Abstract
B cells develop from hematopoietic stem cells (HSC) in the bone marrow (BM) through a number of distinct stages before they migrate to the periphery as naïve mature B lymphocytes. These developmental stages can be identified by expression of cell surface antigens and Ig gene rearrangement status. The aim of this study was to characterize the earliest steps of normal human B cell development by gene expression profiling. Immunomagnetic selection and subsequent fluorescence-activated cell sorting (FACS) were used to isolate five populations from adult human BM: CD34+CD38− (HSC), CD34+CD10+CD19− early lymphoid progenitor cells (ELP), CD34+CD10+CD19+IgM− progenitor B cells (pro-B), CD34−CD10+CD19+IgM− precursor B cells (pre-B) and CD34−CD10+CD19+IgM+ immature B cells (IM). Total RNA was extracted from the purified cell populations, amplified and hybridized to Lymphochip cDNA microarrays. Six independent experiments from different donors were performed for each cell population. Expression of the genes encoding the selection markers confirmed the validity of the approach. Interestingly, genes necessary for the V(D)J-recombination such as RAG-1, RAG-2, TdT and ADA showed higher gene expression in the ELP population than in the HSC. In contrast, the transcription factors E2A, EBF, and Pax-5, which are all essential for early B-cell development, were first turned on in pro-B cells, in accordance with the B-cell lineage commitment. The ELP did not express B, T or NK lineage markers, except for a higher expression level of CD2 in the ELP population than in the four other cell populations. Taken together, the expression pattern of CD2 and the V(D)J-recombination genes in the ELP population, indicate that these cells have developed a lymphocyte potential, but are not fully committed to B-lineage cells. Hierarchical cluster analysis of the 758 differentially expressed genes (differences in relative expression by a factor of two or more and with maximum10% FDR) revealed a pattern that clearly separated the five consecutive cell populations. Furthermore, we created expression signatures based on information from Gene Ontology (GO) http://source.stanford.edu/cgi-bin/source/sourceSearch. One of the clearest distinctions between the gene expressions of the five developmental populations involved genes associated with proliferation, and showed that the HSC and IM populations are relatively indolent while the pro-B and pre-B populations comprised high expression levels of nearly all the proliferation associated genes. Finally, we examined in further detail the transitions between HSC, ELP and pro B cells. We found 25 genes to be differently expressed in the ELP population in comparison to the HSC and pro-B populations, including IGJ, BCL2 and BLNK. To identify combinations of markers that could better discriminate the ELP population, we also performed a gene pair class separation test. This resulted in 68 gene pairs with score above 10 that were denoted very good discriminators. For several of the markers the differences in gene expression were verified at the protein level by five colour FACS analysis. Taken together, these results provide new insight into the molecular processes that take place in the early human B cell differentiation, and in particular provide new information regarding expression of genes in the ELP population.
Human B Cell Populations (Preliminary Pages)