A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells

Journal of Immunology Research, Aug 2018

The selective in vitro generation of rat, mouse, and human terminal deoxynucleotidyl transferase-positive (TdT

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

http://downloads.hindawi.com/journals/jir/1993/013675.pdf

A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells

A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells SEAN D. McKENNA" IRVING GOLDSCHNEIDER The selective in vitro generation of rat, mouse, and human terminal deoxynucleotidyl transferase-positive (TdT lymphoid cells in our long-term xenogeneic bone marrow (BM) culture system is characterized by physical interaction between the developing lymphocytes and mouse BM-adherent stromal cells and macrophages. In the present study, experiments in which micropor)us membrane culture inserts were inoculated with rat BM cells demonstrated that although the generation of primitive B-lineage lymphoid cells requires the presence of a mouse BM feeder layer, cognitive recognition events are not necessary. Similarly, cell-free (and serum-free) medium conditioned with mouse BM (but not thymus or spleen) adherent cells and stromal-cell lines therefrom supported the proliferation of early rat lymphoid cells in a dose-dependent manner. Double immunofluorescence for incorporated bromo-deoxyuridine (BrdU) and early B-lineage markers of rat BM lymphoid cells maintained in culture inserts or conditioned medium (CM), and studies of their in vitro and in vivo developmental potentials, indicated that the lymphoproliferative response resulted from the selective stimulation of lymphoid stem and/or progenitor cells. The most primitive of these target cells had a HIS24 HIS50- TdT- c/- sIg-, pre-pro-B-cell phenotype. Whereas this subset normally constitutes less than 2% of B-lineage BM cells in vivo, it comprises more than 25% of total lymphoid cells in vitro. In addition, the number of TdT cells, predominantly of the early pro-B-cell phenotype (HIS24 HIS50-c/-sIg-), was increased approximately tenfold above input levels. Based on these and previous findings, a schematic model is proposed for the developmental pathway of early B-lineage cells in rat BM from the level of the committed (possibly common) lymphoid stem cell to that of the pre-B-cell. Terminal transferase (TdT); pro-B cell stimulating factor; in vitro culture system; bone marrow stromal cells INTRODUCTION The growth and differentiation of lymphoid cells from stem and progenitor cells are thought to be regulated by cell-cell contact as well as by the release of soluble factors (reviewed in Kincade et al., 1989; Dorshkind, 1990) . A major technical advance in the study of these cellular and biochemical events in lymphopoiesis has been the development of long-term, feeder layer-depetdent, in vitro bone marrow (BM) culture systems, in which manipula"Corresponding author. tion of culture conditions profoundly affects the lineages and/or stages of development that are generated. For example, Dexter-type cultures selectively produce myeloid lineage cells and multipotential stem cells, but few lymphoid cells (Dexter et al., 1977) , whereas the Whitlock-Witte culture system selectively produces large numbers of pre-B cells (c/+, sIg-), but not myeloid or multipotent stem cells (Whitlock and Witte, 1982; Whitlock et al., 1984) . Recently, modifications to the WhitlockWitte culture system have been described that permit the generation of B-lineage cells more primitive than those observed in standard cultures. These modifications include seeding the BM feeder layers with mouse fetal liver cells (Denis et al., 1984, 1987) , transfecting the cultured ells with the BCR/ ABL chimeric oncogene (Scherele et al., 1990), and initiating the culture in the presence of interleukin-4 (Peschel et al., 1989) . The role of oluble factors in the generation of pre-B-cells has been extensively studied using bone marrow stromal cell lines established from the foregoing cultures. In particular, interleukin-7 (IL-7), a cytokine first purified from mouse BM stromal cells, has been found to stimulate the proliferation of pre-B (B220 /) and possibly pro-B (B220-) cells from mouse BM (Namen et al., 1988a,1988b; Henney, 1989; Lee et al., 1989) . However, despite the effect of IL-7, the long-term survival of early B-lineage cells in primary cultures generally is not maintained in the absence of direct contact with BM stromal cells (Kierney and Dorshkind, 1987; Sudo et al., 1989) . This is consistent with the development of foci of proliferating lymphoid precursor cells on and within the adherent BM stromal-cell layer (Whitlock and Witte, 1982; Whitlock et al., 1984; Hayashi et al., 1984; Medlock et al., 1993a, 1993b) . Hardy et al. (1991) have reported that the earliest phenotypically distinct mouse B-lineage cell population, called "pre-pro-" B-cells (B220 $7/ BP-I-, HSA-, Ig genes in germline configuration), requires only contact with BM stromal cells to survive in a 4-day coculture system. However, later stages of lymphopoiesis, marked by expression of HSA and progressive Ig gene rearrangements, were increasingly (...truncated)


This is a preview of a remote PDF: http://downloads.hindawi.com/journals/jir/1993/013675.pdf

Sean D. Mckenna, Irving Goldschneider. A Selective Culture System for Generating Terminal Deoxynucleotidyl Transferase-Positive Lymphoid Cells In Vitro. V. Detection of Stage-Specific Pro-B-Cell Stimulating Activity in Medium Conditioned by Mouse Bone Marrow Stromal Cells, Journal of Immunology Research, 3, DOI: 10.1155/1993/13675