Mesenchymal stem cells and their use in therapy: what has been achieved?

The considerable therapeutic potential of human multipotent mesenchymal stromal cells or mesenchymal stem cells (MSCs) has generated increasing interest in a wide variety of biomedical disciplines. Nevertheless, researchers report studies on MSCs using different methods of isolation and expansion, as well as different approaches to characterize them; therefore, it is increasingly difficult to compare and contrast study outcomes. To begin to address this issue, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed minimal criteria to define human MSCs. First, MSCs must be plastic-adherent when maintained in standard culture conditions (α minimal essential medium plus 20% fetal bovine serum). Second, MSCs must express CD105, CD73 and CD90, and MSCs must lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules. Third, MSCs must differentiate into osteoblasts, adipocytes and chondroblasts in vitro. MSCs are isolated from many adult tissues, in particular from bone marrow and adipose tissue. Along with their capacity to differentiate and transdifferentiate into cells of different lineages, these cells have also generated great interest for their ability to display immunomodulatory capacities. Indeed, a major breakthrough was the finding that MSCs are able to induce peripheral tolerance, suggesting that they may be used as therapeutic tools in immune-mediated disorders. Although no significant adverse events have been reported in clinical trials to date, all interventional therapies have some inherent risks. Potential risks for undesirable events, such as tumor development, that might occur while using these stem cells for therapy must be taken into account and contrasted against the potential benefits to patients.

Nitric oxide: news from stem cells to platelets.

Nitric oxide (NO) is a diffusible, short-lived, diatomic free radical ubiquitously produced by mammalian cells. The generation of NO from L-arginine is enzymatically regulated by three different isoforms of NO synthases. The NO signaling pathway involves mainly the activation of soluble guanylyl cyclase to produce cyclic GMP (cGMP) as a second messenger and downstream mediator. In addition, the free radical activity of NO can cause cellular damage through a phenomenon known as nitrosative stress. NO is a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. In the hematopoietic system, NO is thought to be an autocrine or paracrine messenger but also an intracellular effector molecule. Megakaryopoiesis and subsequent thrombopoiesis occur through complex biologic steps that involve hematopoietic stem cell commitment to megakaryocytic lineage, megakaryocyte maturation and finally, platelet release. Here, we summarize the current knowledge regarding the role of exogenous and endogenous NO in hematopoietic stem cell biology, megakaryocyte development and platelet biogenesis as well as relevance of platelet-derived NO generation on platelet function. Dysregulation of NO synthesis has been observed in several diseases, and the evaluation of a series of pharmacological agents with the ability to modulate the NO/cGMP pathway in platelets will also be discussed.

Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.

Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins.

BACKGROUND: Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures. OBJECTIVES: To explore the effect of higher temperatures on platelet physiology. METHODS: Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)ß(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied. RESULTS: Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion. CONCLUSIONS: Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.

NF-kappaB inhibitors impair platelet activation responses.

BACKGROUND: Although platelets are anucleated cells, they express several transcription factors that exert non-genomic functions, including the positive and negative regulation of platelet activation. NF-kappaB is a major transcriptional regulator of genes involved in survival, proliferation and inflammation. OBJECTIVE: Because platelets play a critical role not only in hemostasis, but also in inflammation and tumor progression, we evaluated the role of NF-kappaB in platelet physiology. RESULTS: Immunofluorescence, Western blotting and ELISA studies revealed that platelets express IkappaBalpha and NF-kappaB, and that stimulation with thrombin triggers IkappaBalpha phosphorylation and degradation and the binding of platelet NF-kappaB p65 subunit to synthetic oligonucleotides containing the consensus sequence for NF-kappaB. Two specific unrelated inhibitors of NF-kappaB activation, BAY 11-7082 and Ro 106-9920, reduced PAC-1 and fibrinogen binding to integrin alpha(IIb)beta3 and restricted platelet spreading on immobilized fibrinogen. Both inhibitors impaired aggregation mediated by ADP, epinephrine, collagen or thrombin, but not arachidonic acid. ATP release, TXB2 formation, P-selectin expression, ERK phosphorylation and cPLA2 activity stimulated by thrombin were reduced in BAY 11-7082- or Ro 106-9920-treated platelets. Although bleeding time was not affected, ADP-induced platelet aggregation was impaired in mice treated with BAY 11-7082. CONCLUSIONS: Our results suggest that NF-kappaB may be a novel mediator of platelet responses. The blockade of platelet function by NF-kappaB inhibitors might be relevant in those clinical situations where these drugs are being considered for anti-tumor and/or anti-inflammatory therapy.