A Liquid Culture System Model For Adult Hematopoiesis At

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The biological implications of gene expression patterns of hematopoietic lineage differentiation is poorly understood. In this study, we examined gene expression patterns and comparative analysis of expressed genes in enriched erythroid and myeloid lineages of human CD133+(stem/progenitor) cells. Total cellular RNA was extracted form 5 cell population pellets, reverse transcribed into cDNA, and subjected to RAGE (rapid-analysis-gene-expression) PCR amplification using 320 primers specific for gene sequences of blood development. PCR products were separated through 8% TBE gel and identified by searching the GeneSystem 320TM database or DNA sequencing. mRNA expression patterns of expressed 266 gene-specific fragments were categorized into 3 groups (11 types): (1) genes expressed specifically in a single cell population (Types I?III), (2) genes expressed in 2 cell populations (Types IV?VII), and (3) genes expressed in 3 or more populations (Types VIII?XI). Of 145 defined cDNAs, 3 (2%) were novel genes. Protein profiles of same populations determined by 2-dimensional gel electrophoresis were in good agreement with overlapped and distinguished gene patterns. Flow cytometry also detected the co-expression of lineage-specific antigens during lineage commitment. Specifically, cell sorting based on CD13 (myeloid) and CD36( erythroid) expression demonstrated the existence of double-positive CD13 and CD36 cells in these lineages. Further clonagenic analysis showed that erythroid burst- and colony-forming units (BFU-E and CFU-E), granulocyte colony-forming units (CFU-G), and mixed colonies (CFU-GE) were induced in CD13+/CD36+, but not in CD13-/CD36- cell fractions with EPO, G-CSF, or EPO plus G-CSF. On the other hand, single-positive CD13 or CD36 population cells generated almost exclusively CFU-G or CFU-E, but no CFU-GE with above cytokines. In addition, the effect of G-CSF on myeloid only and EPO on both erythoid and myeloid progenitors was observed through the experiment. The study suggests that CD13+/CD36+ cells possess the potential for differentiation of myeloid and erythroid lineages even after 4-week culture in a single cytokine. These data support the hypothesis that co-expression of lineage-restrictive antigens on normal hematopoietic progenitors provides a mechanism for lineage plasticity in response to stress. Comparative analysis of genes expressed in erythroid and myeloid lineages using GoSurfer program showed statistically significant differential biological processes and indicated that genes shared in both lineages involved mainly in development, response to stimulus, and signal transduction pathways, while genes specifically expressed in either alone mostly in regulation of biological process and programmed cell death. We conclude that lineage conversion may be a characteristic of normal hematopoiesis, and the co-expression of lineage-specific antigens on progenitors may provide the basis for gene expression overlap and lineage plasticity.