Human Niche Research Articles

The Reemergence of Monkeypox in Nigeria

The emergence and reemergence of infectious diseases caused by viruses continue to be a major public health concern globally, affecting both humans and animals. One such disease is monkeypox, a zoonotic infection caused by the monkeypox virus (MPXV) that has recurred in sub-Saharan Africa over several decades. Notably, the 2022 outbreak of monkeypox in Nigeria follows a deadly outbreak in 2017, which was preceded by the disease’s first recorded outbreak in 1978. Epidemiological investigations in 2017 showed no apparent link between human monkeypox cases and the outbreak that year, indicating the potential existence of multiple sources of the virus and limited human-to-human transmission. This underscores the presence of an alternative ecological niche in humans. Furthermore, in some communities in Nigeria, monkeys are regarded as sacred and not hunted or consumed, leading to their proliferation and increased likelihood of MPXV transmission. This mini-review focuses on the occurrence, epidemiological distribution, geographical distribution, endemicity, and possible solutions to reduce the spread of human monkeypox in Nigeria. The implications of this reemergence and the need for effective public health measures to prevent and control outbreaks of monkeypox are also discussed.

Social cognition and human aesthetic niche—the evolving human cognition as a participant of human niche construction

Aesthetic phenomena have been intertwined with the life of humans to a significant degree that is not observed in non-human animals. The complexity and subtlety of it have long been regarded as one symbol of human’s exceptional cognitive power. This power can sometimes be misrepresented by a view that regards human aesthetic behaviours as innate/encoded and automated settings that are brought along with the human brain. In scenarios like this, the naturally selected brain takes up a role as some super explanator—by appealing to the configurations of the neural connectivity as reasons/causations for observed phenotypic traits. Therefore, this line of thinking can sometimes obscure the role played by the sociocultural background in affecting those configurations. By drawing upon the notion of niche construction, I will propose a nature-nurture coevolving framework for understanding human cognitive evolution. It will be argued that the evolutionary trajectory of human cognition is heavily defined by and is, therefore, better understood through the lens of a human cultural niche and of contextualised/context-dependent expressions of human behavioural traits. This view will be delivered by highlighting the dynamics between selective pressures and the differential expression of human phenotypic traits and acknowledging the evolutionary causal role of human cultural behaviours and practices. Finally, I argue that a major evolution of social cognition was brought about through an aesthetic tradition of the Acheulean and conclude by briefly proposing a potential subject for future study. The basic research method applied in this article is theoretical deduction. Specifically, a restricted interdisciplinary investigation that concerns academic literature from relevant fields (centring on the topic of niche construction) of archaeology, evolutionary biology and human cognition was used. Furthermore, through a process of assessing and identifying of plausible evidence, the abovementioned arguments of this study are generated.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

