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

The development of mature blood cells from haematopoietic stem cells is a process involving a gradual loss of multi-lineage potential and a subsequent gain of lineage restricted cellular features. The maturation process is reflected in surface marker expression allowing for sorting of cells at defined stages of development and for detailed functional and molecular analysis of stage specific events [1, 2]. This has revealed that the differentiation process is critically dependent on a set of transcription factors that appears to act in a hierarchical and coordinated manner to activate the correct genes and allow the developmental pathway to proceed [3]. However, the action of transcription factors and the outcome of the developmental process are also highly dependent on communication with other cells in the bone marrow (BM) micro-environment [4, 5]. One of the most carefully investigated developmental pathways in the BM is the differentiation of B-lymphoid cells. The earliest B cell progenitors are responsive to the stimulatory action of the chemokine CXCL12 (SDF-1) [6, 7] produced by BM stromal cells [8], acting via the CXCR4 receptor on the pro-B cells [9, 10]. CXCL12 is produced by stromal cells scattered in the BM, possibly creating a distinct anatomical niche for the earliest stages of B-lymphoid development [8]. These early cells are also supported by the action of FL-ligand, that via the FLT-3 receptor [11, 12] stimulate lymphoid primed multipotent progenitors (LMPPs [13]) to proceed into the lymphoid lineages [14]. The subsequent developmental stage in B-lymphocyte development display a critical need for the cytokine IL-7 and mice deficient in either the cytokine or the α component (IL-7Rα) of the hetero-dimeric receptor, display disturbances in differentiation in both B and T lymphocyte development [15, 16]. The phenotype observed in these mice is further enhanced by the combined disruption of both the IL-7Rα and FL genes where the block of B cell development is nearly complete [17, 18]. It has been reported that ectopic expression of a second ligand for the IL7Rα subunit, Thymic Stromal Lympho Protein (TSLP) [19], can rescue the B-cell defects in IL-7 deficient mice arguing for partially redundant functions of IL-7 and TSLP [20]. However, mice deficient in TSLP develop an apparently normal B-cell compartment suggesting that the central factor in vivo is IL-7 [21]. Later stages of B cell development has been suggested to be dependent on contact with supporting cells since hematopoietic cells deficient in RANK ligand are impaired in their ability to reconstitute the B220⁺IgM⁺ immature progenitor compartment upon transplantation into wild type mice [22]. Thus, it is apparent that normal B-lymphopoiesis is dependent on communication and contact between the developing cell and surrounding cells like BM stroma cells [5]. Even if a set of critical factors for normal development has been identified, it is reasonable to presume that the cells are exposed to a much larger number of signals that may influence normal as well as malignant B-cell growth and maturation.

In order to identify potential communication pathways involved in the regulation of blood cell development, we have developed a bioinformatic tool, Genomics based Cell-cell Interaction analysis (GCINT) where gene expression microarray data from two different cell types can be used to unravel possible means of signaling in the cellular interphase. The analysis is based on a database containing verified receptor ligand pairs and a second database that allows input of normalized gene expression data from two different cell types. The data is then analyzed so to unravel potential means of communication based on matching expression of receptors and ligands. This allowed us to identify novel potential communication pathways, some of which were functionally investigated by in vitro differentiation of hematopoietic progenitors. In addition, the analysis revealed that the presence of blood cells has a direct impact on the gene expression of the stromal cells suggesting that the interaction involve a complex two-way communication.

