Gene expression analysis during platelet-like particle production in phorbol myristate acetate-treated MEG-01 cells.

A comprehensive gene-expression analysis during platelet (PLT) production from megakaryocytes may give important information on genes involved in the PLT production process. However, the low abundance of primary megakaryocytes makes the gene expression analysis difficult. Therefore, we employed MEG-01 cells, a human megakaryocytic cell line, and confirmed that the cell line produces PLT-like particles by treatment with phorbol myristate acetate (PMA). After treatment of MEG-01 cells with PMA for 8 or 24 h, comprehensive gene expression analysis was carried out using a microarray and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). From the microarray analysis, 141 genes were up-regulated (>2-fold) and 164 genes were down-regulated (<1/2-fold). However, known PLT-related genes were not included in the up- or down-regulated genes. On the other hand, RT-PCR analysis detected increased expression of beta1-tubulin, CD62P, gpIbalpha and gpIII, which are related to PLT function and megakaryocyte differentiation, following PMA treatment for 24 h. These results indicate that the MEG-01 cell may be an alternative model system to study the process of human PLT production from megakaryocytes. The gene-expression analysis might be a powerful tool for identifying genes related to PLT production, if the experimental conditions are optimized.

CD41+/CD45+ cells without acetylcholinesterase activity are immature and a major megakaryocytic population in murine bone marrow.

Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however, their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells, we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)), in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+), with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs, whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions, including beta1-tubulin. In normal mice, the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent, mitomycin-C, Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo.

Characterization of histopathology and gene-expression profiles of synovitis in early rheumatoid arthritis using targeted biopsy specimens.

The disease category of early rheumatoid arthritis (RA) has been limited with respect to clinical criteria. Pathological manifestations of synovitis in patients whose disease is clinically classified as early RA seem to be heterogeneous, with regular variations. To clarify the relation between the molecular and histopathological features of the synovitis, we analyzed gene-expression profiles in the synovial lining tissues to correlate them with histopathological features. Synovial tissues were obtained from knee joints of 12 patients with early RA by targeted biopsy under arthroscopy. Surgical specimens of long-standing RA (from four patients) were examined as positive controls. Each histopathological parameter characteristic of rheumatoid synovitis in synovial tissues was scored under light microscopy. Total RNAs from synovial lining tissues were obtained from the specimens selected by laser capture microdissection and the mRNAs were amplified by bacteriophage T7 RNA polymerase. Their cDNAs were analyzed in a cDNA microarray with 23,040 cDNAs, and the levels of gene expression in multilayered lining tissues, compared with those of normal-like lining tissues in specimens from the same person, were determined to estimate gene-expression profiles characteristic of the synovial proliferative lesions in each case. Based on cluster analysis of all cases, gene-expression profiles in the lesions in early RA fell into two groups. The groups had different expression levels of genes critical for proliferative inflammation, including those encoding cytokines, adhesion molecules, and extracellular matrices. One group resembled synovitis in long-standing RA and had high scores for some histopathological features - involving accumulations of lymphocytes and plasma cells - but not for other features. Possible differences in the histopathogenesis and prognosis of synovitis between the two groups are discussed in relation to the candidate genes and histopathology.