A comparative study of cellular diversity between the Xenopus pronephric and mouse metanephric nephron

The kidney is an essential organ that ensures bodily fluid homeostasis and removes soluble waste products from the organism. The functional units within the kidneys are epithelial tubules called nephrons. These tubules take in filtrate from the blood or coelom and selectively reabsorb nutrients through evolutionarily conserved nephron segments, leaving waste product to be eliminated in the urine. Genes coding for functional transporters are segmentally expressed, enabling nephrons to function as selective filters. The developmental patterning program that generates these segments is of great interest. The Xenopus embryonic kidney, the pronephros, has served as a valuable model to identify genes involved in nephron formation and patterning. Prior work has defined the gene expression profiles of Xenopus epithelial nephron segments via in situ hybridization strategies, but our understanding of the cellular makeup of the Xenopus pronephric kidney remains incomplete. Here, we scrutinize the cellular composition of the Xenopus pronephric nephron through comparative analyses with previous Xenopus studies and single-cell mRNA sequencing of the adult mouse kidney, this study reconstructs the cellular makeup of the pronephric kidney and identifies conserved cells, segments, and expression profiles. The data highlight significant conservation in podocytes, proximal and distal tubule cells and divergence in cellular composition underlying the evolution of the corticomedullary axis, while emphasizing the Xenopus pronephros as a model for physiology and disease.

Application of imprint cytology in assessment of immunological effects of isolated and combined action of selenium and copper nanoparticles

Introduction. Touch Imprint Cytology as the method of impression cytology of smears-prints is of great diagnostic value not only in clinical practice but is also of interest as an express method for assessing the immunological effects of the influence of metal-containing nanoparticles on the tissues of laboratory animals in an experiment. Materials and methods. The study involved the spleen and mesenteric lymph nodes (MLN) of outbred male rats (24 individuals), with an initial weight of 220-230 g, after subchronic intoxication, which was caused by repeated intraperitoneal injections of metal-containing nanoparticles of selenium (SeO) and copper (CuO) nanoparticles (NPs) at a dose of 0.5 mg/kg and their combination three times a week (a total of 18 injections). After sacrificing the rats by decapitation, the spleen and MLN were removed from the animals from each group; made smears were dried at room temperature. Stained according to Leishman. Cell composition and cytological signs were assessed in a light binocular microscope by Carl Zeiss Primo Star with a USCMOS video imaging system at a magnification of 100x and 1000x under cytological criteria. Cell counting in the analysis of spleen and MLN preparations was carried out in percentage - 100 cells from each smear (48 studies), as well as calculating the number of cellular elements per 1 mm2 of the smear surface area, by calculating the absolute amount of each cellular element in the microscope field of view of 0.03 mm2, followed by recalculation per 1 mm2 (the number of studies is 48). Differences between the mean group quantitative results were processed using Student’s criteria using Excel software. Differences between mean values were considered statistically significant if the probability of a random difference did not exceed 5% (p < 0.05). Results. The main results obtained in the study of cytomorphological parameters of smears - spleen prints and MLN of rats after exposure to SeO and CuO NPs, both independently and their combination using two methods for calculating the cellular composition of preparations, are presented. The main changes in the cellular composition during immunological effects are highlighted. Inflammatory reactions of the hyperergic type were revealed when exposed to selenium nanoparticles, both in autonomous action and in combination with copper nanoparticles. The formation of local cellular immunity was noted due to an increase in the level of plasma cells in smears imprints when exposed to copper nanoparticles. Conclusion. Using the impression method of smears-prints in conjunction with the histological examination of tissue preparations allows iimplementing complete cytomorphological parameters in studying the immunological effects of metal-containing nanoparticles.