Abstract 631: Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions

Abstract Background: Renal cell carcinoma (RCC) is the most common renal tumor in adults, and among these clear cell RCC (ccRCC) is most common subtype and accounts for most renal cancer-related deaths. One-third of patients with advanced ccRCC present with bone metastases at the time of diagnosis. Despite therapeutic advances, once cancer has metastasized to the bone, it represents a highly morbid and ultimately lethal disease. Comprehensive genomic studies in ccRCC have provided important insights into the somatic alterations and intra-tumor heterogeneity. However, the bone metastatic niche in humans, including the immune and stromal microenvironments, has not been well-defined. Methods: We collected fresh patient samples directly from the operating room and performed single-cell transcriptomic profiling of human ccRCC tumors (ccRCC primary), solid metastatic tissue (Bone Met), liquid bone marrow at the vertebral level of spinal cord compression (Involved), liquid bone marrow from a different vertebral body distant from the tumor site but within the surgical field (Distal), as well as bone marrow stroma cells from patients undergoing hip replacement surgery (Benign) as control. We provide a comparative single cell transcriptomic analysis between ccRCC primary tumors and bone metastatic lesions. Results: Comparison of malignant cells from primary and metastatic sites reveal a distinct transcriptional signature that is predictive of metastatic potential and patient survival. The bone marrow in metastatic patients has sustained immune-suppressive microenvironment, featuring increased, exhausted CD8+ cytotoxic T-cells, T-regulatory cells and tumor-associated macrophages (TAM) with distinct transcriptional states in metastatic lesions. Bone marrow stroma from tumor samples demonstrated a tumor-associated mesenchymal stromal cell population (TA-MSC) that appears to be supportive of epithelial-to mesenchymal transition (EMT), bone remodeling, and a cancer-associated fibroblast (CAFs) phenotype. This stromal subset is associated with poor progression-free and overall survival, markedly upregulates bone remodeling through the dysregulation of RANK/RANKL/OPG signaling activity in bone cells, ultimately leading to bone resorption. Conclusions: These results provide insights into mechanisms underlying of ccRCC tumors progression leading bone metastasis. We also highlight potential communication channels and therefore therapeutic targets within both the immune and the tumor cell populations. Citation Format: Shenglin Mei, Adele M. Alchahin, Ioanna Tsea, Youmna Kfoury, Taghreed Taghreed, Hirak Sarkar, Shulin Wu, Alexander O. Subtelny, Yida Zhang, Keyan Salari, Chin-Lee Wu, Mark A. Randolph, David T. Scadden, Douglas Dahl, John Shin, Peter Kharchenko, Philip Saylor, David Sykes, Ninib Baryawno. Single-cell analysis of immune and stroma cell remodeling in renal cell carcinoma primary tumors and bone metastatic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 631.

The v8-10 variant isoform of CD44 is selectively expressed in the normal human colonic stem cell niche and frequently is overexpressed in colon carcinomas during tumor development

ABSTRACT CD44 protein and its variant isoforms are expressed in cancer stem cells (CSCs), and various CD44 isoforms can have different functional roles in cells. Our goal was to investigate how different CD44 isoforms contribute to the emergence of stem cell (SC) overpopulation that drives colorectal cancer (CRC) development. Specific CD44 variant isoforms are selectively expressed in normal colonic SCs and become overexpressed in CRCs during tumor development. We created a unique panel of anti-CD44 rabbit genomic antibodies to 16 specific epitopes that span the entire length of the CD44 molecule. Our panel was used to comprehensively investigate the expression of different CD44 isoforms in matched pairs (n = 10) of malignant colonic tissue and adjacent normal mucosa, using two (IHC & IF) immunostaining approaches. We found that: i) CD44v8–10 is selectively expressed in the normal human colonic SC niche; ii) CD44v8–10 is co-expressed with the SC markers ALDH1 and LGR5 in normal and malignant colon tissues; iii) colon carcinoma tissues frequently (80%) stain for CD44v8–10 while staining for CD44v6 was less frequent (40%). Given that CD44v8–10 expression is restricted to cells in the normal human colonic SC niche and CD44v8–10 expression progressively increases during CRC development, CD44v8–10 expression likely contributes to the SC overpopulation that drives the development and growth of colon cancers. Since the CD44 variant v8–10 epitope is located on CD44’s extracellular region, it offers great promise for targeted anti-CSC treatment approaches.

Development and characterization of fermented soy beverages containing encapsulated or non-encapsulated vaginal probiotics

Human microbial niches such as the healthy vagina, are recently emerging as “unconventional” sources of candidate probiotics capable of preventing from different vaginal diseases. These microorganisms could be provided as oral preparations since they can reach the vaginal niche passing through the gastrointestinal tract. However, their use in food would be challenging. The aim of this work was to develop and characterize fermented soy beverages with encapsulated and non-encapsulated vaginal lactobacilli, namely Lactobacillus crispatus BC4 and Lactobacillus gasseri BC9, as future dietary strategies for vaginal dysbiosis. The viability of vaginal strains remained stable at 7 log CFU/mL of product during the entire 28 days of storage, despite the use of encapsulated or non-encapsulated bacteria. Samples containing encapsulated bacteria, especially E-BC4+BC9, showed higher Water Holding Capacity (62.29%), lactic acid content (1.43%), and a remarkable antagonistic activity against enteropathogens. Moreover, encapsulation protected the strains from simulated GIT conditions (>1 Log) but reduced the acceptability of the final products. Overall, strain BC4 and BC9, alone or in mix, demonstrated to be promising co-starter cultures providing a characteristic flavor (pleasant smell and taste) and aroma (lower hexanal, benzaldehyde and higher diacetyl, and 2,3-pentanedione, compared to control) to the fermented soy beverages.

