We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

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5-HT4 receptor pre-mRNA is alternatively spliced in human (h) tissue to produce several splice variants, called 5-HT4(a) to 5-HT4(h) and 5-HT4(n). Polymerase chain reaction (PCR) with primers designed to amplify both 5-HT4(a) and 5-HT4(b) amplified three additional bands in different tissues, two representing different mRNA species both encoding 5-HT4(g) and one representing mRNA for a novel splice variant named 5-HT4(i), cloned from testis and pancreas respectively. Primary and nested PCR detected both 5-HT4(g) and 5-HT4(i) in multiple tissues. Whereas 5-HT4(i), was found in all cardiovascular tissues analysed, 5-HT4(g) was mainly present in atria. However, quantitative RT-PCR indicated 5-HT4(g) expression also in cardiac ventricle. The pharmacological profiles and ability to activate adenylyl cyclase (AC) were compared between four recombinant h5-HT4 splice variants (a, b, g and i) expressed transiently and stably in HEK293 cells. Displacement of [(3)H]GR113808 with ten ligands revealed identical pharmacological profiles (affinity rank order: GR125487, SB207710, GR113808>SB203186>serotonin, cisapride, tropisetron>renzapride, 5-MeOT>5-CT). In transiently transfected HEK293 cells cisapride was a partial agonist compared to serotonin at 5-HT4(b), 5-HT4(g) and 5-HT4(i) receptors. In membranes from HEK293 cells stably expressing 5-HT4(g) (3,000 fmol/mg protein) or 5-HT4(i) (500 fmol/mg protein), serotonin and 5-MeOT were full agonists while cisapride was full agonist at 5-HT4(g) and partial agonist at 5-HT4(i), probably due to different receptor expression levels. At both 5-HT4(g) and 5-HT4(i), the behaviour of 5-HT4 receptor antagonists was dependent on receptor level. At high receptor levels, tropisetron and SB207710 and to a variable extent SB203186 and GR113808 displayed some partial agonist activity, whereas GR125487 and SB207266 reduced the AC activity below basal, indicating both receptors to be constitutively active. We conclude that the novel 5-HT4(i) receptor splice variant is pharmacologically indistinguishable from other 5-HT4 splice variants and that the 5-HT4(i) C-terminal tail does not influence coupling to AC.

Advanced therapy medicinal products (ATMPs), which include gene therapy medicinal products, somatic cell therapy medicinal products and tissue-engineered products, are at the cutting edge of innovation and offer a major hope for various diseases for which there are limited or no therapeutic options. They have therefore been subject to considerable interest and debate. Following the European regulation on ATMPs, a consolidated regulatory framework for these innovative medicines has recently been established. Central to this framework is the Committee for Advanced Therapies (CAT) at the European Medicines Agency (EMA), comprising a multidisciplinary scientific expert committee, representing all EU member states and European Free Trade Association countries, as well as patient and medical associations. In this article, the CAT discusses some of the typical issues raised by developers of ATMPs, and highlights the opportunities for such companies and research groups to approach the EMA and the CAT as a regulatory advisor during development.