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Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stressrelated parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets. [ABSTRACT FROM AUTHOR]

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the antiinflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms. [ABSTRACT FROM AUTHOR]

Background and aimsA complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape.MethodsHere we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB’s from decompensated cirrhosis patients (n = 3).ResultsWe confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters.ConclusionOur results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-β signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-β neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-β-dependent mechanisms.

Preterm birth was previously identified as a high-risk factor for the long-term development of chronic kidney disease. However, the detailed pattern of podocyte (PD) changes caused by preterm birth and the potential mechanism underlying this process have not been well clarified. In present study, a rat model of preterm birth was established by delivery of pups 2 days early and podometric methods were applied to identify the changes in PDs number caused by preterm birth. In addition, single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatic analysis were performed in the preterm rat kidney to explore the possible mechanism caused by preterm birth. As results, when the kidney completely finished nephrogenesis at the age of 3 weeks, a reduction in the total number of differentiated PDs in kidney sections was detected. In addition, 20 distinct clusters and 12 different cell types were identified after scRNA-seq in preterm rats (postnatal day 2) and full-term rats (postnatal day 0). The numbers of PDs and most types of inherent kidney cells were decreased in the preterm birth model. In addition, 177 genes were upregulated while 82 genes were downregulated in the PDs of full-term rats compared with those of preterm rats. Further functional GO analysis revealed that ribosome-related genes were enriched in PDs from full-term rats, and kidney development-related genes were enriched in PDs from preterm rats. Moreover, known PD-specific and PD precursor genes were highly expressed in PDs from preterm rats, and pseudotemporal analysis showed that PDs were present earlier in preterm rats than in full-term rats. In conclusion, the present study showed that preterm birth could cause a reduction in the number of differentiated PDs and accelerate the differentiation of PDs.

Background: Calcific aortic valve disease (CAVD) is a progressive fibrocalcific disease that can be treated only through valve replacement. This study aimed to determine the role of hub genes and immune cell infiltration in CAVD progression. Methods: In this study, bioinformatics analysis was used to identify hub genes involved in CAVD. The datasets were downloaded from the Gene Expression Omnibus (GEO) database. Gene expression differences were evaluated via pathway and Gene Ontology analyses. Weighted gene co-expression network analysis (WGCNA) and differentially expressed genes were used to screen hub genes. The CIBERSORT algorithm was used to compare immune infiltration into the calcified aortic valve based on the hub genes between high- and lowexpression groups. We also performed single-cell RNA sequencing based on six different human aortic valve leaflets. The expression of hub genes was identified in human and mouse samples through quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry, immunofluorescence, and ELISA, and clinical features of the patients were investigated. Results: In total, 454 differentially expressed genes were obtained from the GEO database. WGCNA was used to find 12 co-expression modules in the Array Express database, of which one hub module (brown module) was most correlated with CAVD. Two hub genes were identified after combining the differentially expressed genes S100A8 and S100A9. Regarding these genes, the immune infiltration profiles varied between high- and low-expression groups. Compared with that in the low hub gene expression group, the high hub gene expression group had a higher proportion of activated NK cells (p < 0.01) and M1 macrophages (p < 0.05). The expression of S100A8 and S100A9 was consistent with single-gene RNA sequencing results, confirming that the expression levels of these two hub genes are significantly upregulated in patients with CAVD (p < 0.01). Furthermore, these results were verified using mouse and human samples by performing immunofluorescence, immunohistochemistry, qPCR, and ELISA analyses. Finally, the localization of S100A8 and S100A9 in monocytes and macrophages was confirmed via immunofluorescence using human aortic valves. Conclusion: These results demonstrate that S100A8 and S100A9 are two hub genes involved in CAVD, which might play an important role in its development through immune-related signaling pathways. [ABSTRACT FROM AUTHOR]