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Hematopoietic stem cells (HSCs) are stem cells that can differentiate into various blood cells and have long-term self-renewal capacity. At present, HSC transplantation is an effective therapeutic means for many malignant hematological diseases, such as aplastic hematological diseases and autoimmune diseases. The hematopoietic microenvironment affects the proliferation, differentiation, and homeostasis of HSCs. The regulatory effect of the hematopoietic microenvironment on HSCs is complex and has not been thoroughly studied yet. In this study, we focused on mononuclear cells (MNCs), which provided an important microenvironment for HSCs and established a methodological system for identifying cellular composition by means of multiple technologies and methods. First, single-cell RNA sequencing (scRNA-seq) technology was used to investigate the cellular composition of cells originating from different microenvironments during different stages of hematopoiesis, including mouse fetal liver mononuclear cells (FL-MNCs), bone marrow mononuclear cells (BM-MNCs), and in vitro-cultured fetal liver stromal cells. Second, bioinformatics analysis showed a higher proportion and stronger proliferation of the HSCs in FL-MNCs than those in BM-MNCs. On the other hand, macrophages in in vitro-cultured fetal liver stromal cells were enriched to about 76%. Differential gene expression analysis and Gene Ontology (GO) functional enrichment analysis demonstrated that fetal liver macrophages have strong cell migration and actin skeleton formation capabilities, allowing them to participate in the hematopoietic homeostasis through endocytosis and exocytosis. Last, various validation experiments such as quantitative real-time PCR (qRT-PCR), ELISA, and confocal image assays were performed on randomly selected target genes or proteins secreted by fetal liver macrophages to further demonstrate the potential relationship between HSCs and the cells inhabiting their microenvironment. This system, which integrates multiple methods, could be used to better understand the fate of these specific cells by determining regulation mechanism of both HSCs and macrophages and could also be extended to studies in other cellular models. [ABSTRACT FROM AUTHOR]

Pathogenic variants of zinc finger C4H2-type containing (ZC4H2) on the X chromosome cause a group of genetic diseases termed ZC4H2-associated rare disorders (ZARD), including Wieacker-Wolff Syndrome (WRWF) and Female-restricted Wieacker-Wolff Syndrome (WRWFFR). In the current study, a de novo c.352C>T (p.Gln118*) mutation in ZC4H2 (NM_018684.4) was identified in a female neonate born with severe arthrogryposis multiplex congenita (AMC) and Pierre-Robin sequence (cleft palate and micrognathia). Plasmids containing the wild-type (WT), mutant-type (MT) ZC4H2, or GFP report gene (N) were transfected in 293T cell lines, respectively. RT-qPCR and western blot analysis showed that ZC4H2 protein could not be detected in the 293T cells transfected with MT ZC4H2. The RNA seq results revealed that the expression profile of the MT group was similar to that of the N group but differed significantly from the WT group, indicating that the c.352C>T mutation resulted in the loss of function of ZC4H2. Differentially expressed genes (DEGs) enrichment analysis showed that c.352C>T mutation inhibited the expression levels of a series of genes involved in the oxidative phosphorylation pathway. Subsequently, expression levels of ZC4H2 were knocked down in neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) by lentiviral-expressed small hairpin RNAs (shRNAs) against ZC4H2. The results also demonstrated that decreasing the expression of ZC4H2 significantly reduced the growth of NSCs by affecting the expression of genes related to the oxidative phosphorylation signaling pathway. Taken together, our results strongly suggest that ZC4H2 c.352C>T (p.Gln118*) mutation resulted in the loss of protein function and caused WRWFFR. [ABSTRACT FROM AUTHOR]

Inflammation and fibrosis in human liver are often precursors to hepatocellular carcinoma (HCC), yet none of them is easily modeled in animals. We previously generated transgenic mice with hepatocyte-specific expressed herpes simplex virus thymidine kinase (HSV-tk). These mice would develop hepatitis with the administration of ganciclovir (GCV). However, our HSV-tk transgenic mice developed hepatitis and HCC tumor as early as six months of age even without GCV administration. We analyzed the transcriptome of the HSV-tk HCC tumor and hepatitis tissue using microarray analysis to investigate the possible causes of HCC. Gene Ontology (GO) enrichment analysis showed that the up-regulated genes in the HCC tissue mainly include the immune-inflammatory and cell cycle genes. The down-regulated genes in HCC tumors are mainly concentrated in the regions related to lipid metabolism. Gene set enrichment analysis (GSEA) showed that immune-inflammatory-related signals in the HSV-tk mice are up-regulated compared to those in Notch mice. Our study suggests that the immune system and inflammation play an important role in HCC development in HSV-tk mice. Specifically, increased expression of immune-inflammatory-related genes is characteristic of HSV-tk mice and that inflammation-induced cell cycle activation maybe a precursory step to cancer. The HSV-tk mouse provides a suitable model for the study of the relationship between immune-inflammation and HCC, and their underlying mechanism for the development of therapeutic application in the future. [ABSTRACT FROM AUTHOR]