We study differentiation of lymphoid cells as a model system for analyzing stem cell propagation and differentiation. This model allows us to examine the roles of cell-cell contact and exogenous growth factor signaling between a hematopoietic stem or progenitor B cell population and a stromal cell population of mesenchymal origin. We also study the process of cellular transformation in vitro and in vivo. These studies will help CBER regulate products such as hematopoietic, mesenchymal, neuronal, and more pluripotent stem cells. We have discovered and are elucidating the role of the dlk molecule in cell-cell interactions between stromal cells and progenitor B (proB) cells. Dlk is a member of the EGF-like homeotic gene family which influences cell fate during development. Our work has shown that modulation of the level of dlk expression on stromal cell surfaces can change the growth requirements and differentiation of proB cells in contact with them. Removal of IL-7 from proB cells interacting with normal storma with normal dlk levels leads to rapid differentiation followed by apoptotic cell death. In contrast, interaction with stromal cells with decreased dlk expression allows continued proB cell growth and does not lead to cell differentiation in the absence of IL-7. We are currently studying how cell-cell contact changes the growth factor requirements of proB cells, how the dlk molecule on the surface of the stromal cell changes gene expression in the proB cells, and how this interaction changes the apoptotic signal pathway that is normally induced in proB cells after removal of the growth factor IL-7. RT-PCR assays show increases in Notch-3 gene expression in proB cells grown without IL-7 but on stroma with low levels of dlk. This result suggests that dlk affects Notch signal transduction pathways in progenitor cells in the hematopoietic lineage. In addition, Dr. Bauer's group has recently used gene knock-out technology to derive a mouse strain lacking dlk. The dlk knock-out strain is of small stature from birth. Since the dlk gene is imprinted and fetal expression is paternally controlled, even heterozygous animals carrying the paternal knockout allele are affected. Since in vitro studies showed that dlk affects proB cell growth requirements, an investigation of B cell development and function in the knock-out mice is underway. Knock-out mice show expanded populations of several B-cell differentiation stages and serum immunoglobulin levels are elevated. Currently, a study of immune responses to T-dependent and T-independent antigens is underway to assess the function of the B-cell compartment. This animal model will provide much insight into cell-cell interactions in fetal development. The influence of growth factors on stem cell populations is also studied in this model system. We are currently studying the effects of TGF-beta in our progenitor cell culture system. TGF-beta is a member of a gene family that profoundly affects differentiation and includes the activins, inhibin, and bone morphogenic proteins (BMPs). We observed that normal proB cells express TGF-beta receptors and are inhibited by exogenous TGF-beta. TGF-beta causes accumulation of proB cells in G1 stage of the cell cycle, stops proliferation, but does not induce apoptosis or differentiation. IL-7 can overcome these effects even in the continued presence of TGF-beta. We will continue to study this as a useful model relevant to the complex patterns of growth and differentiation control exercised by exogenous growth factors in stem cell cultures. Our model system is also being used to study the role of oncogene dysregulation and anti-oncogenes in malignant transformation of a progenitor cell population. We have demonstrated that proB cells can be infected and transformed in vitro by viruses carrrying c-myc with either v-abl or v-raf. We have also examined the role of oncogene mediated subversion of IL-7 signal transduction and found that, in contrast to abl-myc virus, the raf-myc virus can transform pre-B cells without constitutive activation of IL-7 JAK-STAT signaling. ProB cells transformed by either virus lose sensitivity to TGF-beta despite continued expression of receptors. These results show that abrogation of normal signals from both positive and negative growth factors can contribute to malignant transformation.
