Ross S. Basch

Immortalized Multipotential Mesenchymal Cells and the Hematopoietic Microenvironment

In an attempt to analyze the cellular and molecular basis of the capacity of bone marrow stromal cells to support hematopoiesis in culture, we developed a series of murine stromal cell lines from a single long-term bone marrow culture (BMC). The cytokines produced by these cells were analyzed using immunohistochemical techniques, ribonuclease protection assays (RPA) and RT-PCR. We examined the capacity of these cloned cell lines to replace primary bone marrow-derived stromal cells in long-term bone marrow cultures (LT-BMC) and sought correlations between the capacity to support hematopoiesis in culture with the production of known cytokines. These immortalized lines replicate many of the functions of the hematopoietic microenvironment. They express cytokines known to play a role in hematopoiesis. All of the lines constitutively express mRNA for PBSF (SDF-1), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), FLT-3, thrombopoietin (TPO), interleukin 7 (IL-7), leukemia inhibitory factor (LIF), tumor necrosis factor-beta (TNF-beta), and interferon-gamma (IFN-gamma). Most lines also express granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF. They vary in their expression of IL-6, tumor growth factor-beta1 (TGF-beta1), TGF-beta2, and TNF-alpha. Growing these lines in the presence of cytokines that influence hematopoiesis alters the levels of cytokine message. The most striking effects were produced by TNF-alpha. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as osteoblast-specific factor-2 (OSF-2) and bone morphogenetic protein-1 (BMP-1). They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Several of the lines can maintain hematopoiesis in culture, as measured by the continuous production of myeloid colony-forming cells (CFU-c), for months. This capacity to support hematopoiesis does not correlate with any pattern of cytokine expression. Several of these lines also support the growth of human hematopoietic cells, and human CFU-c can be detected in the cultures in which CD34(+) bone marrow cells (BMC) are cultured on murine stromal cells. No correlation between the production of any of the known cytokines and the ability to support murine hematopoiesis was detected. In addition, there was no correlation between the capacity to support murine hematopoiesis and the capacity to maintain human HSC. Despite repeated cloning, the lines remain heterogeneous and are capable of producing cells with the properties of fibroblasts, osteoblasts, adipocytes, and myoblasts. In addition to the cytokine mRNAs, the cell lines express factors associated with bone formation such as OSF-2 and BMP-1. They also express the neural cell-adhesion molecule neuropilin and neurotrophic factors including NGF and BDNF.

The Growth Arrest-Specific Gene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-l-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.

Hematopoietic thymocyte precursors: III. A population of thymocytes with the capacity to return (“home”) to the thymus☆

Abstract A subpopulation of thymocytes with the capacity to return (“home”) to and repopulate the thymus of an irradiated mouse has been identified. These cells differ from the majority of cortical thymocytes in that they are of a lower buoyant density and have higher cortisone and X-ray resistance. They also bear an antigen common to mouse brain but not found on the majority of cortical thymocytes. This subpopulation is derived from a hematopoietic thymocyte progenitor and provides an intrathymic reserve of thymocyte precursors. It appears to be a non self-sustaining “transit” population in the pathway of T-cell development.

Amplification of the biotin-avidin immunofluorescence technique

An amplification of the immunofluorescence technique which uses biotinylated antibody and fluoresceinated avidin is described. By introducing a sandwich technique using fluorescein-conjugated goat anti-avidin, a 5-fold enhancement of staining over the conventional immunofluorescence method is achieved, and the brightness is more than twice that achieved with the simple biotin-fluoresceinated avidin reaction.

ANTIGENIC AND FUNCTIONAL EVIDENCE FOR THE IN VITRO INDUCTIVE ACTIVITY OF THYMOPOIETIN (THYMIN) ON THYMOCYTE PRECURSORS

Thymopoietin, a polypeptide hormone isolated from bovine thymus, induced in vitro the differentiation of prothymocytes to cells with the antigenic and functional characteristics of intrathymic thymocytes. These changes included the acquisition of the differentiation antigens TL and Thy-1 (theta) and the ability to respond to the mitogen Con-A. Thymopoietin appears to be highly speicfic in inducing the prothymocyte to be highly specific in inducing the prothymocyte to thymocyte differentiation and does not affect the pluripotential stem cell measured by the colony forming assay (CFU-S), the erythropoietin-sensitive cell or B-cells. Experiments are in progress to determine whether additional hormonal inductive signals are required to complete the differentiation of an immunologically competent T-cell.