Comparative assessment of the pulmonary effect in rats to a single intratracheal administration of selenium or copper oxide nanoparticles

Introduction. Professional contact with selenium, copper and their compounds, including nanoscale forms, occurs in the metallurgical processes of copper sludge processing, copper pyrite roasting, manganese, selenium and tellurium production. The wide prevalence of selenium and copper oxide nanoparticles (SeO and CuO NPs) necessitates a comparative experimental assessment of its toxicity. Materials and methods. The copper or selenium oxide nanoparticle suspensions or a deionized water were intratracheally administered to female rats at single time. The bronchoalveolar lavage fluid (BALF) was obtained 24 hours after administration. There were evaluated the cellular composition and the biochemical parameters of the BALF. Results. The changes in the cellular composition of BALF demonstrate the SeO-NP and CuO-NP have a cytotoxic effect. The BALF biochemical indices were changed to a greater extent under CuO-NP. However, the phagocytic capacity of alveolar macrophages is activated under the SeO-NP. Conclusion. The SeO-NP and CuO-NP have a cytotoxic effect. SeO-NP have a positive effect on pulmonary phagocytosis, which can presumably be associated with selenium is a biomicroelement.

Analysis of the cellular composition of the native auto-transplantation mixture used for plastics of bone tissue defects

The cell composition of native transplant autosmes (NTA) used for bone plastics was studied. The histological examination showed the fragments of bone beams with preserved osteoblasts, the foci of myeloid and lymphoid hematopoiesis and the fibrin deposits, which suggested the presence of MMSCs. Immunophenotyping of the NTA cell population revealed a high level of expression of the surface markers CD105, CD73, and CD90 characteristic for MMSC. DNA-flow cytometry of the bone dust confirmed almost complete preservation of graft viability on the 3rd day of culturing (97.7 % of live cells). The data of this study confirm the presence of the osteogenic, osteoinductive, and osteoconductive properties of the bone dust and emphasize the importance of a further study of this-type bone graft for use in surgical interventions.

The Tumor Immune Landscape and Architecture of Tertiary Lymphoid Structures in Urothelial Cancer

Candidate immune biomarkers have been proposed for predicting response to immunotherapy in urothelial cancer (UC). Yet, these biomarkers are imperfect and lack predictive power. A comprehensive overview of the tumor immune contexture, including Tertiary Lymphoid structures (TLS), is needed to better understand the immunotherapy response in UC. We analyzed tumor sections by quantitative multiplex immunofluorescence to characterize immune cell subsets in various tumor compartments in tumors without pretreatment and tumors exposed to preoperative anti-PD1/CTLA-4 checkpoint inhibitors (NABUCCO trial). Pronounced immune cell presence was found in UC invasive margins compared to tumor and stroma regions. CD8+PD1+ T-cells were present in UC, particularly following immunotherapy. The cellular composition of TLS was assessed by multiplex immunofluorescence (CD3, CD8, FoxP3, CD68, CD20, PanCK, DAPI) to explore specific TLS clusters based on varying immune subset densities. Using a k-means clustering algorithm, we found five distinct cellular composition clusters. Tumors unresponsive to anti-PD-1/CTLA-4 immunotherapy showed enrichment of a FoxP3+ T-cell-low TLS cluster after treatment. Additionally, cluster 5 (macrophage low) TLS were significantly higher after pre-operative immunotherapy, compared to untreated tumors. We also compared the immune cell composition and maturation stages between superficial (submucosal) and deeper TLS, revealing that superficial TLS had more pronounced T-helper cells and enrichment of early TLS than TLS located in deeper tissue. Furthermore, superficial TLS displayed a lower fraction of secondary follicle like TLS than deeper TLS. Taken together, our results provide a detailed quantitative overview of the tumor immune landscape in UC, which can provide a basis for further studies.

Regenerated crustacean limbs are precise replicas

Animals can regenerate complex organs, yet this frequently results in imprecise replicas of the original structure. In the crustacean Parhyale, embryonic and regenerating legs differ in gene expression dynamics but produce apparently similar mature structures. We examine the fidelity of Parhyale leg regeneration using complementary approaches to investigate microanatomy, sensory function, cellular composition and cell molecular profiles. We find that regeneration precisely replicates the complex microanatomy and spatial distribution of external sensory organs, and restores their sensory function. Single-nuclei sequencing shows that regenerated and uninjured legs are indistinguishable in terms of cell type composition and transcriptional profiles. This remarkable fidelity highlights the ability of organisms to achieve identical outcomes via distinct processes.

