Neuroprotection of host cells by human central nervous system stem cells in a mouse model of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1(-/)(-) mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology.

Engraftment of sorted/expanded human central nervous system stem cells from fetal brain.

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for >10 passages.

Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture.

We have investigated the ability of several cytokine combinations to improve retrovirus-mediated transduction of human primitive hematopoietic progenitors (PHPs) from mobilized peripheral blood (MPB). Retroviral infection of CD34+ cells was performed by culture on fibronectin fragment CH-296 (RetroNectin, RN), using the truncated human nerve growth factor receptor (NGFR) as the transgene reporter. Transgene expression among progeny of PHPs was assayed by FACS analysis after long-term stromal culture (LTC).

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line.

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures.

Recombinant human interleukin-3 (rhIL-3) enhances the mobilization of peripheral blood progenitor cells by recombinant human granulocyte colony-stimulating factor (rhG-CSF) in normal volunteers.

To identify a precisely timed and safe protocol for progenitor cell mobilization, we studied the effects of rhIL-3 and rhG-CSF administration to normal volunteers. rhG-CSF 5 micrograms/kg/d was administered subcutaneously (s.c.

Enrichment of human hematopoietic stem cell activity in the CD34+Thy-1+Lin- subpopulation from mobilized peripheral blood.

The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization.

The effects of interleukin 7 (IL-7) on human bone marrow in vitro.

Interleukin 7 (IL-7) stimulates the proliferation of pre-B cells from long-term murine lymphoid cultures and normal bone marrow. In addition, IL-7 stimulates the proliferation of murine T cells, including fetal and adult thymocytes as well as peripheral T cells. Flow cytometry and cell enumeration analyses were carried out on light-density human bone marrow cells incubated in the presence or absence of IL-7.

Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24.

Human mast cell growth factor (MGF) complementary DNAs (cDNAs) were cloned from HeLa cells using the polymerase chain reaction with oligonucleotides corresponding to murine and human MGF sequences. Sequencing of the cloned human MGF polymerase chain reaction products revealed two types of cDNA: a full length form corresponding in size to the murine cDNA, and an alternately spliced clone with a deletion of the sixth exon of the gene. Since membrane-bound MGF is predicted to be proteolytically cleaved within the sequences encoded by exon 6 to generate a soluble protein, this alternately spliced cDNA would likely encode a noncleavable, membrane-bound form of MGF.

Interleukin 4 alone or in combination with interleukin 1 stimulates 3T3 fibroblasts to produce colony-stimulating factors.

Colony-stimulating activity (CSA) can be produced by fibroblasts when stimulated by interleukin 1 (IL-1). We show that like IL-1, interleukin 4 (IL-4) can stimulate 3T3 fibroblasts to produce CSA. Biological and molecular analyses show that a significant portion of the CSA is colony-stimulating factor 1 (CSF-1) and granulocyte colony-stimulating factor (G-CSF).

Induction of the granulocyte-macrophage colony-stimulating factor (CSF) receptor by granulocyte CSF increases the differentiative options of a murine hematopoietic progenitor cell.

32DC13(G) is an interleukin-3-dependent murine hematopoietic precursor cell line which differentiates into neutrophilic granulocytes upon exposure to granulocyte colony-stimulating factor (G-CSF) but ceases to proliferate and dies when exposed to granulocyte-macrophage (GM)-CSF. Surface receptors for GM-CSF are undetectable on 32DC13(G) cells but can be induced by priming the cells with G-CSF. Exposure of the G-CSF-primed cells to GM-CSF then results in the generation of monocytes as well as granulocytes.

Deletion of carboxy-terminal residues of murine granulocyte-macrophage colony-stimulating factor results in a loss of biologic activity and altered glycosylation.

A deletion mutant of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) which differs in primary structure from native GM-CSF in the carboxy-terminal 11 amino acids was prepared. Four amino acid residues are mutated and the seven terminal residues including Cys-118 are deleted. Supernatants from COS-1 cells transfected with this deletion mutant (GM-CSF(del] showed a 3000-fold decrease in the ability to stimulate bone marrow stem cells to proliferate and differentiate into granulocytes and macrophages in vitro.

Interleukin 1 plus interleukin 3 plus colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid bone marrow cells.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.

Synergistic effects of purified recombinant human and murine B cell growth factor-1/IL-4 on colony formation in vitro by hematopoietic progenitor cells. Multiple actions.

Purified recombinant human B cell growth factor-1/IL-4 was evaluated, alone and in combination, with purified preparations of recombinant human (rhu) CSF or erythropoietin (Epo) for effects on colony formation by human bone marrow CFU-GM progenitor cells (GM) and burst forming unit-E progenitor cells. rhu IL-4 synergized with rhu G-CSF to enhance granulocyte colony formation, but had no effect on CFU-GM colony formation stimulated by rhu GM-CSF, rhu IL-3, or rhu CSF-1. Rhu IL-4 synergized with Epo to enhance BFU-E colony formation equal to that of Epo plus either rhu IL-3, rhu GM-CSF, or rhu G-CSF.

Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein.

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol.

Human macrophage-colony stimulating factor: alternative RNA and protein processing from a single gene.

Macrophage-colony stimulating factor (M-CSF, CSF-1) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, two human M-CSF cDNA clones were isolated encoding proteins of 256 and 554 amino acids. We report here the isolation of a third M-CSF cDNA that encodes a protein of 438 amino acids.

Interleukin 1 regulates hematopoietic activity, a role previously ascribed to hemopoietin 1.

A murine in vitro assay was developed to measure potentiation of a proliferative response to suboptimal concentrations of the hematopoietic regulatory molecule granulocyte/macrophage colony-stimulating factor by an immature bone marrow population. The assay, designated the 5-fluorouracil bone marrow proliferation assay, was used to characterize potentiating activity in serum-free culture supernatants of the human tumor cell line HBT 5637. Molecular and biochemical analyses indicated that the HBT 5637-derived potentiating activity could be attributed to interleukin 1 alpha.

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3.

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375.

Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor.

Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages. In this report we have used a mouse GM-CSF cDNA clone to isolate human GM-CSF clones from libraries made from HUT-102 messenger RNA and mitogen-stimulated T-lymphocyte messenger RNA. The human cDNA clones contained a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and showed 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF.

The regulation of mononuclear phagocyte entry into S phase by the colony stimulating factor CSF-1.

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. Populations of adherent bone marrow-derived macrophages (BMM) devoid of CSF-1 producing cells were used to study regulation by CSF-1 of macrophage entry into S phase. More than 95% of BMM possess the CSF-1 receptor.

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1).

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.

Ribosomal proteins are synthesized preferentially in cells commencing growth.

Mouse 3T3 cells, in stationary phase because of serum deprivation, have only half the ribosome content of growing cells. Furthermore, the proportion of protein synthesis devoted to ribosomal proteins is only half that in growing cells. On addition of serum the synthesis of each ribosomal protein increases threefold, demonstrating the coordination of the synthesis of the ribosomal proteins.

Survival of mononuclear phagocytes depends on a lineage-specific growth factor that the differentiated cells selectively destroy.

CSF-1 is a hemopoietic growth factor that specifically causes the proliferation and differentiation of mononuclear phagocytic cells. Receptors for CSF-1 occur exclusively on cells of the mononuclear phagocytic series (precursor leads to monoblast leads to promonocyte leads to monocyte leads to macrophage). Studies of the actions of CSF-1 on freshly explanted macrophages have been complicated by contamination of the primary cell isolates with CSF-1-producing cells and by the heterogeneity of the proliferative responses of individual macrophages.