Eph/ephrin Signaling and Biology of Mesenchymal Stromal/Stem Cells

Mesenchymal stromal/stem cells (MSCs) have emerged as important therapeutic agents, owing to their easy isolation and culture, and their remarkable immunomodulatory and anti-inflammatory properties. However, MSCs constitute a heterogeneous cell population which does not express specific cell markers and has important problems for in vivo homing, and factors regulating their survival, proliferation, and differentiation are largely unknown. Accordingly, in the present article, we review the current evidence on the relationships between Eph kinase receptors, their ephrin ligands, and MSCs. These molecules are involved in the adult homeostasis of numerous tissues, and we and other authors have demonstrated their expression in human and murine MSCs derived from both bone marrow and adipose tissue, as well as their involvement in the MSC biology. We extend these studies providing new results on the effects of Eph/ephrins in the differentiation and immunomodulatory properties of MSC

The fish spleen

2023 Acuerdos transformativos CRUEIn the present study, we review the structure and function of fish spleen with special emphasis on its condition in Elasmobranchs, Teleosts and Lungfish. Apart from the amount of splenic lymphoid tissue, the histological organization of the organ ensures the existence of areas involved in antigen trapping, the ellipsoids, and exhibit numerous melano-macrophages which appear isolated or forming the so-called melano-macrophage centres. An extensive discussion on the functional significance of these centres conclude that they are mere accumulations of macrophages consequence of tissue homeostasis rather than primitive germinal centres, as proposed by some authors.Depto. de Biología CelularFac. de Ciencias BiológicasTRUEpubAPC financiada por la UC

FoxN1 mediates thymic cortex–medulla differentiation through modifying a developmental pattern based on epithelial tubulogenesis

The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros−/− thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex–medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros−/− mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros−/− and WT thymi after arrival of lymphoid progenitor cells

Peripheral T‑cell responses of EphB2‑ and EphB3‑deficient mice in a model of collagen‑induced arthritis

2024 Acuerdos transformativos CRUE-CSICBoth EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3−/− mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2−/− T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3−/− lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions.Unión Europea. NextGenerationEU. Plan de Recuperación Transformación y ResilienciaMinisterio de Ciencia, Innovación y UniversidadesInstituto de Salud Carlos IIIDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Eph/ephrinB signalling is involved in the survival of thymic epithelial cells

The signals that determine the survival/death of the thymic epithelial cells (TECs) component during embryonic development of the thymus are largely unknown. In this study, we combine different in vivo and in vitro experimental approaches to define the role played by the tyrosine kinase receptors EphB2 and EphB3 and their ligands, ephrinsB, in the survival of embryonic and newborn (NB) TECs. Our results conclude that EphB2 and EphB3 are involved in the control of TEC survival and that the absence of these molecules causes increased apoptotic TEC proportions that result in decreased numbers of thymic cells and a smaller-sized gland. Furthermore, in vitro studies using either EphB2-Fc or ephrinB1-Fc fusion proteins demonstrate that the blockade of Eph/ephrinB signalling increases TEC apoptosis, whereas its activation rescues TECs from cell death. In these assays, both heterotypic thymocyte–TEC and homotypic TEC–TEC interactions are important for Eph/ephrinB-mediated TEC survival.Ministerio de Educación y Ciencia (España)Ministerio de Salud y Consumo (España)Comunidad de MadridUniversidad Complutense de MadridDepto. de Biología CelularFac. de Ciencias BiológicasTRUEpu

Mesenchymal stromal cells derived from the bone marrow of acute lymphoblastic leukemia patients show altered BMP4 production: correlations with the course of disease.