Differentiation of Peritubular Myoid-Like Cells from Human Induced Pluripotent Stem Cells.

Infertility affects 10-15% of couples, with half attributed to male factors. An improved understanding of the cell-type-specific dysfunction contributing to male infertility is needed to improve available therapies; however, human testicular tissues are difficult to obtain for research purposes. To overcome this, researchers have begun to use human induced pluripotent stem cells (hiPSCs) to generate various testis-specific cell types in vitro. Peritubular myoid cells (PTMs) are one such testicular cell type that serves a critical role in the human testis niche but, to date, have not been derived from hiPSCs. This study set forth to generate a molecular-based differentiation method for deriving PTMs from hiPSCs, mirroring in vivo patterning factors. Whole transcriptome profiling and quantitative polymerase chain reaction (qPCR) show that this differentiation method is sufficient to derive cells with PTM-like transcriptomes, including upregulation of hallmark PTM functional genes, secreted growth and matrix factors, smooth muscle, integrins, receptors, and antioxidants. Hierarchical clustering shows that they acquire transcriptomes similar to primary isolated PTMs, and immunostaining shows the acquisition of a smooth muscle phenotype. Overall, these hiPSC-PTMs will allow in vitro study of patient-specific PTM development and function in spermatogenesis and infertility.

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.

Adult hippocampal neurogenesis in Alzheimer's disease: A roadmap to clinical relevance.

Adult hippocampal neurogenesis (AHN) drops sharply during early stages of Alzheimer's disease (AD), via unknown mechanisms, and correlates with cognitive status in AD patients. Understanding AHN regulation in AD could provide a framework for innovative pharmacological interventions. We here combine molecular, behavioral, and clinical data and critically discuss the multicellular complexity of the AHN niche in relation to AD pathophysiology. We further present a roadmap toward a better understanding of the role of AHN in AD by probing the promises and caveats of the latest technological advancements in the field and addressing the conceptual and methodological challenges ahead.

Organoid modeling of human fetal lung alveolar development reveals mechanisms of cell fate patterning and neonatal respiratory disease.

Variation in lung alveolar development is strongly linked to disease susceptibility. However, underlying cellular and molecular mechanisms are difficult to study in humans. We have identified an alveolar-fated epithelial progenitor in human fetal lungs, which we grow as self-organizing organoids that model key aspects of cell lineage commitment. Using this system, we have functionally validated cell-cell interactions in the developing human alveolar niche, showing that Wnt signaling from differentiating fibroblasts promotes alveolar-type-2 cell identity, whereas myofibroblasts secrete the Wnt inhibitor, NOTUM, providing spatial patterning. We identify a Wnt-NKX2.1 axis controlling alveolar differentiation. Moreover, we show that differential binding of NKX2.1 coordinates alveolar maturation, allowing us to model the effects of human genetic variation in NKX2.1 on alveolar differentiation. Our organoid system recapitulates key aspects of human fetal lung stem cell biology allowing mechanistic experiments to determine the cellular and molecular regulation of human development and disease.

Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.

The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cellscrosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.

Ostensive communication, market exchange, mindshaping, and elephants.

Heintz & Scott-Phillips's hypothesis that the topic range and type diversity of human expressive communication gains support from consilience with prior accounts of market exchange as fundamental to unique human niche construction, and of mindshaping as much more important than mindreading. The productivity of the idea is illustrated by the light it might shed on why elephants seem to engage in continuous social communication for little evident purpose.

Extended Spectrum β-Lactamase-Producing Escherichia coli from Poultry and Wild Birds (Sparrow) in Djelfa (Algeria), with Frequent Detection of CTX-M-14 in Sparrow.

