Akaitz Dorronsoro

The Achilles' heel of senescent cells: from transcriptome to senolytic drugs

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age‐related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro‐survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL‐xL, or plasminogen‐activated inhibitor‐2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM‐MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation‐exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/∆ mice, delaying age‐related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

IL1β Induces Mesenchymal Stem Cells Migration and Leucocyte Chemotaxis Through NF-κB

Mesenchymal stem cells are often transplanted into inflammatory environments where they are able to survive and modulate host immune responses through a poorly understood mechanism. In this paper we analyzed the responses of MSC to IL-1β: a representative inflammatory mediator. Microarray analysis of MSC treated with IL-1β revealed that this cytokine activateds a set of genes related to biological processes such as cell survival, cell migration, cell adhesion, chemokine production, induction of angiogenesis and modulation of the immune response. Further more detailed analysis by real-time PCR and functional assays revealed that IL-1β mainly increaseds the production of chemokines such as CCL5, CCL20, CXCL1, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11 and CX3CL1, interleukins IL-6, IL-8, IL23A, IL32, Toll-like receptors TLR2, TLR4, CLDN1, metalloproteins MMP1 and MMP3, growth factors CSF2 and TNF-α, together with adhesion molecules ICAM1 and ICAM4. Functional analysis of MSC proliferation, migration and adhesion to extracellular matrix components revealed that IL-1β did not affect proliferation but also served to induce the secretion of trophic factors and adhesion to ECM components such as collagen and laminin. IL-1β treatment enhanced the ability of MSC to recruit monocytes and granulocytes in vitro. Blockade of NF-κβ transcription factor activation with IκB kinase beta (IKKβ) shRNA impaired MSC migration, adhesion and leucocyte recruitment, induced by IL-1β demonstrating that NF-κB pathway is an important downstream regulator of these responses. These findings are relevant to understanding the biological responses of MSC to inflammatory environments.

Regulation of chronic inflammatory and immune processes by extracellular vesicles

Almost all cell types release extracellular vesicles (EVs), which are derived either from multivesicular bodies or from the plasma membrane. EVs contain a subset of proteins, lipids, and nucleic acids from the cell from which they are derived. EV factors, particularly small RNAs such as miRNAs, likely play important roles in cell-to-cell communication both locally and systemically. Most of the functions associated with EVs are in the regulation of immune responses to pathogens and cancer, as well as in regulating autoimmunity. This Review will focus on the different modes of immune regulation, both direct and indirect, by EVs. The therapeutic utility of EVs for the regulation of immune responses will also be discussed.

Regulation of human bone marrow stromal cell proliferation and differentiation capacity by glucocorticoid receptor and AP-1 crosstalk.

Although marrow adipocytes and osteoblasts derive from a common bone marrow stromal cells (BMSCs), the mechanisms that underlie osteoporosis‐associated bone loss and marrow adipogenesis during prolonged steroid treatment are unclear. We show in human BMSCs (hBMSCs) that glucocorticoid receptor (GR) signaling in response to high concentrations of glucocorticoid (GC) supports adipogenesis but inhibits osteogenesis by reducing c‐Jun expression and hBMSC proliferation. Conversely, significantly lower concentrations of GC, which permit hBMSC proliferation, are necessary for normal bone mineralization. In contrast, platelet‐derived growth factor (PDGF) signaling increases both JNK/c‐Jun activity and hBMSC expansion, favoring osteogenic differentiation instead of adipogenesis. Indeed, PDGF antagonizes the proadipogenic qualities of GC/GR signaling. Thus our results reveal a novel c‐Jun‐centered regulatory network of signaling pathways in differentiating hBMSCs that controls the proliferation‐dependent balance between osteogenesis and adipogenesis.