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated.This work was supported by grands BFU2009-10315, SAF2012-33180, and BFU2010-18250 from the Spanish Ministry of Science and Innovation, Spanish Association Against Cancer 2010 AECC; P2010/BMD-2420-CellCAM from Regional Government of Madrid and RD12/0019/0007-TerCell Network from the Health Institute Carlos III. MNVG contract UCM number 50053735 by Comunidad de Madrid. AE grant number AP2010-0795 of Ministry of Education, Culture and Sports. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Mesenchymal stem cells derived from low risk acute lymphoblastic leukemia patients promote NK cell antitumor activity

Mesenchymal stem cells (MSCs) are key components of the bone marrow microenvironment which contribute to the maintenance of the hematopoietic stem cell niche and exert immunoregulatory functions in innate and adaptive immunity. We analyze the immunobiology of MSCs derived from acute lymphoblastic leukemia (ALL) patients and their impact on NK cell function. In contrast to the inhibitory effects on the immune response exerted by MSCs from healthy donors (Healthy-MSCs), we demonstrate that MSCs derived from low/intermediate risk ALL patients at diagnosis (ALL-MSCs) promote an efficient NK cell response including cytokine production, phenotypic activation and most importantly, cytotoxicity. Longitudinal studies indicate that these immunostimulatory effects of ALL-MSCs are progressively attenuated. Healthy-MSCs adopt ALL-MSC-like immunomodulatory features when exposed to leukemia cells, acquiring the ability to stimulate NK cell antitumor function. The mechanisms underlying to these functional changes of ALL-MSCs include reduced production of soluble inhibitory factors, differential expression of costimulatory and coinhibitory molecules, increased expression of specific TLRs and Notch pathway activation. Collectively our findings indicate that, in response to leukemia cells, ALL-MSCs could mediate a host beneficial immunomodulatory effect by stimulating the antitumor innate immune response

Relevance of BMP4 signaling in proliferation and viability in ALL-MSCs.

<p>(A) Effects of different doses of dorsomorphin, an inhibitor of the canonical BMP signaling pathway, on the viability and proliferation of Healthy-MSCs and ALL- MSCs. BM-MSCs were cultured for six days in Mesen PRO-RS™ medium alone (no treatment) or containing different doses of dorsomorphin. Cultures treated with dorsomorphin 10 µM were supplied with 0.01 ng/mL BMP4 to avoid non-specific effects of the drug. Data represent the percentage of proliferation of treated Healthy-MSC or ALL-MSC-Diagnosis cultures with respect to that of non-treated ones in five independent experiments. (B) Annexin V staining was measured by flow cytometry in Healthy-MSC and ALL-MSC-Diagnosis cells harvested from control and dorsomorphin-treated cultures on day 6. The percentages of Annexin V+ cells in the iodide-negative population are indicated in each histogram. Data are representative of three independent results. (C) Cell proliferation measured as BrdU incorporation in newly synthesized ADN of Healthy-MSCs cultured for six days in the presence of increasing concentrations of BMP4. Note the low proliferation in cultures treated with high doses of BMP4 (100 ng/mL) and its similarity to that found in non-treated cultures of ALL-MSC-Diagnosis endogenously producing high amounts of the morphogen. Data represent the mean ±SD of five independent experiments, each with three cultures per time point. (D) Correlations between the proliferative rate of Healthy-MSCs and ALL-MSCs at different times in the course of disease and BMP4 production assessed in the 6 day culture supernatants. Note the gradual increase of MSC proliferation in correlation with the decreased production of BMP4. (E) Viability of Healthy-MSCs treated six days with different doses of BMP4 analyzed by Annexin V and propidium iodide staining. Results are representative of three independent experiments.</p

BMP2/4 signaling pathway in ALL-MSCs in the course of disease.

<p>Expression of different components of the BMP2/4 signaling pathway as determined by quantitative PCR: (A) receptors, (B) R-SMADs and Co-SMADs, (C) ID proteins and (D) inhibitors of BMP signaling. PCR data are represented as arbitrary units of gene expression. Asterisks indicate statistical significance between Healthy-MSCs (n = 6) and ALL-MSCs (n = 11) or OTT-MSCs (n = 3) (*<i>P</i>≤0.05). (E) MSCs were cultured for six days in Mesen PRO-RS™ medium alone (light gray histogram) or supplemented with BMP4 (100 ng/mL) (open histogram) for the last 30 minutes. Detection of intracellular SMAD1 phosphorylation was performed by flow cytometry. Background staining is shown by isotype-matched control Abs (dark gray histogram). The results are representative of three different experiments.</p

Clinic data of patients.

<p>Note: M: male; F: female; ND: no determined; L: Low risk; I: Intermediate risk; H: High risk.</p