Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Hematopoietic stem and progenitor cells integrate microbial signals to promote post-inflammation gut tissue repair.

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated following bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we show that acute gut inflammation expands HSPCs in the BM and directs them to inflamed mesenteric lymph nodes through GM-CSFR activation for further expansion and potential differentiation into Ly6C+ /G+ myeloid cells specialized in gut tissue repair. We identified this process to be mediated by Bacteroides, a commensal gram-negative bacteria that activates innate immune signaling. These findings establish cross-organ communication between the BM and distant inflamed sites, whereby a certain subset of multipotent progenitors is specified to respond to imminent hematopoietic demands and to alleviate inflammatory symptoms.

Akkermansia muciniphila induces slow extramedullary hematopoiesis via cooperative IL-1R/TLR signals.

Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the spleen, a process termed extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria regulate not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbes on hematopoietic pathology remains unclear. Here, we find that systemic single injections of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium, rapidly activate BM myelopoiesis and slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we term delayed EMH. Mechanistically, delayed EMH triggered by A. m. is mediated entirely by the MYD88/TRIF innate immune signaling pathway, which persistently stimulates splenic myeloid cells to secrete interleukin (IL)-1α, and in turn, activates IL-1 receptor (IL-1R)-expressing splenic HSPCs. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) or IL-1α partially diminishes A. m.-induced delayed EMH, while inhibition of both pathways alleviates splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Phospholipid metabolic adaptation promotes survival of IDH2 mutant acute myeloid leukemia cells.

Genetic mutations in the isocitrate dehydrogenase (IDH) gene that result in a pathological enzymatic activity to produce oncometabolite have been detected in acute myeloid leukemia (AML) patients. While specific inhibitors that target mutant IDH enzymes and normalize intracellular oncometabolite level have been developed, refractoriness and resistance has been reported. Since acquisition of pathological enzymatic activity is accompanied by the abrogation of the crucial WT IDH enzymatic activity in IDH mutant cells, aberrant metabolism in IDH mutant cells can potentially persist even after the normalization of intracellular oncometabolite level. Comparisons of isogenic AML cell lines with and without IDH2 gene mutations revealed two mutually exclusive signalings for growth advantage of IDH2 mutant cells, STAT phosphorylation associated with intracellular oncometabolite level and phospholipid metabolic adaptation. The latter came to light after the oncometabolite normalization and increased the resistance of IDH2 mutant cells to arachidonic acid-mediated apoptosis. The release of this metabolic adaptation by FDA-approved anti-inflammatory drugs targeting the metabolism of arachidonic acid could sensitize IDH2 mutant cells to apoptosis, resulting in their eradication in vitro and in vivo. Our findings will contribute to the development of alternative therapeutic options for IDH2 mutant AML patients who do not tolerate currently available therapies.

IL-1 mediates microbiome-induced inflammaging of hematopoietic stem cells in mice.

Aging is associated with impaired hematopoietic and immune function caused in part by decreased fitness in the hematopoietic stem cell (HSC) population and an increased myeloid differentiation bias. The reasons for this aging-associated HSC impairment are incompletely understood. Here we demonstrate that older specific pathogen free (SPF) wild-type (WT) mice in contrast to young SPF mice produce more interleukin-1a and interleukin-1b (IL-1a/b) in steady-state bone marrow (BM), with most of the IL-1a/b being derived from myeloid BM cells. Furthermore, blood from steady-state older SPF WT mice contains higher levels of microbe-associated molecular patterns, specifically TLR4 and TLR8 ligands. In addition, BM myeloid cells from older mice produce more IL-1b in vitro, and older mice show higher and more durable IL-1a/b responses upon stimulation with lipopolysaccharide in vivo. To test whether HSC aging is driven by IL-1a/b, we evaluated HSCs from IL-1 receptor 1 (IL-1R1) knockout (KO) mice. Indeed, older HSCs from IL-1R1KO mice show significantly mitigated aging-associated inflammatory signatures. Moreover, HSCs from older IL-1R1KO and from germ-free mice maintain unbiased lymphomyeloid hematopoietic differentiation upon transplantation, thus resembling this functionality of young HSCs. Importantly, in vivo antibiotic suppression of microbiota or pharmacologic blockade of IL-1 signaling in older WT mice was similarly sufficient to reverse myeloid-biased output of their HSC populations. Collectively, our data define the microbiome/IL-1/IL-1R1 axis as a key, self-sustaining and also therapeutically partially reversible driver of HSC inflammaging.

