Skeletal stem/progenitor cells provide the niche for extramedullary hematopoiesis in spleen.

In bone marrow, the niche which supports hematopoiesis and nurtures hematopoietic stem cells (HSCs) contains perivascular reticular cells representing a subset of skeletal stem/progenitor cells (SSPCs). These stromal cells which provide the niche are lost or become inadequate during stress, disease or ageing, such that HSCs leave bone marrow and enter spleen and other peripheral sites to initiate extramedullary hematopoiesis and particularly myelopoiesis. Spleen also maintains niches for HSCs under steady-state conditions, evident since neonatal and adult spleen contain HSCs in low number and provide low-level hematopoiesis. In spleen, HSCs are found in the sinusoidal-rich red pulp region also in the vicinity of perivascular reticular cells. These cells resemble to some extent the known stromal elements reflecting HSC niches in bone marrow, and are investigated here for their characteristics as a subset of SSPCs. The isolation of spleen stromal subsets and the generation of cell lines which support HSCs and myelopoiesis in vitro has led to the identification of perivascular reticular cells which are unique to spleen. Analysis of gene and marker expression, as well as differentiative potential, identifies an osteoprogenitor cell type, reflective of one of several subsets of SSPCs described previously in bone, bone marrow and adipose tissue. The combined information supports a model for HSC niches in spleen involving perivascular reticular cells as SSPCs having osteogenic, stroma-forming capacity. These associate with sinusoids in red pulp to form niches for HSCs and to support the differentiation of hematopoietic progenitors during extramedullary hematopoiesis.

Androgens alter the heterogeneity of small extracellular vesicles and the small RNA cargo in prostate cancer.

Proliferation and survival of prostate cancer cells are driven by the androgen receptor (AR) upon binding to androgen steroid hormones. Manipulating the AR signalling axis is the focus for prostate cancer therapy; thus, it is crucial to understand the role of androgens and AR on extracellular vesicle (EV) secretion and cargo. In this study, we report that plasma-derived circulating vesicles consisting of CD9 and double-positive for CD9 and Prostate Specific Membrane Antigen (PSMA) are increased in patients with advanced metastatic prostate cancer, whereas double positives for CD9 and CD63 small extracellular vesicles (S-EVs) are significantly higher in patients with localised prostate cancer. Androgen manipulation by dihydrotestosterone (DHT) and the clinical antagonist enzalutamide (ENZ) altered the heterogeneity and size of CD9 positive S-EVs in AR expressing prostate cancer cells, while assessment of the total number and protein cargo of total S-EVs was unaltered across different treatment groups. Furthermore, hormone stimulation caused strong and specific effects on the small RNA cargo of S-EVs. A total of 543 small RNAs were found to be regulated by androgens including miR-19-3p and miR-361-5p. Analysis of S-EVs heterogeneity and small RNA cargo may provide clinical utility for prostate cancer and be informative to understand further the mechanism of resistance to androgen targeted therapy in castration-resistant prostate cancer.

Pathogenesis of Viral Hepatitis-Induced Chronic Liver Disease: Role of Extracellular Vesicles.

Extracellular vesicles are encapsulated lipid nanoparticles secreted by a variety of cell types in living organisms. They are known to carry proteins, metabolites, nucleic acids, and lipids as their cargoes and are important mediators of intercellular communication. The role of extracellular vesicles in chronic liver disease has been reported. Chronic liver disease such as viral hepatitis accounts for a significant mortality and morbidity burden worldwide. Hepatic fibrosis has been commonly associated with the chronic form of viral hepatitis, which results in end-stage liver disease, including cirrhosis, liver failure, and carcinoma in some patients. In this review, we discuss the potential role of extracellular vesicles in mediating communication between infectious agents (hepatitis B and C viruses) and host cells, and how these complex cell-cell interactions may facilitate the development of chronic liver disease. We will further discuss how understanding their biological mechanism of action might be beneficial for developing therapeutic strategies to treat chronic liver disease.

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

Identification of Stromal Cells in Spleen Which Support Myelopoiesis.

Stromal cells in spleen organize tissue into red pulp, white pulp and marginal zone, and also interact with hematopoietic cells to regulate immune responses. This study has used phenotypic information of a previously described spleen stromal cell line called 5G3, which supports restricted hematopoiesis in vitro, to identify an equivalent stromal cell subset in vivo and to test its capacity to support hematopoiesis. Using stromal cell fractionation, phenotypic analysis, as well as cell growth and hematopoietic support assays, the Sca-1+gp38+Thy1.2+CD29+CD51+ fraction of spleen stroma has been identified as an equivalent stromal subset resembling the 5G3 cell counterpart. While heterogeneity may still exist within that subset, it has been shown to have superior hematopoietic support capacity compared with the 5G3 cell line, and all other spleen stromal cell fractions tested.

In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line.

There are very few model systems which demonstrate hematopoiesis in vitro. Previously, we described unique splenic stromal cell lines which support the in vitro development of hematopoietic cells and particularly myeloid cells. Here, the 5G3 spleen stromal cell line has been investigated for capacity to support the differentiation of hematopoietic cells from progenitors in vitro. Initially, 5G3 was shown to express markers of mesenchymal but not endothelial or hematopoietic cells and to resemble perivascular reticular cells in the bone marrow through gene expression. In particular, 5G3 resembles CXCL12-abundant reticular cells or perivascular reticular cells, which are important niche elements for hematopoiesis in the bone marrow. To analyse the hematopoietic support function of 5G3, specific signaling pathway inhibitors were tested for the ability to regulate cell production in vitro in cocultures of stroma overlaid with bone marrow-derived hematopoietic stem/progenitor cells. These studies identified an important role for Wnt and Notch pathways as well as tyrosine kinase receptors like c-KIT and PDGFR. Cell production in stromal cocultures constitutes hematopoiesis, since signaling pathways provided by splenic stroma reflect those which support hematopoiesis in the bone marrow.