Hypoxia-inducible factor 1 alpha contributes to cardiac healing in mesenchymal stem cells-mediated cardiac repair

Mesenchymal stem cells (MSC) are effective in treating myocardial infarction (MI) and previous reports demonstrated that hypoxia improves MSC self-renewal and therapeutics. Considering that hypoxia-inducible factor-1 alpha (HIF-1α) is a master regulator of the adaptative response to hypoxia, we hypothesized that HIF-1α overexpression in MSC could mimic some of the mechanisms triggered by hypoxia and increase their therapeutic potential without hypoxia stimulation. Transduction of MSC with HIF-1α lentivirus vectors (MSC-HIF) resulted in increased cell adhesion and migration, and activation of target genes coding for paracrine factors. When MSC-HIF were intramyocardially injected in infarcted nude rats, significant improvement was found (after treatment of infarcted rats with MSC-HIF) in terms of cardiac function, angiogenesis, cardiomyocyte proliferation, and reduction of fibrotic tissue with no induction of cardiac hypertrophy. This finding provides evidences for a crucial role of HIF-1α on MSC biology and suggests the stabilization of HIF-1α as a novel strategy for cellular therapies.

Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes

Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.

Identification of HSP90 inhibitors as a novel class of senolytics

Aging is the main risk factor for many chronic degenerative diseases and cancer. Increased senescent cell burden in various tissues is a major contributor to aging and age-related diseases. Recently, a new class of drugs termed senolytics were demonstrated to extending healthspan, reducing frailty and improving stem cell function in multiple murine models of aging. To identify novel and more optimal senotherapeutic drugs and combinations, we established a senescence associated β-galactosidase assay as a screening platform to rapidly identify drugs that specifically affect senescent cells. We used primary Ercc1−/− murine embryonic fibroblasts with reduced DNA repair capacity, which senesce rapidly if grown at atmospheric oxygen. This platform was used to screen a small library of compounds that regulate autophagy, identifying two inhibitors of the HSP90 chaperone family as having significant senolytic activity in mouse and human cells. Treatment of Ercc1−/∆ mice, a mouse model of a human progeroid syndrome, with the HSP90 inhibitor 17-DMAG extended healthspan, delayed the onset of several age-related symptoms and reduced p16INK4a expression. These results demonstrate the utility of our screening platform to identify senotherapeutic agents as well as identified HSP90 inhibitors as a promising new class of senolytic drugs.The accumulation of senescent cells is thought to contribute to the age-associated decline in tissue function. Here, the authors identify HSP90 inhibitors as a new class of senolytic compounds in an in vitro screening and show that administration of a HSP90 inhibitor reduces age-related symptoms in progeroid mice.

Human mesenchymal stromal cells modulate T‐cell responses through TNF‐α‐mediated activation of NF‐κB

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF‐κB) in human MSCs. This pathway is activated by TNF‐α that is generated following TCR stimulation of T cells. Inhibition of NF‐κB through silencing of IκB kinase β or the TNF‐α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC‐associated NF‐κB activation primarily leads to inhibition of T‐cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF‐α/NF‐κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF‐κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.

Human mesenchymal stromal cell-mediated immunoregulation: mechanisms of action and clinical applications.

Mesenchymal stromal cells (MSCs) are multipotent cells found in connective tissues that can differentiate into bone, cartilage, and adipose tissue. Interestingly, they can regulate immune responses in a paracrine way and allogeneic MSCs do not elicit immune response. These properties have encouraged a number of clinical trials in a broad range of regenerative therapies. Although these trials were first focused on their differentiation properties, in the last years, the immunosuppressive features have gained most of the attention. In this review, we will summarize the up-to-date knowledge about the immunosuppressive mechanisms of MSCs in vivo and in vitro and the most promising approaches in clinical investigation.

Histone Deacetylase 3 Modulates the Expansion of Human Hematopoietic Stem Cells

Epigenetic changes are regarded as emerging major players for hematopoietic stem cell (HSC) biology. Although some histone deacetylase (HDAC) inhibitors, such as valproic acid (VA), induce differentiation and apoptosis in a variety of leukemic cells in vitro, they produce a favorable effect on the expansion of normal HSCs. In this study, we have identified the VA target HDAC3 as a negative regulator of umbilical cord blood HSC expansion. We demonstrate that knockdown of the transcript dramatically improves CD34+ cell expansion, which correlates with a higher potential to generate colony-forming units in functional assays. We show that this effect is mediated at the level of primitive hematopoietic cells and that it is not due to negative effects on specific cell commitment or alterations in the cell cycle. HDAC3 inhibition does not block commitment to the monocytic lineage and the maturation of monocyte precursors, which are the main inhibited pathways in the presence of VA. Therefore, our results identify HDAC3 as a promising target for therapies aiming to expand HSCs.