During the past decade there has been a substantial advance in our understanding of estrogen signaling both from a clinical as well as a preclinical perspective. Estrogen signaling is a balance between two opposing forces in the form of two distinct receptors (ERα and ERβ) and their splice variants. The prospect that these two pathways can be selectively stimulated or inhibited with subtype-selective drugs constitutes new and promising therapeutic opportunities in clinical areas as diverse as hormone replacement, autoimmune diseases, prostate and breast cancer, and depression. Molecular biological, biochemical, and structural studies have generated information which is invaluable for the development of more selective and effective ER ligands. We have also become aware that ERs do not function by themselves but require a number of coregulatory proteins whose cell-specific expression explains some of the distinct cellular actions of estrogen. Estrogen is an important morphogen, and many of its proliferative effects on the epithelial compartment of glands are mediated by growth factors secreted from the stromal compartment. Thus understanding the cross-talk between growth factor and estrogen signaling is essential for understanding both normal and malignant growth. In this review we focus on several of the interesting recent discoveries concerning estrogen receptors, on estrogen as a morphogen, and on the molecular mechanisms of anti-estrogen signaling.

Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in a diverse range of biological processes. For future therapeutic applications and for EV biology research in general, understanding the in vivo fate of EVs is of utmost importance. Here we studied biodistribution of EVs in mice after systemic delivery. EVs were isolated from 3 different mouse cell sources, including dendritic cells (DCs) derived from bone marrow, and labelled with a near-infrared lipophilic dye. Xenotransplantation of EVs was further carried out for cross-species comparison. The reliability of the labelling technique was confirmed by sucrose gradient fractionation, organ perfusion and further supported by immunohistochemical staining using CD63-EGFP probed vesicles. While vesicles accumulated mainly in liver, spleen, gastrointestinal tract and lungs, differences related to EV cell origin were detected. EVs accumulated in the tumour tissue of tumour-bearing mice and, after introduction of the rabies virus glycoprotein-targeting moiety, they were found more readily in acetylcholine-receptor-rich organs. In addition, the route of administration and the dose of injected EVs influenced the biodistribution pattern. This is the first extensive biodistribution investigation of EVs comparing the impact of several different variables, the results of which have implications for the design and feasibility of therapeutic studies using EVs.

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes1,2. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells3. Here, we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF/KAT8/MYST1, and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone posttranslational modification during autophagy, affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

Mesenchymal stromal cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells of innate and adaptive immune systems. As MSCs become accepted as a therapeutic option for the treatment of immunological disorders such as Graft versus Host Disease, our need to understand the intricate details by which they exert their effects is crucial. Programmed death‐1 (PD‐1) is an important regulator in T cell activation and homeostatic control. It has been reported that this pathway may be important in contact‐dependent mediated immunomodulation by MSCs. The aim of this study was to establish whether MSCs, in addition to their cell‐surface expression, are able to secrete PD‐1 ligands (PD‐L1 and PD‐L2) and their potential importance in modulating contact‐independent mechanisms of MSC immunosuppression. Here we report that MSCs express and secrete PD‐L1 and PD‐L2 and that this is regulated by exposure to interferon γ and tumor necrosis factor α. MSCs, via their secretion of PD‐1 ligands, suppress the activation of CD4+ T cells, downregulate interleukin‐2 secretion and induce irreversible hyporesponsiveness and cell death. Suppressed T cells demonstrated a reduction in AKT phosphorylation at T308 and a subsequent increase in FOXO3 expression that could be reversed with blockade of PD‐L1. In conclusion, we demonstrate for the first time, that MSCs are able to secrete PD‐1 ligands, with this being the first known report of a biological role for PD‐L2 in MSCs. These soluble factors play an important role in modulating immunosuppressive effects of MSCs directly on T cell behavior and induction of peripheral tolerance. Stem Cells 2017;35:766–776

The therapeutic potential of mesenchymal stromal cells (MSCs) is evident by the number of new and ongoing trials targeting an impressive variety of conditions. In bone and cartilage repair, MSCs are expected to replace the damaged tissue, while in other therapies they modulate a therapeutic response by the secretion of bioactive molecules. MSCs possess a phenotypic plasticity and harbor an arsenal of bioactive molecules that can be released upon sensing signals in the local milieu either directly or packaged in extracellular vesicles (EVs). The reported paracrine effects comprise many of the important functions of MSCs, including supporting hematopoietic stem cells in the bone marrow, promoting angiogenesis, and modulating the immune system. The major drawback in MSC therapy is the incomplete understanding of cell fate following systemic administration as well as the mechanisms by which these cells correct disease. In this review we discuss what is known about MSC engraftment, hemocompatibility, and immunomodulation, as well as the potential of bringing the MSC-EV field toward a clinical translation.

A detailed analysis was performed of the immunomodulatory and proteomic profiles of mesenchymal stromal cells (MSCs) administered to two patients with refractory acute respiratory distress syndrome (ARDS). The respiratory, hemodynamic, and multiorgan failure resolved and multiple pulmonary and systemic inflammation markers decreased. These observations highlight the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects.

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens – genetically governed antigenic determinants – at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome.We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products.We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.