Antimicrobial resistance is a global threat that is spreading more and more in both human and animal niches. This study investigates the antimicrobial resistance and virulence threats of Escherichia coli isolates recovered from intestinal and fecal samples of 100 chickens, 60 turkeys, and 30 sparrows. Extended spectrum β-lactamase (ESBL) producing E. coli isolates were recovered in 12 of the animals tested, selecting one isolate per positive animal: sparrow (eight isolates, 26.7%), turkey (three isolates, 5%), and chicken (one isolate, 1%). The E. coli isolates were ascribed to B1 and D phylogenetic groups. The blaCTX-M-14 gene was detected in all ESBL-producing E. coli isolates from sparrow. The blaCTX-M-15 (two isolates) and blaCTX-M-14 genes (one isolate) were detected in the isolates of turkey, and the blaCTX-M-1 gene in one isolate from broiler. Three lineages were revealed among the tested isolates (ST/phylogenetic group/type of ESBL/origin): ST117/D/CTX-M-1/broiler, ST4492 (CC405)/D/CTX-M-15/turkey, and ST602/B1/CTX-M-14/sparrow. All isolates were negative for stx1, sxt2, and eae virulence genes. Our findings provide evidence that the sparrow could be a vector in the dissemination of ESBL-producing E. coli isolates to other environments. This study also reports, to our knowledge, the first detection of blaCTX-M-14 from sparrow at a global level and in turkey in Algeria.

A universal stress protein upregulated by hypoxia has a role in Burkholderia cenocepacia intramacrophage survival: Implications for chronic infection in cystic fibrosis.

Universal stress proteins (USPs) are ubiquitously expressed in bacteria, archaea, and eukaryotes and play a lead role in adaptation to environmental conditions. They enable adaptation of bacterial pathogens to the conditions encountered in the human niche, including hypoxia, oxidative stress, osmotic stress, nutrient deficiency, or acid stress, thereby facilitating colonization. We previously reported that all six USP proteins encoded within a low-oxygen activated (lxa) locus in Burkholderia cenocepacia showed increased abundance during chronic colonization of the cystic fibrosis(CF) lung. However, the role of USPs in chronic cystic fibrosis infection is not well understood. Structural modeling identified surface arginines on one lxa-encoded USP, USP76, which suggested it mediated interactions with heparan sulfate. Using mutants derived from the B. cenocepacia strain, K56-2, we show that USP76 is involved in host cell attachment. Pretreatment of lung epithelial cells with heparanase reduced the binding of the wild-type and complement strains but not the Δusp76 mutant strain, indicating that USP76 is directly or indirectly involved in receptor recognition on the surface of epithelial cells. We also show that USP76 is required for growth and survival in many conditions associated with the CF lung, including acidic conditions and oxidative stress. Moreover, USP76 also has a role in survival in macrophages isolated from people with CF. Overall, while further elucidation of the exact mechanism(s) is required, we can conclude that USP76, which is upregulated during chronic infection, is involved in bacterial survival within CF macrophages, a hallmark of Burkholderia infection.

Effects of Variation in Urine Sample Storage Conditions on 16S Urogenital Microbiome Analyses.

Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 adult females, we investigated the microbiome preservation efficacy of AssayAssure Genelock (Genelock), compared with no preservative, under different temperature conditions. We varied temperature over 48 h in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: -20°C, ambient temperature, 4°C, freeze-thaw cycle, and heat cycle. At 48 h, all samples were stored at -80°C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing Genelock versus no preservative, we did observe a higher concordance with Genelock samples shipped at colder temperatures (-20°C and 4°C) when compared with the samples shipped at -20°C without preservative. Our results indicate that Genelock does not introduce a significant amount of microbial bias when used on a range of temperatures and is most effective at colder temperatures. IMPORTANCE The urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile; urinary tract microbes have been linked to health problems, including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion, respectively, spent in the United States. annually. Given the low biomass of urine, novelty of the field, and limited reproducibility evidence, it is critical to study urine sample storage conditions to optimize scientific rigor. Efficient and reliable preservation methods inform methods for home self-sample collection and shipping, increasing the potential use in larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.