Unique molecular and functional features of extramedullary hematopoietic stem and progenitor cell reservoirs in humans.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and ß-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells.

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such "hu-NK mice" rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

IL-6 Levels Correlate with Prognosis and Immunosuppressive Stromal Cells in Patients with Colorectal Cancer.

BACKGROUND: The prognosis for patients with colorectal cancer (CRC) is determined by tumor characteristics as well as the host immune response. This study investigated the relationship between an immunosuppressive state and patient prognosis by evaluating the systemic and tumor microenvironment (TME) interleukin (IL)-6 levels. METHODS: Preoperative serum IL-6 levels were measured using an electrochemiluminescence assay. Expression of IL-6 in tumor and stromal cells was evaluated immunohistochemically in 209 patients with resected CRC. Single-cell analysis of tumor-infiltrating immune cells was performed using mass cytometry in 10 additional cases. RESULTS: Elevated serum IL-6 levels were associated with elevated stromal IL-6 levels and a poor prognosis for patients with CRC. High IL-6 expression in stromal cells was associated with low-density subsets of CD3+ and CD4+ T cells as well as FOXP3+ cells. Mass cytometry analysis showed that IL-6+ cells among tumor-infiltrating immune cells were composed primarily of myeloid cells and rarely of lymphoid cells. In the high-IL-6-expression group, the percentages of myeloid-derived suppressor cells (MDSCs) and CD4+FOXP3highCD45RA- effector regulatory T cells (eTreg) were significantly higher than in the low-IL-6-expression group. Furthermore, the proportion of IL-10+ cells in MDSCs and that of IL-10+ or CTLA-4+ cells in eTregs correlated with IL-6 levels. CONCLUSION: Elevated serum IL-6 levels were associated with stromal IL-6 levels in CRC. High IL-6 expression in tumor-infiltrating immune cells also was associated with accumulation of immunosuppressive cells in the TME.

Impact of cerebrovascular comorbidity on prognosis in Japanese patients undergoing PCI: 1-year data from Japanese multicenter registry (KICS).

Cardiovascular and cerebrovascular diseases are considered the principal cause of morbidity and mortality worldwide; the effect of stroke-induced cardiac manifestations is well recognized; however, not enough clinical data have been found about the impact of stroke with underlying cardiac disease. This study's objective is to assess the impact of stroke on the prognosis of patients with underlying IHD, who underwent PCI treatment. This was a multicenter, 1-year observational study in patients undergoing PCI in one of the 17 participating centers across Japan. 18,495 patients were registered on the PCI list; 2481 patients had a prior stroke experience, whereas 15,979 were stroke-free. Our study revealed that stroke patients were significantly older (mean age 73.5 ± 9.6, 69.7(± 11.5), respectively), and suffered from more comorbidities (diabetes, hypertension, and chronic kidney disease, p < 0.0001). During the 1-year period, subjects with stroke showed higher incidence of clinical events compared to those without stroke; to illustrate, all-cause death accounted for 6.2% in patients with stroke, in contrast to only 2.8% in stroke-free patients (p < 0.0001), cardiac death amounted for 2.2 and 1.2%, respectively (p < 0.0001), recurrent stroke for 3.1% and 1.2% (p < 0.0001), non-cardiac death for 3.6 and 1.54% (p < 0.0001), and finally, hemorrhagic complications with 2.6 and 1.3% (p < 0.0001). Kaplan-Meier analysis revealed that stroke patients had a higher probability of all-cause mortality, cardiac death, and recurrent stroke (log-rank p < 0.0001). Cox hazard analysis also showed that the presence of stroke is a significant indicator in determining the outcome of cardiac death (HR = 1.457, 95% CI 1.036-2.051, p = 0.031); hence, proving it to be a crucial predictor on cardiac prognosis. History of prior stroke was common in PCI patients, and independently associated with a higher rate of subsequent cardiovascular and cerebrovascular events recurrence. Thus, highlighting an urgent need for comprehensive prevention of cardiac and cerebrovascular diseases.

Inflammation Regulates Haematopoietic Stem Cells and Their Niche.

