A. Microenvironmental guidance of stem cell self renewal, differentiation, localization, plasticity and de-differentiation
Stem
cell fate decisions are influenced by the microenvironment in which the stem
cell resides. One key component of such microenvironments is the mesenchymal
stroma. Our studies focus on the interactions between hemopoietic stem cells
and the mesenchymal stroma of the bone marrow and other tissue. Our past
studies have highlighted the plasticity of the mesenchymal cell phenotype.
These cells not only form the regulatory microenvironment of the bone marrow,
they also constitute a remarkable pool of stem cells by themselves, in the
embryo and in the adult. They can give rise to a vast spectrum of mature cell
types, and are, therefore, multipotent. Our studies are therefore directed towards the analysis of the molecular
constitution of multipotent stromal cells (MSC) and the mechanistic basis of
their plasticity and capacity to de-differentiate.. Furthermore, we examine the mode of action by which MSC
affect the fate of other cells and influence the development of cancer.
As indicated above, one major function of MSC is to produce the stromal microenvironment that supports hemopoiesis. The mechanism of this stromal cell function is not yet fully understood. We are studying the interactions of stromal cells with stem cells and the subsequent differentiation of these stem cells into maturing B lymphocytes. Based on in vitro studies, evidence was generated in our laboratory implicating stromal activin A, a transforming growth factor (TGF)β family member, in negative regulation of B lymphopoiesis. Activin A reduced the differentiation of bone marrow derived cells towards to B lineage. Accumulation of pre-proB and proB cells occurred and the generation of preB cells was reduced. We are now examining (a) whether activin A operates at the HSC level or downstream in the cascade and (b) how would modification of the microenvironment in vivo, by use of genetically manipulated mesenchymal stem cells, overexpressing different members of the TGFβ superfamily, affect B cell generation .
One immediate consequence of this study is the implication that activin A may affect not only normal B cells but also tumor B lineage cells. Multipe myeloma is a human disease of this cell lineage which is incurable to date. The pathogenesis of multiple myeloma in men shows that the tumor cells are dependent on the bone marrow mesenchymal microenvironment. This is due, in part, to interleukin (IL)-6, a survival factor for myeloma cells, produced by the mesenchymal stroma. We previously showed that activin A is highly potent in blocking cell cycle progression and in inducing apoptotic death of myeloma cells. We further showed that activin A is an antagonist of IL-6 and thereby kills myeloma cells by depriving them of their survival signals. We propose that overexpression of activin A in mesenchymal cells, within the microenvironment of the hemopoietic organs, may lead to regression of myeloma. We noted that mouse myeloma (MM) tumors formed by RPC-5 cells in BALB/c mice, regressed spontaneously. We used bicistronic retroviral vectors for introduction of activin A and green fluorescence protein (GFP) cDNAs into mouse mesenchymal cells. We are now using a model of MM which resembles human MM in tropism to the bone marrow and in inducing bone damage, as a preclinical animal model to determine whether cell therapy using activin A modified mesenchyme may be used in humans.
Activin A is thus one mesenchymal molecule that mediates several major functions relating to the regulation of blood cell proliferation and differentiation.
B. The stem state
Some of the molecules expressed by the mesenchyme are surprisingly shared by mature cells from unexpected lineages. In a recent study performed in our laboratory, we identified a novel T cell receptor (TCR)β transcript that lacks the variable region and possesses an extra 5’ sequence derived from the presumptive intronic Jβ2.6 sequence (hence intronic J2β.6). This transcript is expressed by mesenchymal cells, specifically by mouse embryonic fibroblasts (MEFs) and by stromal cell lines. Additional studies have shown that a recombinant GFP-Jint-Jβ2.6-Cβ2-TCR fusion protein can be overexpressed in 293T cells and in MEFs. Such overexpression leads to apoptotic death of the cells. It is not yet known what might be the role of this protein in those cells. However, our recent studies indicate that the mesenchymal TCR may be involved with cell growth regulation and tumorigenesis. The promiscuous gene expression is not restricted to the TCR. In fact, our studies show that MSCs express many gene families that supposedly "belong" to other lineages. This may put them in a stand-by state, ready at any time to differentiate and give rise to many mature sell types. Based on these studies, the theory of the "stem state" was formulated.
Current projects in our laboratory:
- A cell and gene therapy model for multiple myeloma.
- Mesenchymal stem cell (MSC) differentiation.
- Regulatory T cells in multiple myeloma.
- Control of MSC growth by toll-like receptor/ligand interactions.
- Incomplete TCR: expression in mesenchyme and functional significance in T Cells.
- MSC plasticity and de-differentiation.
Selected publications:
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