Stroke and Neurogenesis: Bridging Clinical Observations to New Mechanistic Insights from Animal Models.

Stroke was the 2nd leading cause of death and a major cause of morbidity. Unfortunately, there are limited means to promote neurological recovery post-stroke, but research has unearthed potential targets for therapies to encourage post-stroke neurogenesis and neuroplasticity. The occurrence of neurogenesis in adult mammalian brains, including humans, was not widely accepted until the 1990s. Now, adult neurogenesis has been extensively studied in human and mouse neurogenic brain niches, of which the subventricular zone of the lateral ventricles and subgranular zone of the dentate gyrus are best studied. Numerous other niches are under investigation for neurogenic potential. This review offers a basic overview to stroke in the clinical setting, a focused summary of recent and foundational research literature on cortical neurogenesis and post-stroke brain plasticity, and insights regarding how the meninges and choroid plexus have emerged as key players in neurogenesis and neuroplasticity in the context of focal cerebral ischemia disrupting the anterior circulation. The choroid plexus and meninges are vital as they are integral sites for neuroimmune interactions, glymphatic perfusion, and niche signaling pertinent to neural stem cells and neurogenesis. Modulating neuroimmune interactions with a focus on astrocyte activity, potentially through manipulation of the choroid plexus and meningeal niches, may reduce the exacerbation of stroke by inflammatory mediators and create an environment conducive to neurorecovery. Furthermore, addressing impaired glymphatic perfusion after ischemic stroke likely supports a neurogenic environment by clearing out inflammatory mediators, neurotoxic metabolites, and other accumulated waste. The meninges and choroid plexus also contribute more directly to promoting neurogenesis: the meninges are thought to harbor neural stem cells and are a niche amenable to neural stem/progenitor cell migration. Additionally, the choroid plexus has secretory functions that directly influences stem cells through signaling mechanisms and growth factor actions. More research to better understand the functions of the meninges and choroid plexus may lead to novel approaches for stimulating neuronal recovery after ischemic stroke.

Abstract IA008: Modeling immune-epithelial interactions in health and disease using human intestinal enteroid co-cultures

Abstract Human intestinal enteroids derived from Lgr5+ actively dividing adult stem cells offer a relevant ex vivo system to study biological processes of the human small intestine and colon. While these primary cultures recreate cellular and functional features of the intestinal epithelium of the small intestine (enteroids) or colon (colonoids), they are limited by the lack of associated cell types, including immunological cells, nerves, stromal cells that help maintain tissue homeostasis and respond to external challenges. In the gut, innate immune cells interact with the epithelium by conducting lumial surveillance, support barrier function, and deploy effector functions in response to injury/damage. We have established a co-culture system of enteroid/colonoid monolayers and underlying macrophages and polymorphonuclear neutrophils to recapitulate the cellular framework of the human intestinal epithelial niche. Human intestinal enteroids can generated from biopsies or resected tissue from any segment of the human intestine/colon and maintained in long-term cultures in a basement membrane-rich matrix supplemented with growth factor-conditioned media necessary to maintain intestinal stem cell homeostasis. Immune cells are isolated from fresh human whole blood or frozen peripheral blood mononuclear cells (PBMC) and co-cultured with human intestinal enteroids/colonoids grown as confluent monolayers on permeable supports (e.g., Transwells). This configuration allows for controlled access to the apical side of the epithelium to mimic the gut luminal environment as well as the basolateral side occupied by immune cells to mimic the mucosal environment. Enteroid-immune co-cultures enable multiple outcome measures, including transepithelial resistance, production of cytokines/chemokines, phenotypic analysis of immune cells, tissue immunofluorescence imaging, protein or mRNA expression, antigen or microbe uptake, and other cellular functions. Citation Format: Nicholas C. Zachos. Modeling immune-epithelial interactions in health and disease using human intestinal enteroid co-cultures [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr IA008.