Structural Features of Glands in the Sphincter Zones of the Large Intestine of an Adult

The aim is to study macro- and microscopic structure, as well as the cellular composition of the glands of the sphincter zones of large intestine of adults of different age groups.Material and methods. On autopsy material obtained from 30 people, without signs of pathology of the digestive tract of three age groups: 20–29 years, 50–59 years, 90–99 years, the structure of the glandular apparatus of the sphincter zones was studied. The areas of the Gerlach flap, Girsch sphincters, Payr–Strauss, Bally, O'bern–Pirogov–Moutier were considered. Quantitative morphometry was performed on histological preparations stained with methylene blue, followed by fixation in a saturated solution of ammonium molybdenum (picric acid), hematoxylin-eosin, picrofuchsin according to Van Gieson. Methods of parametric statistics based on the Statistica 6.0 program were used for statistical data processing.Results. The analysis of the number, size and cellular composition of the glands of the sphincter zones of the large intestine revealed an increase in both the number of glands and their size in all age groups compared to the proximally adjacent areas of the intestine, on average by 1.3–1.5 times. In a similar range, individual indicators of the number of epithelial cells in the glands of the sphincter zones of the colon increased. At the same time, the cellular composition of the glands of the sphincter zones and adjacent areas of the intestinal wall was similar to neighboring areas, with predominant goblet-shaped epithelial cells (52.9–54.2% of cells on the longitudinal section of the gland) and the presence of absorption cells (29.9–31.2%), undifferentiated – 11.9–13.2% and argyrophilic endocrinocytes – 1.4–5.3%.Conclusion. Against the background of narrowing of the lumen of the large intestine in the area of the sphincters and changes in the nature of the mucous membrane, there is an increase in the size and density of the localization of glands in all age groups. This confirms the thesis about the formation of a protective barrier that provides a local adaptive potential of this area of the intestinal wall, against the background of increased mechanical effects of intestinal masses.

The single-cell transcriptional landscape of lung carcinoid tumors

Lung carcinoid tumors, also referred to as pulmonary neuroendocrine tumors or lung carcinoids, are rare neoplasms of the lung with a more favorable prognosis than other subtypes of lung cancer. Still, some patients suffer from relapsed disease and metastatic spread while no consensus treatment exists for metastasized carcinoids. Several recent single-cell studies have provided detailed insights into the cellular heterogeneity of more common lung cancers, such as adeno- and squamous cell carcinoma. However, the characteristics of lung carcinoids on the single-cell level are yet completely unknown.To study the cellular composition and single-cell gene expression profiles in lung carcinoids, we applied single-cell RNA sequencing to three lung carcinoid tumor samples and normal lung tissue. The single-cell transcriptomes of carcinoid tumor cells reflected intertumoral heterogeneity associated with clinicopathological features, such as tumor necrosis and proliferation index. The immune microenvironment was specifically enriched in noninflammatory monocyte-derived myeloid cells. Tumor-associated endothelial cells were characterized by distinct gene expression profiles. A spectrum of vascular smooth muscle cells and pericytes predominated the stromal microenvironment. We found a small proportion of myofibroblasts exhibiting features reminiscent of cancer-associated fibroblasts. Stromal and immune cells exhibited potential paracrine interactions which may shape the microenvironment via NOTCH, VEGF, TGFβ and JAK/STAT signaling. Moreover, single-cell gene signatures of pericytes and myofibroblasts demonstrated prognostic value in bulk gene expression data.Here, we provide first comprehensive insights into the cellular composition and single-cell gene expression profiles in lung carcinoids, demonstrating the non-inflammatory and vessel-rich nature of their tumor microenvironment, and outlining relevant intercellular interactions which could serve as future therapeutic targets.