We here report of how gene expression data can be used to identify molecules involved in cellular synapses. This type of analysis is most helpful for the understanding of complex systems in which cell-cell contact is crucial for the function or development of cells. Our analysis using data from a model stromal cell line and primary hematopoietic cell populations representing different lineages and developmental stages, suggests that even though several communication pathways are shared, we were able to identify potential stage and lineage restricted pathways that may be involved in events such as lineage restriction. Some of these involve known factors such as the IL-7 and CXCL12 pathways, both reported to be important for B cell development [7, 15, 16, 37–39] but the approach taken also allows for the identification of novel factors of potential interest. The methodology opens for a possibility to identify cytokines or surface proteins involved in the regulation of hematopoiesis through comparative analysis of gene expression patterns in functionally different support cells. In this paper we report of such an analysis using fibroblastic NIH3T3 cells, unable to support B-lymphoid development from multipotent progenitor cells, and OP9 cells that are supportive of this process. This allowed us to identify two factors expressed by OP9 but not NIH3T3 cells. Among these has CXCL12 previously been reported to stimulate B cell growth and development [6, 7], however, in our co-culture system, the second candidate, BMP4, were the more potent stimulator of B-cell development (Figure 2). A role for BMP4 in hematopoiesis was has been suggested from studies in xenopus embryos as this factor is important for the specification of the first hematopoietic progenitors [40, 41]. BMP4 has also been shown to stimulate embryonal stem cells from both mouse [42] and humans [43] to undergo development into hematopoietic lineages. BMP proteins have also been shown to stimulate the growth of primitive human hematopoietic stem cells [44] arguing for a functional role on stem cell expansion. Our data suggest that BMP4 also possess the ability to stimulate development of adult progenitors into B-lymphoid cells. We have not investigated the mechanisms involved in effects of BMP4 or selective abilities of the investigated cytokines to support development and expansion of specific cell types but our findings suggest that our genomics approach reveals relevant functional interactions.

The normal BM stroma is composed of a large variety of cell types with potentially highly specific functions in the regulation of hematpoiesis. In these initial studies we have used a model stromal cell line, OP9, with a high potential to stimulate B-lymphocyte development. This has allowed us to obtain ideas about signaling pathways in the bone marrow, however, in order to fully understand the signaling processes we would need to investigate relevant primary stroma cells from mouse BM. The BM is thought to be organized into anatomical niches harboring blood cells of specific developmental stages and possibly lineages. The most immature cells has been suggested to reside in the endosteum where osteoblasts presumably are involved in the regulation and maintenance of the stem cell pool while more mature cells reside closer to the central region of the bone marrow cavity [4, 45–47]. In the case of B-cell progenitors, it has been suggested that they reside in close contact with adventitial reticular cells near to the BM capillaries [8]. Interestingly, it does not appear as if the same stromal cells produce both CXCL12 and IL-7 but that these cytokoines rather are produced by different cells [8]. Thus, in order for the B-cell progenitor to obtain the proper stage specific signals there would exist need for a movement of the developing cells in the BM. This could well be one of the driving forces of cell maturation and niche organization, however, our data suggesting that the stromal cells respond to signals from the hematopoietic cells opens for an alternative or possibly complementary explanation where the cells that in direct contact with the stromal cell stimulates activation of a certain cytokine profile. As the cell mature, the signals from the developing cell changes resulting in new instructions to the stromal cells creating a dynamic system where the progenitors and the stroma directly communicates in order to drive the development of a hematopoietic cell as well as the stroma. In this scenario there would exist plasticity within a certain niche to ensure that all progenitors that enter the compartment are driven to differentiation. Such mechanism could operate on a single cell level, however, since any given stromal cell is likely to be in contact with several progenitor cells at a given time, this would require a synchronized differentiation process and an ability of the stromal cell to return to a ground stage when the hematopoietic cells have left to enter another developmental niche. However, there exist a large need to investigate the plasticity of primary stromal cells or potentially mesenchymal stem cells (MSCs) from the BM. MSCs are multipotent cells capable to differentiate along several pathways to produce for instance adipocytes, chondrocytes and osteoblasts in vitro [48]. The different cell fates can be regulated through modifications of tissue culture conditions and it has been reported that HSCs have an ability to induce osteoblast differentiation of multipotent mesenchymal stem cells, thus contributing to the creation of the stem cell niche [49].

Even if the information generated by genomics based cell interaction analysis represents an approximation, we believe that we have been able to show that this approach allows for the generation of biologically relevant and useful information. The development of protocols that allows for the purification of specific stromal cell populations directly from the bone marrow will allow us to feed even more relevant information into the program and thereby increase our possibilities to identify novel stage and lineage specific cell-cell communication pathways in vivo.