In vitro-derived platelets: the challenges we will have to face to assess quality and safety.

Platelet transfusions are given to patients in hospital who have a low blood platelet count (thrombocytopenia) either because of major bleeding (following trauma or surgery) or because the bone marrow production of platelets is impaired often due to chemotherapy, infiltration with malignant cells, fibrosis or genetic disorders. We are currently entirely reliant on blood donors as a source of platelets in transfusion medicine. However, the demand for platelets continues to rise, driven by an aging population, advances in medical procedures and ever more aggressive cancer therapies, while the supply of blood donors continues to remain static. In recent years, several groups have made major advances toward the generation of platelets in vitro for human transfusion. Recent successes include results in both generating mature human megakaryocytes as well as in developing bioreactors for extracting platelets from these megakaryocytes. Platelets made in vitro could address several issues inherent to platelets derived from blood donors - the ability to scale up/down more flexibly according to demand and therefore less precarious supply line, reduction of the risk of exposure to infectious agents and finally the possibility of engineering stem cells to reduce immunogenicity. Here we define the quality control tools and suggest measures for implementation across the field for in vitro platelet genesis, to aid collaboration between laboratories and to aid production of the burdens of proof that will eventually be required by regulators for efficacy and biosafety. We will do this firstly, by addressing the quality control of the nucleated cells used to make the platelets with a particular emphasis to safety issues and secondly, we will look at how platelet function measurement are addressed particularly in the context of platelets derived in vitro.

Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells.

BACKGROUND: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. OBJECTIVE: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. METHODS: Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. RESULTS: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. CONCLUSION: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.

Cloning and expression in murine erythroleukemia cells: the soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.

We have cloned, expressed, and purified the extracellular domains of types I and II human tumor necrosis factor receptors. Both proteins were expressed in and secreted by murine erythroleukemia cells under the control of the human beta-globin promoter placed down-stream from the human globin locus control region. Secretion of both proteins was directed by the respective tumor necrosis factor receptor signal sequence. Each tumor necrosis factor receptor extracellular domain was expressed as a chimeric protein, fused to a carboxy terminal flexible peptide linker and an antigenic affinity tag. Secretion of both proteins into the growth medium in a hollow fiber bioreactor was achieved. A monoclonal antibody generated against the affinity tag allowed the purification of both proteins. These were isolated as biologically active products in that they bound human tumor necrosis factor-alpha in a 125I-radioiodinated ligand binding assay. The two proteins also bound tumor necrosis factor-alpha at approximately equimolar ratios as demonstrated by BIAcore sensorgram analysis.