy-dependent manner into functional niche cells in the host that include mesenchymal stromal cells and endothelial cells, previously regarded as "nonhematopoietic" sources. Our findings thus identify young donor HSCs as a primary parental source of the niche, thereby suggesting a clinical solution to revitalizing aged or damaged bone marrow hematopoietic niche.

Macrophage-stem cell crosstalk: regulation of the stem cell niche.

The cells of the innate immune system are the sentinels of tissue homeostasis, acting as 'first responders' to cellular damage and infection. Although the complex interplay of different immune cells during the initial inflammatory phases of infection and repair has been documented over many decades, recent studies have begun to define a more direct role for specific immune cells in the modulation of tissue repair. One particular cell of the innate immune system, the macrophage, has emerged as a central integrator of the complex molecular processes that drive tissue repair and, in some cases, the development of specific cell types. Although macrophages display directed orchestration of stem cell activities, bidirectional cellular crosstalk mechanisms allow stem cells to regulate macrophage behaviour within their niche, thus increasing the complexity of niche regulation and control. In this Review, we characterize the roles of macrophage subtypes in individual regenerative and developmental processes and illustrate the surprisingly direct role for immune cells in coordinating stem cell formation and activation.

Smooth muscle contributes to the development and function of a layered intestinal stem cell niche.

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here, we identify the mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle populations. In young and adult mice, diverse sub-cryptal cells provide redundant ISC-supportive factors; few of these are restricted to single cell types. Niche functions refine during postnatal crypt morphogenesis, in part to oppose the dense aggregation of differentiation-promoting BMP+ sub-epithelial myofibroblasts at crypt-villus junctions. Muscularis mucosae, a specialized muscle layer, first appears during this period and supplements neighboring RSPO and BMPi sources. Components of this developing niche are conserved in human fetuses. The in vivo ablation of mouse postnatal smooth muscle increases BMP signaling activity, potently limiting a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal cells together create the milieu that is required to propagate crypts during rapid organ growth and to sustain adult ISCs.

Canonical and non-canonical functions of STAT in germline stem cell maintenance.

BACKGROUND: Maintenance of the Drosophila male germline stem cells (GSCs) requires activation of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway by niche signals. The precise role of JAK/STAT signaling in GSC maintenance, however, remains incompletely understood. RESULTS: Here, we show that, GSC maintenance requires both canonical and non-canonical JAK/STAT signaling, in which unphosphorylated STAT (uSTAT) maintains heterochromatin stability by binding to heterochromatin protein 1 (HP1). We found that GSC-specific overexpressing STAT, or even the transcriptionally inactive mutant STAT, increases GSC number and partially rescues the GSC-loss mutant phenotype due to reduced JAK activity. Furthermore, we found that both HP1 and STAT are transcriptional targets of the canonical JAK/STAT pathway in GSCs, and that GSCs exhibit higher heterochromatin content. CONCLUSIONS: These results suggest that persistent JAK/STAT activation by niche signals leads to the accumulation of HP1 and uSTAT in GSCs, which promote heterochromatin formation important for maintaining GSC identity. Thus, the maintenance of Drosophila GSCs requires both canonical and non-canonical STAT functions within GSCs for heterochromatin regulation.

Identification of phenotypically, functionally, and anatomically distinct stromal niche populations in human bone marrow based on single-cell RNA sequencing.

Hematopoiesis is regulated by the bone marrow (BM) stroma. However, cellular identities and functions of the different BM stromal elements in humans remain poorly defined. Based on single-cell RNA sequencing (scRNAseq), we systematically characterized the human non-hematopoietic BM stromal compartment and we investigated stromal cell regulation principles based on the RNA velocity analysis using scVelo and studied the interactions between the human BM stromal cells and hematopoietic cells based on ligand-receptor (LR) expression using CellPhoneDB. scRNAseq led to the identification of six transcriptionally and functionally distinct stromal cell populations. Stromal cell differentiation hierarchy was recapitulated based on RNA velocity analysis and in vitro proliferation capacities and differentiation potentials. Potential key factors that might govern the transition from stem and progenitor cells to fate-committed cells were identified. In situ localization analysis demonstrated that different stromal cells were localized in different niches in the bone marrow. In silico cell-cell communication analysis further predicted that different stromal cell types might regulate hematopoiesis through distinct mechanisms. These findings provide the basis for a comprehensive understanding of the cellular complexity of the human BM microenvironment and the intricate stroma-hematopoiesis crosstalk mechanisms, thus refining our current view on human hematopoietic niche organization.

A mysterious triangle of blood, bones, and nerves.

The relationship between bone tissue and bone marrow, which is responsible for hematopoiesis, is inseparable. Osteoblasts and osteocytes, which produce and consist of bone tissue, regulate the function of hematopoietic stem cells (HSC), the ancestors of all hematopoietic cells in the bone marrow. The peripheral nervous system finely regulates bone remodeling in bone tissue and modulates HSC function within the bone marrow, either directly or indirectly via modification of the HSC niche function. Peripheral nerve signals also play an important role in the development and progression of malignant tumors (including hematopoietic tumors) and normal tissues, and peripheral nerve control is emerging as a potential new therapeutic target. In this review, we summarize recent findings on the linkage among blood system, bone tissue, and peripheral nerves.

Utilizing CyTOF to Examine Hematopoietic Stem and Progenitor Phenotype.

Regulation of hematopoiesis is dependent upon interactions between hematopoietic stem/progenitor cells and niche components, requiring a highly diverse array of different cell-cell interactions and cell signaling events. The overwhelming diversity of the components that can regulate hematopoiesis, especially when factoring in how the cell surface and intracellular protein expression profiles of hematopoietic stem/progenitor cells and niche components differ between homeostatic conditions and stressed conditions such as aging and irradiation, can make utilizing techniques like flow cytometry daunting, particularly while examining small cell populations such as hematopoietic stem cells (HSCs). Due to the complexity of the hematopoietic system, high-dimensional single-cell genomics and proteomics are constantly performed to understand the heterogeneity and expression profiles within this system. This chapter describes one such single-cell assay, which utilizes mass cytometry Time of Flight (CyTOF) technology to determine differences in expression profile within HSC, using changes in HSC populations due to gender and aging.

The emergence of the stem cell niche.

Stem cell niches are composed of dynamic microenvironments that support stem cells over a lifetime. The emerging niche is distinct from the adult because its main role is to support the progenitors that build organ systems in development. Emerging niches mature through distinct stages to form the adult niche and enable proper stem cell support. As a model of emerging niches, this review highlights how differences in the skeletal muscle microenvironment influence emerging versus satellite cell (SC) niche formation in skeletal muscle, which is among the most regenerative tissue systems. We contrast how stem cell niches regulate intrinsic properties between progenitor and stem cells throughout development to adulthood. We describe new applications for generating emerging niches from human pluripotent stem cells (hPSCs) using developmental principles and highlight potential applications for regeneration and therapeutics.

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.