The mechanisms underlying lineage-specific differentiation of erythroid and myeloid cells at molecular level remain unclear. To gain insight into this process, differentiation of human bone marrow AC133+ cells seeded in methylcellulose or liquid medium was induced with EPO or G-CSF for 14-day in primary culture, and continued with the alternate cytokine for another 14-day in secondary cultures. Analysis of primitive hematopoietic markers CD34 and Notch1 or lineage-specific markers EPO-R and CD13 by single-cell RT-PCR showed individual colonies of 2 to 16 cells co-expressed EPO-R and CD13 on either CD34-positive or CD34-negative cells induced by EPO or G-CSF during primary and secondary semisolid cultures. In situ hybridization with the same cell surface markers in population of cells confirmed the single cell data. Gene expression patterns of 266 human genes during erythroid and myeloid lineages development at the RNA level by Rapid analysis of gene expression (RAGE) technique showed that 3% of the genes were expressed in D0 AC133+ cells, 5.6% in erythroid and 8.6% in myeloid lineage, respectively. Erythroid cells share 3% and myeloid cells share 2.6% gene expression pattern with D0 cells. More than 50% of these genes co-expressed in both erythroid and myeloid lineages. A total 107 sequences of these genes have been identified thus far using GeneBank and Celera databases as reference sources, with 77(72%) representing known genes including cell cycle genes, transcription factors, and lineage specific genes, 23(21.5%) previously identified genes with unknown function, and 6 (5.6%) novel genes. Proteomicanalysis with two-dimensional gel electrophoresis (2-DE) revealed several interesting results. First, among the detectable 688 protein spots of D0 cells, only 8.6% and 8.4% can be matched to E14 and G14 samples, respectively. However, 60.9% and 46.8% can be matched with E14/G14 and G14/E14, respectively. Second, the match rate between E14 and G14 is around 40%, while E14/G14 and G14/E14 share around 70% proteins qualitatively. Some protein spots of D0 gel disappeared during the differentiation procedure, and never come back, while some protein spots presented in D0 disappeared in E14 and G14 reemerged again in E14/G14 and G14/E14. Further experiments are in progress to characterize these specific spots by MALDI-MS coupled with peptide mass fingerprinting database. The studies indicate that co-expression of lineage-specific gene markers strongly suggests that different lineage-associated genes co-exist within the same cell prior to exclusive commitment to either erythroid or myeloid lineage, indicating that these two lineages originate from the same precursors. Discrete gene and protein changes between erythroid and myeloid development in this culture system may reveal a mechanistic basis for lineage-specific differentiation. Taken together, the results indicate that there are both discrete and common events that occur during erythroid and myeloid development that can be resolved by differential gene and protein expression profiling. A further characterization of the factors leading to lineage-specific differentiation will likely greatly improve our understanding of disorders associated with disruptions in progenitor cell proliferation and differentiation, such as the myeloproliferative and myelodysplastic syndromes.
