Origin and Heterogeneity of Tissue Myeloid Cells: A Focus on GMP-Derived Monocytes and Neutrophils.

Myeloid cells are a significant proportion of leukocytes within tissues, comprising granulocytes, monocytes, dendritic cells, and macrophages. With the identification of various myeloid cells that perform separate but complementary functions during homeostasis and disease, our understanding of tissue myeloid cells has evolved significantly. Exciting findings from transcriptomics profiling and fate-mapping mouse models have facilitated the identification of their developmental origins, maturation, and tissue-specific specializations. This review highlights the current understanding of tissue myeloid cells and the contributing factors of functional heterogeneity to better comprehend the complex and dynamic immune interactions within the healthy or inflamed tissue. Specifically, we discuss the new understanding of the contributions of granulocyte-monocyte progenitor-derived phagocytes to tissue myeloid cell heterogeneity as well as the impact of niche-specific factors on monocyte and neutrophil phenotype and function. Lastly, we explore the developing paradigm of myeloid cell heterogeneity during inflammation and disease.

Hematopoietic stem cell niche generation and maintenance are distinguishable by an epitranscriptomic program.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Early rhombic lip Protogenin+ve stem cells in a human-specific neurovascular niche initiate and maintain group 3 medulloblastoma.

We identify a population of Protogenin-positive (PRTG+ve) MYChigh NESTINlow stem cells in the four-week-old human embryonic hindbrain that subsequently localizes to the ventricular zone of the rhombic lip (RLVZ). Oncogenic transformation of early Prtg+ve rhombic lip stem cells initiates group 3 medulloblastoma (Gr3-MB)-like tumors. PRTG+ve stem cells grow adjacent to a human-specific interposed vascular plexus in the RLVZ, a phenotype that is recapitulated in Gr3-MB but not in other types of medulloblastoma. Co-culture of Gr3-MB with endothelial cells promotes tumor stem cell growth, with the endothelial cells adopting an immature phenotype. Targeting the PRTGhigh compartment of Gr3-MB in vivo using either the diphtheria toxin system or chimeric antigen receptor T cells constitutes effective therapy. Human Gr3-MBs likely arise from early embryonic RLVZ PRTG+ve stem cells inhabiting a specific perivascular niche. Targeting the PRTGhigh compartment and/or the perivascular niche represents an approach to treat children with Gr3-MB.

A synthetic differentiation circuit in Escherichia coli for suppressing mutant takeover.

Differentiation is crucial for multicellularity. However, it is inherently susceptible to mutant cells that fail to differentiate. These mutants outcompete normal cells by excessive self-renewal. It remains unclear what mechanisms can resist such mutant expansion. Here, we demonstrate a solution by engineering a synthetic differentiation circuit in Escherichia coli that selects against these mutants via a biphasic fitness strategy. The circuit provides tunable production of synthetic analogs of stem, progenitor, and differentiated cells. It resists mutations by coupling differentiation to the production of an essential enzyme, thereby disadvantaging non-differentiating mutants. The circuit selected for and maintained a positive differentiation rate in long-term evolution. Surprisingly, this rate remained constant across vast changes in growth conditions. We found that transit-amplifying cells (fast-growing progenitors) underlie this environmental robustness. Our results provide insight into the stability of differentiation and demonstrate a powerful method for engineering evolutionarily stable multicellular consortia.

The local microenvironment drives activation of neutrophils in human brain tumors.

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

Cellular architecture of human brain metastases.

Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.

Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis.

Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Neurobiology, Stem Cell Biology, and Immunology: An Emerging Triad for Understanding Tissue Homeostasis and Repair.

The peripheral nervous system (PNS) endows animals with the remarkable ability to sense and respond to a dynamic world. Emerging evidence shows the PNS also participates in tissue homeostasis and repair by integrating local changes with organismal and environmental changes. Here, we provide an in-depth summary of findings delineating the diverse roles of peripheral nerves in modulating stem cell behaviors and immune responses under steady-state conditions and in response to injury and duress, with a specific focus on the skin and the hematopoietic system. These examples showcase how elucidating neuro-stem cell and neuro-immune cell interactions provides a conceptual framework that connects tissue biology and local immunity with systemic bodily changes to meet varying demands. They also demonstrate how changes in these interactions can manifest in stress, aging, cancer, and inflammation, as well as how these findings can be harnessed to guide the development of new therapeutics.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Cell Types Promoting Goosebumps Form a Niche to Regulate Hair Follicle Stem Cells.