Environmental biodiversity, human microbiota and association with allergies

Background. The article presents modern views on the role of microbiota in the occurrence of atopic pathology. The reasons for the decline in biodiversity, changes in the dynamic balance between macro-microorganisms, violation of the colonization of human ecological niches, which are important in the formation of local and systemic immunity, are considered; the main causes of dysbiosis at an early age, which precedes the formation of allergic diseases (food allergy, atopic dermatitis, allergic rhinitis, asthma), a significant increase in which has been noted in recent decades. Aim: to analyze reviews, articles devoted to the study of the role of the human microbiota, a decrease in its biodiversity in the formation of allergic pathology. Methods: the use of analytical reviews, articles posted in electronic databases: PubMed, Scopus, Web of Science, position of the European Academy of Allerogology and Clinical Immunology (EAACI). Conclusion: the analysis made it possible to present the modern role of microorganisms inhabiting the ecological niches of the human body, their dynamic balance on the function of all organs and systems, with consideration of the reasons for the decrease in biodiversity and the subsequent development of allergic diseases; the need to further study the role of not individual pathogens, but the general properties of the microbial population, which will help to change the approach to disease therapy, based not on replacing individual microorganisms, but on restoring the all population.

Generation of Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life. Three dimensional in vitro models to study spatio-temporal control of intrinsic and/or extrinsic factors orchestrating hematopoietic fate decisions in health and disease are needed. Here we present a method to generate a complex bone marrow-like organoid (BMO) from human induced pluripotent stem cells (iPSCs), consisting of hematopoietic, mesenchymal and vascular cells. Our approach expands current co-culture-based models of isolated primary endothelial and stromal cells, or bioengineered microfluidic chips and overcomes some of their intrinsic limitations. We devised a protocol of stepwise mesodermal progenitor differentiation through hemogenic endothelium to generate a complex heterocellular organoid: After embryoid body formation, mesoderm was induced and patterned by a sequential combination of Wnt activation, Nodal inhibition, BMP4, VEGF, bFGF and SCF. Embryoid bodies were embedded into a three-dimensional matrix and stimulated with defined cytokine cocktails in a timely fashion to generate hematopoietic progenitor cells while maintaining endothelial cell generation. At day 10 of differentiation, sprouted embryoid bodies were separated and transferred into a 96-well plate to promote full organoid maturation until day 17. Flow cytometric analysis of dissociated BMOs revealed three main cellular compartments, consisting of hematopoietic (CD45+), mesenchymal (CD45-CD31-CD34-CD271+) and endothelial cells (CD45-CD31+). Analysis of the cellular composition yielded an average content of~40,6% hematopoietic cells, ~39,5% mesenchymal cells, ~6% endothelial cells, ~1,6% HSPCs and ~0,9% MSCs per BMO. Single-cell RNA sequencing of dissociated BMOs confirmed the presence of these distinct cell clusters. Sorted hematopoietic (CD45+CD11b-CD34+-HSPCs) and mesenchymal (CD45-CD31-CD271+CD105+CD90+CD73+-MSCs) stem and progenitor cells showed multilineage differentiation potential in colony-forming-unit (CFU) and trilineage-differentiation assays, respectively. Confocal imaging of whole organoids showed that CD31+ endothelial cells formed a vessel-like network covered by a Collagen IV+ basement membrane and PDGFR-β+ murals cells, resembling pericytes. Ultrastructural analysis by transmission electron microscopy (TEM) revealed capillary-like structures of endothelial cells enclosed by pericytes, which were connected by tight junctions. Importantly, we also detected CXCL12+- CAR-like cells in close association to endothelial cells in our BMOs. Whereas CD45 expressing cells were distributed throughout the organoid, Runx1 expressing cells were found to be localized in distinct clusters. We examined whether the BMO niche promotes maturation of blood progenitors into mature cells of the myeloid lineage. We identified neutrophil progenitor stages from ProNeu to matureNeus by flow cytometry, S100A8/A9 and MPO expressing cells by confocal imaging and cells with characteristic features of neutrophil granulocytes by TEM, showing that granulopoiesis proceeds without the addition of lineage-instructing cytokines as G-CSF or GM-CSF. Next, we tested whether BMOs can be used as a model system to recapitulate the phenotype of monogenic bone marrow failure syndromes. BMOs from iPSCs edited to express a hypomorphic variant of VPS45 showed increased formation of reticulin fibers and a higher abundance of SMA expressing pericytes, reminiscent of increased myelofibrosis in bone marrow biopsies of VPS45 deficient patients. We also started to show that BMOs can be transplanted into immunodeficient NSG-mice with further BMO growth in vivo for up to four months. In ongoing experiments we analyze the contribution of human hematopoietic cells to blood cell chimerism in the host mice. In summary, iPSC-derived BMOs offer a new tool to study hematopoietic development and disease evolution within a complex three-dimensional human environment exhibiting key cellular and structural features of the human bone marrow niche.