Haematopoietic stem cells (HSCs) reside in the bone marrow and are supported by the specialised microenvironment, a niche to maintain HSC quiescence. To deal with haematopoietic equilibrium disrupted during inflammation, HSCs are activated from quiescence directly and indirectly to generate more mature immune cells, especially the myeloid lineage cells. In the process of proliferation and differentiation, HSCs gradually lose their self-renewal potential. The extensive inflammation might cause HSC exhaustion/senescence and malignant transformation. Here, we summarise the current understanding of how HSC functions are maintained, damaged, or exhausted during acute, prolonged, and pathological inflammatory conditions. We also highlight the inflammation-altered HSC niche and its impact on escalating the insults on HSCs.

[Innate immune signal-mediated regulation and alterations in hematopoiesis].

Hematopoietic stem cells (HSCs) are maintained in steady state in the bone marrow (BM); these cells are capable of continuous self-renewal and have the potential for multilineage-differentiation into all blood cell lineages. The BM has long been considered as an immune-privilege organ with little immunological reactions. However, recent findings have revealed that immunological/hematopoietic challenges such as infection or inflammation induce broad spectrum of immune and inflammatory responses in BM. While these responses play a beneficial role to boost immune activation and blood production, chronic challenge might lead to BM pathology and dysregulation, including hematopoietic aplasia or neoplasia. We will introduce recent findings focused on hematopoietic activation induced by existing outside of our body or co-existing with us, and discuss to what degree and how function of HSCs and progenitors is regulated and altered by bacterial insult.

Enhanced thrombopoietin but not G-CSF receptor stimulation induces self-renewing hematopoietic stem cell divisions in vivo.

In steady-state adult hematopoiesis, most hematopoietic stem cells (HSCs) are in the resting phase of the cell cycle. Upon enhanced hematopoietic demand, HSCs can be induced to divide and self-renew or differentiate. However, the cell-extrinsic signals inducing HSC cycling remain to be elucidated. Using in vivo high-resolution single HSC divisional tracking, we directly demonstrate that clinically applied thrombopoietin receptor but not granulocyte colony-stimulating factor (G-CSF) receptor agonists drive HSCs into self-renewing divisions leading to quantitative expansion of functional HSC as defined by their in vivo serial multilineage and long-term repopulating potential. These results suggest that thrombopoietin mimetics might be applicable to expand HSCs in vivo and to sensitize thrombopoietin receptor-expressing HSCs to cell cycle-dependent cytotoxic agents.

MPL expression on AML blasts predicts peripheral blood neutropenia and thrombocytopenia.

Although the molecular pathways that cause acute myeloid leukemia (AML) are increasingly well understood, the pathogenesis of peripheral blood cytopenia, a major cause of AML mortality, remains obscure. A prevailing assumption states that AML spatially displaces nonleukemic hematopoiesis from the bone marrow. However, examining an initial cohort of 223 AML patients, we found no correlation between bone marrow blast content and cytopenia, questioning the displacement theory. Measuring serum concentration of thrombopoietin (TPO), a key regulator of hematopoietic stem cells and megakaryocytes, revealed loss of physiologic negative correlation with platelet count in AML cases with blasts expressing MPL, the thrombopoietin (scavenging) receptor. Mechanistic studies demonstrated that MPLhi blasts could indeed clear TPO, likely therefore leading to insufficient cytokine levels for nonleukemic hematopoiesis. Microarray analysis in an independent multicenter study cohort of 437 AML cases validated MPL expression as a central predictor of thrombocytopenia and neutropenia in AML. Moreover, t(8;21) AML cases demonstrated the highest average MPL expression and lowest average platelet and absolute neutrophil counts among subgroups. Our work thus explains the pathophysiology of peripheral blood cytopenia in a relevant number of AML cases.

[Inflammation and early hematopoiesis].

Lifelong, self-renewing, and, multilineage-differentiating hematopoietic stem cells (HSCs) gradually divide in steady-state bone marrow (BM). Conversely, in cases of hematopoietic stress, including infection and inflammation, hematopoiesis is highly demanded due to massive cell consumption in the stressed tissues and involves HSC recruitment to fulfil the hematopoietic demand. Accumulating evidence indicates that infection-related inflammation acts on blood-forming HSCs and progenitors within the BM to facilitate hematopoiesis for self-defense. In this review, we discuss the mechanisms used by various inflammatory responses involving not only HSCs but also the niche cells in the BM, a site that has long been considered an immune-privileged organ.

The analysis, roles and regulation of quiescence in hematopoietic stem cells.

