Genomics based analysis of interactions between developing B-lymphocytes and stromal cells reveal complex interactions and two-way communication

Abstract

Background: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow.

Results: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development), OP-9 cells (strongly supportive of B cell development), pro-B and pre-B cells as well as mature peripheral B-lineage cells, we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further, the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells.

Conclusions: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways.

Figure 1

Differential ability of OP9 and NIH3T3 cells to stimulate B-cell development from hematopoetic progenitor cells. Representative FACS plots of the cells generated after 14 days of incubation of 100 LSK cells on OP9 or NIH3T3 cells with the indicated addition of cytokines. The cells were analyzed with regard to the expression of CD19, B220, and MAC1 as indicated in the diagrams. The numbers in the panels represents average percentages of different cell subsets obtained under the defined conditions after analysis of at least three independent experiments.

Figure 2

BNP4 stimulates B-cell development from early bone marrow progenitor cells. Diagrams displaying the cellular composition of cultures as judged by FACS analysis of B220, CD19 and MAC-1 surface expression obtained by in vitro differentiation of 100 LSK cells. The progenitors were incubated for 14 days with either OP9 or NIH3T3 cells and the indicated cytokines. Each dot in the diagrams represents one well and data are collected from at least three independent experiments (n = 13-18). Line indicates the average of all the analyzed wells and the P values has been calculated as compared to the cellular composition obtained on NIH3T3 cells with the addition of FL and IL7.

Figure 3

B-cell progenitors modulate the behavior of OP9 stromal cells in vitro. Panel (A-B) display pictures of OP-9 cells grown in parallel cultures with either conditioned media (A) or in direct contact with pro-B cells (B) generated by in vitro differentiation of LSK cells. The pictures are taken from the same six well tissue culture plates with 200 times amplification and under the same light conditions (Leica DFC290). The black bar indicates 100 μM. Panel (C) shows a representative sorting experiment where trypsin/EDTA treated co-cultures of OP9S and pro-B cells are separated based on forward scatter and Strawberry expression. Panel (D) display a cluster analysis of RMA normalized gene expression patterns in OP9S cells incubated with either conditioned media or the pre-B cell line 230-238. The analysis displays genes with an expression level above 100 and with at least 5-fold difference in expression in the two experiments with P-value < 0.05. Red indicates high and blue low expression.

Figure 4

Co-culture of cell lines representing different hematopoietic lineages induces differential gene expression patterns in OP9 cells. The diagrams display HPRT normalized Q-PCR data from OP9S cells generated by co-incubation of hematopoietic cell lines or filtered media from the parallel co-culture experiments (OP9S). The expression pattern in the control cells were set to one for each of the Nov (Black bar), SpiB (White bar) or Cxcl10 (Striped bars) and the relative induction/reduction is indicated. Error bars indicate experimental variation in one out of two or three experiments.