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10.

Brain metastasis (br-met) develops in an immunologically unique br-met niche. Central nervous system-native myeloid cells (CNS-myeloids) and bone-marrow-derived myeloid cells (BMDMs) cooperatively regulate brain immunity. The phenotypic heterogeneity and specific roles of these myeloid subsets in shaping the br-met niche to regulate br-met outgrowth have not been fully revealed. Applying multimodal single-cell analyses, we elucidated a heterogeneous but spatially defined CNS-myeloid response during br-met outgrowth. We found Ccr2+ BMDMs minimally influenced br-met while CNS-myeloid promoted br-met outgrowth. Additionally, br-met-associated CNS-myeloid exhibited downregulation of Cx3cr1. Cx3cr1 knockout in CNS-myeloid increased br-met incidence, leading to an enriched interferon response signature and Cxcl10 upregulation. Significantly, neutralization of Cxcl10 reduced br-met, while rCxcl10 increased br-met and recruited VISTAHi PD-L1+ CNS-myeloid to br-met lesions. Inhibiting VISTA- and PD-L1-signaling relieved immune suppression and reduced br-met burden. Our results demonstrate that loss of Cx3cr1 in CNS-myeloid triggers a Cxcl10-mediated vicious cycle, cultivating a br-met-promoting, immune-suppressive niche.

A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia.

Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis, and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA sequencing (scRNA-seq) to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes spanning new fibroblastic and osteoblastic subpopulations including distinct osteoblast differentiation trajectories. Emerging acute myeloid leukemia impaired mesenchymal osteogenic differentiation and reduced regulatory molecules necessary for normal hematopoiesis. These data suggest that tissue stroma responds to malignant cells by disadvantaging normal parenchymal cells. Our taxonomy of the stromal compartment provides a comprehensive bone marrow cell census and experimental support for cancer cell crosstalk with specific stromal elements to impair normal tissue function and thereby enable emergent cancer.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

Deep Profiling of Mouse Splenic Architecture with CODEX Multiplexed Imaging.

A highly multiplexed cytometric imaging approach, termed co-detection by indexing (CODEX), is used here to create multiplexed datasets of normal and lupus (MRL/lpr) murine spleens. CODEX iteratively visualizes antibody binding events using DNA barcodes, fluorescent dNTP analogs, and an in situ polymerization-based indexing procedure. An algorithmic pipeline for single-cell antigen quantification in tightly packed tissues was developed and used to overlay well-known morphological features with de novo characterization of lymphoid tissue architecture at a single-cell and cellular neighborhood levels. We observed an unexpected, profound impact of the cellular neighborhood on the expression of protein receptors on immune cells. By comparing normal murine spleen to spleens from animals with systemic autoimmune disease (MRL/lpr), extensive and previously uncharacterized splenic cell-interaction dynamics in the healthy versus diseased state was observed. The fidelity of multiplexed spatial cytometry demonstrated here allows for quantitative systemic characterization of tissue architecture in normal and clinically aberrant samples.

Identification of the Human Skeletal Stem Cell.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel Disease.

Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6.

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.

Specialized Intercellular Communications via Cytonemes and Nanotubes.

In recent years, thin membrane protrusions such as cytonemes and tunneling nanotubes have emerged as a novel mechanism of intercellular communication. Protrusion-based cellular interactions allow for specific communication between participating cells and have a distinct spectrum of advantages compared to secretion- and diffusion-based intercellular communication. Identification of protrusion-based signaling in diverse systems suggests that this mechanism is a ubiquitous and prevailing means of communication employed by many cell types. Moreover, accumulating evidence indicates that protrusion-based intercellular communication is often involved in pathogenesis, including cancers and infections. Here we review our current understanding of protrusion-based intercellular communication.