Tissue homeostasis requires the presence of multipotent adult stem cells that are capable of efficient self-renewal and differentiation; some of these have been shown to exist in a dormant, or quiescent, cell cycle state. Such quiescence has been proposed as a fundamental property of hematopoietic stem cells (HSCs) in the adult bone marrow, acting to protect HSCs from functional exhaustion and cellular insults to enable lifelong hematopoietic cell production. Recent studies have demonstrated that HSC quiescence is regulated by a complex network of cell-intrinsic and -extrinsic factors. In addition, detailed single-cell analyses and novel imaging techniques have identified functional heterogeneity within quiescent HSC populations and have begun to delineate the topological organization of quiescent HSCs. Here, we review the current methods available to measure quiescence in HSCs and discuss the roles of HSC quiescence and the various mechanisms by which HSC quiescence is maintained.

LPS-stimulated human bone marrow stroma cells support myeloid cell development and progenitor cell maintenance.

The nonhematopoietic bone marrow (BM) microenvironment provides a functional niche for hematopoietic cell maintenance, recruitment, and differentiation. It consists of multiple cell types including vasculature, bone, adipose tissue, and fibroblast-like bone marrow stromal cells (BMSC), which can be summarized under the generic term niche cells. BMSC express Toll-like receptors (TLRs) and are capable to respond to TLR-agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis. Here, we show that in addition to enhanced myeloid colony formation from human CD34+ cells, lipopolysaccharide (LPS) stimulation retains overall higher numbers of CD34+ cells in co-culture assays using BMSC, with eightfold more CD34+ cells that underwent up to three divisions as compared to non-stimulated assays. When subjected to cytokine-supplemented myeloid colony-forming unit (CFU) assays or transplanted into newborn RAG2(-/-) γc (-/-) mice, CD34(+) cells from LPS-stimulated BMSC cultures give rise to the full spectrum of myeloid colonies and T and B cells, respectively, thus supporting maintenance of myeloid and lymphoid primed hematopoietic progenitor cells (HPCs) under inflammatory conditions. Collectively, we suggest that BMSC enhance hematopoiesis during inflammatory conditions to support the replenishment of innate immune effector cells and to prevent the exhaustion of the hematopoietic stem and progenitor cell (HSPC) pool.

Engineering of a functional bone organ through endochondral ossification.

Embryonic development, lengthening, and repair of most bones proceed by endochondral ossification, namely through formation of a cartilage intermediate. It was previously demonstrated that adult human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) can execute an endochondral program and ectopically generate mature bone. Here we hypothesized that hMSCs pushed through endochondral ossification can engineer a scaled-up ossicle with features of a "bone organ," including physiologically remodeled bone, mature vasculature, and a fully functional hematopoietic compartment. Engineered hypertrophic cartilage required IL-1ß to be efficiently remodeled into bone and bone marrow upon subcutaneous implantation. This model allowed distinguishing, by analogy with bone development and repair, an outer, cortical-like perichondral bone, generated mainly by host cells and laid over a premineralized area, and an inner, trabecular-like, endochondral bone, generated mainly by the human cells and formed over the cartilaginous template. Hypertrophic cartilage remodeling was paralleled by ingrowth of blood vessels, displaying sinusoid-like structures and stabilized by pericytic cells. Marrow cavities of the ossicles contained phenotypically defined hematopoietic stem cells and progenitor cells at similar frequencies as native bones, and marrow from ossicles reconstituted multilineage long-term hematopoiesis in lethally irradiated mice. This study, by invoking a "developmental engineering" paradigm, reports the generation by appropriately instructed hMSC of an ectopic "bone organ" with a size, structure, and functionality comparable to native bones. The work thus provides a model useful for fundamental and translational studies of bone morphogenesis and regeneration, as well as for the controlled manipulation of hematopoietic stem cell niches in physiology and pathology.

Hematopoietic stem cell response to inflammation.

Hematopoietic stem cells (HSCs) have unique functional properties, including self-renewal and multi-lineage differentiation potential, and are thought to be fully responsible for lifelong hematopoiesis. However, recent studies have shown that HSCs divide much more slowly than thought, and, therefore, that daily hematopoiesis is maintained not by HSCs but by hematopoietic progenitors with limited self-renewal. When hematopoietic stress such as an infection occurs, hematopoietic production is at high demand at the site of infection. To meet hematopoietic needs, HSCs are also presumably recruited to orchestrate hematopoiesis. The beneficial and detrimental effects of inflammation on HSC function and the associated hematopoietic regulation are discussed herein, by summarizing recent findings.