Single-cell characterization of monolayer cultured human dental pulp stem cells with enhanced differentiation capacity

Human dental pulp stem cells (hDPSCs) are easily obtained multipotent cells, however, their potential value in regenerative medicine is hindered by the phenotypic and functional changes after conventional monolayer expansion. Here, we employed single-cell RNA sequencing (scRNA-seq) to comprehensively study the transcriptional difference between the freshly isolated and monolayer cultured DPSCs. The cell cluster analysis based on our scRNA-seq data showed that monolayer culture resulted in a significant cellular composition switch compared to the freshly isolated DPSCs. However, one subpopulation, characterized as MCAM(+)JAG(+)PDGFRA(−), maintained the most transcriptional characteristics compared to their freshly isolated counterparts. Notably, immunofluorescent staining revealed that the MCAM(+)JAG(+)PDGFRA(−) hDPSCs uniquely located in the perivascular region of human dental pulp tissue. Flow-cytometry analysis confirmed that their proportion remained relatively stable (~2%) regardless of physiological senescence or dental caries. Consistent with the annotation of scRNA-seq data, MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed higher proliferation capacity and enhanced in vitro multilineage differentiation potentials (osteogenic, chondrogenic and adipogenic) compared with their counterparts PDGFRA(+) subpopulation. Furthermore, the MCAM(+)JAG(+)PDGFRA(−) hDPSCs showed enhanced bone tissue formation and adipose tissue formation after 4-week subcutaneous implantation in nude mice. Taken together, our study for the first time revealed the cellular composition switch of monolayer cultured hDPSCs compared to the freshly isolated hDPSCs. After in vitro expansion, the MCAM(+)JAG(+)PDGFRA(−) subpopulation resembled the most transcriptional characteristics of fresh hDPSCs which may be beneficial for further tissue regeneration applications.

EXTH-40. MULTIDIMENSIONAL INTERROGATION OF GLIOBLASTOMA MICROENVIRONMENT TREATMENT RESPONSE IN EXPLANT CULTURES

The immune tumor microenvironment (iTME) of glioblastoma (GBM) contains microglial, macrophage, other myeloid cell populations and as adaptive immune cells. Recent therapeutic strategies for GBM aim at targeting iTME components to induce antitumoral immunity. A patient-tailored, ex vivo drug testing and response analysis platform would facilitate personalized therapy planning, provide insights into treatment-induced immune mechanisms in the iTME, and enable the discovery of biomarkers of response and resistance. Here, we generated patient-derived, live 3D GBM bioreactors from different tumor regions to assess iTME treatment responses to microglia modulators and immune checkpoint inhibitors. Intact GBM tissue specimens from the tumor center and periphery were cultured for 7 days in the presence or absence of anti-PD1, anti-CD47 antibodies or their combination. Tissues were analyzed by CODEX highly multiplexed microscopy using an immune-centered 54-marker panel, and changes in cytokine and chemokine levels in culture supernatants were investigated. A computational pipeline for integrative therapy response assessment was implemented. Explant cultures from n=8 IDH wt GBM were subjected to this integrative personalized analysis. Tissue integrity after 3D bioreactor cultures was comparable to tissue taken directly after surgery. FFPE CODEX workflow was feasible with adequate staining quality in bioreactor cultures. 850'000 single cells were segmented and clustered. Cellular composition between tumor center and the peripheral invasion zone differed significantly in immune phenotypes, cytokine profile and response to innate, adaptive or combinatorial local immunotherapies. Multiplexed cytokine analysis revealed IFNγ response signatures in a subset of center samples, whereas the peripheral invasion zone displayed a blunted cytokine response. This cytokine signature corresponded to cellular composition shifts within specific cellular neighborhoods. CD4 and CD8 T cells were invigorated and left their vascular niche. Our study demonstrates that local immunotherapies enable an active antitumoral immune response within the tumor center, and provides a multidimensional personalized framework for immunotherapy response assessment.