Stem cells can respond to growth factors from surrounding fluids or signals from molecules on the surface of adjacent cells. These interactions can cause stem cell self-renewal or initiate differentiation of stem cells into many different cell and tissue types. Our laboratory investigates the role of cell-cell communication in progenitor-B cells which can self-renew, differentiate, or undergo programmed cell death in response to secreted factors or contact-mediated signals coming from adjacent stromal cells. Soluble growth factors and cell-cell contact with stromal cells are both required to maintain viable progenitor-B cells. We are examining effects on progenitor-B cell growth, differentiation, and neoplastic transformation mediated by the antagonistic signals from two secreted factors, IL-7 (cell growth) and TGF-beta (cell cycle arrest). We also focus on a stromal cell surface molecule, dlk, which affects how the precursor-B cells respond to growth factors and programmed cell death. Stromal dlk molecules appear to modulate the Notch-Delta signaling pathway in the precursor B cells. In diverse organisms from mouse to fruit fly, this pathway is involved in cell fate determination. We also use knock-out mice with a homozygous deletion in the dlk gene to study effects on fetal development and lymphoid cell differentiation. In addition, previous experiments have suggested that CD45 may be involved in macrophage and neutrophil cell migration. We have been exploring methods such as RNAi to modulate CD45 expression to evaluate its role in and cell migration. Finally, we have developed methods for the generation of purified, synthetic RNA for evaluation as a reference material for microarray analysis. The goal is to develop robust, reproducible techniques for gene expression measurements in cell product development and characterization. Growth factors and cell-cell interactions governing growth, differentiation, and transformation of B-lineage progenitor population. We study differentiation of lymphoid 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. Finally, we study the role of cell surface molecules in chemotaxis of macrophages as a model for cell-cell interaction effects on chemotaxis. 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, we have 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. Immune responses to T-dependent antigens are abnormal. This result also stimulated investigations into T-cell development and function. 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 carrying 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. Role of CD45 in Phagocyte Motility and Signal Transduction. The leukocyte common antigen (CD45) family is a group of high molecular weight single transmembrane glycoproteins with cytoplasmic protein tyrosine phosphatase domains expressed on the plasma membranes of all leukocytes. Recent information regarding CD45 function indicates that antibody reagents to certain CD45 isoforms have potent immunosuppressive activities and may contribute to the immunosuppressive action of therapeutic agents such as anti-thymocyte globulin which is administered to transplant recipients for the prevention of graft-versus-host-disease and graft rejection. We have previously shown that certain extracellular domain epitopes of CD45 are involved in human neutrophil chemotaxis. We have pursued these observations using a genetic approach and mutagenized a murine macrophage cell line to obtain variant clones with low or undetectable CD45 expression. We found that cell line variants lacking CD45 expression were defective in their chemotactic response to recombinant human C5a, however, the cells exhibited normal random motility. These observations suggested that CD45 may be involved in directional (chemotactic) migration which is an essential host defense response to inflammatory stimuli. To clarify the role of CD45 in phagocyte chemotaxis, we are attempting to isolate a CD45 knockout in the RAW264 macrophage cell line. We initially constructed a targeting vector in which exon 12 was disrupted by a neomycin gene having its own heterologous promoter and polyadenylation signal. PCR screening of over 3000 clones indicated that no homologous recombination event had occurred. We have now constructed a promoterless construct and have shown that transfectants can be isolated. The transfectants are observed at >100-fold lower frequency, demonstrating that a more stringent selection has been achieved. PCR screening of pools of transfectants generated from the promoterless construct indicated that two pools contained clones with homologous recombinants. Clones from one of the pools have been isolated and positive clones identified by PCR. The positive clones are now being analyzed by Southern blotting to confirm the correct insertion into the genome. We plan to use a similar approach to isolate a double knockout and to compare chemotactic and other properties of the parent, single knockout and double knockout cells. The knockout and parent cells may be used to evaluate expression systems for cell activation and/or motility genes.