Thymopoietin-induced acquisition of responsiveness to T cell mitogens

Abstract A population of murine spleen cells, enriched by flotation in discontinuous bovine serum albumin gradients, was induced to differentiate in vitro by incubation with the purified thymic polypeptide hormone thymopoietin. These cells, normally unresponsive to both T and B cell mitogens, acquired the capacity to respond to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) but remained unresponsive to the B cell mitogen lipopolysaccharide from Escherichia coli . The acquisition of responsiveness to mitogens was not impaired by treatment with anti-Thy-1 serum + complement before induction but was prevented by this treatment after induction; thus the cells acquiring the functional capacity to respond to T cell mitogens had also been induced to express the T cell alloantigen Thy-1. Like the expression of T cell alloantigens, the capacity to respond to Con A developed rapidly and reached its maximum within 6 hr. Responses to Con A always greatly exceeded those to PHA. Our data suggest that committed precursor cells, which we believe to be prothymocytes, are induced by thymopoietin to differentiate to cells with an antigenic phenotype and mitogen responsiveness similar to cortical thymocytes.

TBLR1 regulates the expression of nuclear hormone receptor co-repressors.

BackgroundTranscription is regulated by a complex interaction of activators and repressors. The effectors of repression are large multimeric complexes which contain both the repressor proteins that bind to transcription factors and a number of co-repressors that actually mediate transcriptional silencing either by inhibiting the basal transcription machinery or by recruiting chromatin-modifying enzymes.ResultsTBLR1 [GenBank: NM024665] is a co-repressor of nuclear hormone transcription factors. A single highly conserved gene encodes a small family of protein molecules. Different isoforms are produced by differential exon utilization. Although the ORF of the predominant form contains only 1545 bp, the human gene occupies ~200 kb of genomic DNA on chromosome 3q and contains 16 exons. The genomic sequence overlaps with the putative DC42 [GenBank: NM030921] locus. The murine homologue is structurally similar and is also located on Chromosome 3. TBLR1 is closely related (79% homology at the mRNA level) to TBL1X and TBL1Y, which are located on Chromosomes X and Y. The expression of TBLR1 overlaps but is distinct from that of TBL1. An alternatively spliced form of TBLR1 has been demonstrated in human material and it too has an unique pattern of expression. TBLR1 and the homologous genes interact with proteins that regulate the nuclear hormone receptor family of transcription factors. In resting cells TBLR1 is primarily cytoplasmic but after perturbation the protein translocates to the nucleus. TBLR1 co-precipitates with SMRT, a co-repressor of nuclear hormone receptors, and co-precipitates in complexes immunoprecipitated by antiserum to HDAC3. Cells engineered to over express either TBLR1 or N- and C-terminal deletion variants, have elevated levels of endogenous N-CoR. Co-transfection of TBLR1 and SMRT results in increased expression of SMRT. This co-repressor undergoes ubiquitin-mediated degradation and we suggest that the stabilization of the co-repressors by TBLR1 occurs because of a novel mechanism that protects them from degradation. Transient over expression of TBLR1 produces growth arrest.ConclusionTBLR1 is a multifunctional co-repressor of transcription. The structure of this family of molecules is highly conserved and closely related co-repressors have been found in all eukaryotic organisms. Regulation of co-repressor expression and the consequent alterations in transcriptional silencing play an important role in the regulation of differentiation.

Quantitative studies on histocompatibility antigens of the mouse.

A substantial fraction of the H‐2 determined isoantigens of the C57BL/6 mouse have been found to be closely associated with the acid phosphatase‐rich lysosomal fraction of the cell. These antigens occur in high concentration in organs of the RES and undergo marked increase during “activation” of this system. A hypothesis is proposed that these antigens are especially abundant in a system of organelles which mediate the secretory and phagocytic functions of various cells. Antigen was detectable by a serologic technique in the tissues of newborn animals. On the first day of life the antigen concentration of the liver exceeded that of the spleen, but the more rapid increase in splenic activity resulted in a reversal of these relative specific activities on the 5th‐7th days. Adult levels were not attained until the 28th day of life.

Expression of CD41 and c‐mpl does not indicate commitment to the megakaryocyte lineage during haemopoietic development

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+CD41+) were isolated from normal human bone marrow or induced in culture from CD34+CD41− bone marrow cells by treatment with thrombopoietin (TPO) or IL‐3. We found that although this population included the majority of cells that can form CFU‐MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+CD41+‐induced cells was greater than that of isolated CD34+CD41+ cells in that the isolated cells only formed CFU‐MK and BFU‐e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU‐e. A major portion of the cells that expressed CD34+CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c‐mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+CD38− cells), expressed c‐mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c‐mpl was predictive of commitment to the MK lineage.

Complementary and antagonistic effects of IL‐3 in the early development of human megakaryocytes in culture

The effect of IL‐3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41+ MK from pre‐MK, the number of cells produced was greatly augmented by the addition of IL‐3 and SCF. IL‐3 was also able to support the growth of MK colonies in semi‐solid media (CFU‐MK). The CD41+ cells that developed in suspension cultures containing IL‐3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL‐3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb‐IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL‐3. The inhibitory effects of IL‐3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL‐3 did not stimulate MK growth. The results obtained in these cultures suggest that IL‐3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL‐3 results in the appearance of cells that do not mature normally.