A Functional in Vitro 3D Model of Human Hematopoietic Niches

Niches in the bone marrow (BM) regulate hematopoietic stem, progenitor and precursor cell fate and behaviour through cell-cell interactions and soluble factor secretion. Niche biology and niche-hematopoietic crosstalk are very complex processes not completely elucidated yet, due essentially to human material accessibility difficulties and limits of existing in vitro culture models. Here, we develop a 3D fully human bone marrow model recapitulating all hematopoietic and non-hematopoietic niche elements to aid further investigation of normal and pathological haematopoiesis and to move away from expensive, time-consuming and imperfect animal models. At first, we have developed a protocol to in vitro production and culture of all human main cellular non-hematopoietic components of bone marrow microenvironment. This comprehensive stroma is generated from human primary cells from femoral head, without any external cell lines contribution, and it reflects accurately the characteristics of the donor. In a 2-step culture process, mesenchymal stromal cells and endothelial cells are first selected and amplified simultaneously in an amplification medium, then they are cultured in the amplification medium as control or in a specific differentiation medium patented by the authors in conventional 2D culture plates or in 3D cultures: in spheroids, by self-organization in ultra-low-adherence plates or on a biomaterial (β-TriCalcium Phosphate), in the U-CUP perfused bioreactor. The resulting cellular stroma composition is analysed by quantifying the gene expression of markers of the osteoblastic (RUNX2, OSX, BSP), adipocytic (LPL, PPARγ, ADIPOQ) and endothelial (CD31) lineages . Moreover, specific markers of hematopoietic niche as ANG1, SCF, CXCL12, LEPR, VCAM, OPN are investigated by RT-qPCR. The spatial organization and the proportion of the different cellular stroma components are analysed by immunofluorescence (Osteoblastic cells: OSX marker, adipocytic cells: Bodipy probe, endothelial cells: CD31). The resulting tissues exhibited all compositional and structural features of human physiological BM: all stroma cells are presents and they express the main niche soluble factors. So, we explore if medullary stroma differentiated using our protocol is able to support the maintenance of HSPCs, to do this we performed coculture experiments in 2D with CD34+ cells sorted from umbilical cord blood. After 14 and 21 days of coculture, hematopoietic cells are recovered, counted and analysed by flow cytometry. Results show that our differentiated stroma is capable of maintaining, expanding and functionally regulated healthy human cord blood-derived HSPCs. An immature compartment (CD34+CD38-) is still present at day 21. Importantly, our primary in vitro niche model supports HSPCs with no cytokine addition. The same results are obtained with healthy human peripheral blood-derived HSPCs cocultured in 3D with the stroma organised in spheroids. Moreover, we demonstrated that our BM model is capable not only to maintain HSPC compartment but also to drive hematopoietic differentiation. In 2D coculture we detected mature blood cells and in 3D cultures, peripheral blood-derived HSPCs cocultured with spheroids are able to engage in erythroid lineage and recapitulate the early phases of erythropoiesis. These results demonstrate the fully complexity and huge versatility of our functional in vitro 3D model of human hematopoietic niches. Our exhaustive fully human hematopoietic niche model prepared from the primary cells of single donor, enables the design of advanced, tuneable in vitro BM proxies for the study of human physiological and pathological haematopoiesis and for screening molecules for predictive purposes in personalized medicine.