Human T-lymphocyte products stimulate human hemopoietic progenitor cell proliferation in diffusion chambers in vivo

Human myeloid colony formation in diffusion chambers in mice (CFU-DG) was enhanced following administration of a human T-cell-line-derived conditioned medium (Mo). The Mo cell line also elaborates activities stimulating human myeloid colony formation in vitro in agar (CSF) and potentiating erythroid colony formation in vitro in methylcellulose (EPA). Depletion of CSF from Mo conditioned medium by heat inactivation or gel exclusion chromatography did not affect CFU-DG formation. EPA and CFU-DG stimulating activities are heat stable and have approximately the same molecular weight. Culture of human bone marrow cells in diffusion chambers in mice for 4 days under the influence of Mo conditioned medium resulted in significant increment of BFU-E and CFU-DG as judged by subculture of diffusion chamber contents. No effect on CFU-C could be detected.     

Spleen and hematopoiesis

In order to investigate the role of the human spleen on hematopoiesis, hematopoietic stem cells and stimulates were evaluated in fetal and adult spleens. BFU-E and CFU-C were existed in 20 weeks and 23 weeks fetal spleens (BFU-E 145 +/- 45/10(5) mononuclear cells, CFU-C 55 +/- 6/10(5) mononuclear cells). In adult spleen, a few stem cells were recognized, which may be contaminated from peripheral blood in sinus of the spleen. We tested conditioned media from adult spleen cells for the stimulative activity on the in vitro growth of BFU-E and CFU-C from bone marrow mononuclear cells. Spleen conditioned medium stimulated proliferation of these precursor cells. It seemed that PHA-stimulated spleen conditioned medium augmented BFU-E, whereas CFU-C growth was suppressed. Adult and fetal spleens were studied immunohistochemically using anti-G-CSF, GM- CSF and erythropoietin antibodies. The cells with G-CSF and GM-CSF were shown in fetal spleens. In adult spleens, however, only GM-CSF was detected.     

Two different types of granulocyte colony-stimulating factor produced by bovine lung tissue

Summary Bovine lung tissue produces two different types of granulocyte colony-stimulating factor (CSF). The high molecular weight (MW) type (CSF-F) of 70,000 d by Sephadex G-100 gel filtration is only found in conditioned medium of homogenized tissue indubated in sealed glass bottles. This species of CSF exclusively stimulates CFU-C of mouse bone marrow, human bone marrow only hardly. The low MW type CSF (CSF-M) of approximately 29,000 d by gel filtration is found mainly in conditioned medium of slightly minced tissue incubated in Petri dishes. It stimulates both human and mouse CFU-C. Methods to prepare both types of CSF are described. By propagating a fibroblast cell line from bovine lung tissue it was found that fibroblasts are the source of the 70,000 d CSF. Indirect evidence suggests that macrophages produce the 29,000 d CSF species.Abbreviations BLCM bovine lung conditioned medium - CFU-C colony forming units in culture - CM conditioned medium - CSF colony stimulating factor - CSF-F colony stimulating factor produced by fibroblasts - CSF-M colony stimulating factor produced by macrophages - MLCM mouse lung conditioned medium - MW molecular weight - RPMI Roswell Park Memorial Institute (medium) - PBS phosphate balanced salt (solution) - SDS sodium dodecyl sulfate     

Characterization of factor-producing and factor-dependent clones from long-term hamster bone marrow suspension cultures

Long-term hamster bone marrow (BM) cultures produce stem cells that can be grown in the absence of an adherent layer and without addition of exogenous growth factors or hormones. Cloning of these three- to five-month-old suspension cultures by the limiting dilution method generated both factor-dependent (FD) and factor-producing (FP) cell lines. The FP clones have been in culture for two years and are composed of macrophages by morphologic and histochemical criteria. FP serum-free conditioned medium (FPCM) produces both stimulatory and inhibitory effects on hematopoiesis. Addition of 1%-5% FPCM to fresh or cultured hamster BM cells stimulates both CFU-C and erythropoietin (epo)-dependent BFU-E colony formation but not CFU-GEMM in semisolid media, as well as increased numbers of differentiating myeloid and erythroid cells in suspension. In contrast, addition of 10%-15% FPCM produces substantial inhibition of epo-stimulated erythropoiesis. FD cells have been in culture for over 12 months. They exhibit an absolute requirement for the continued presence of 5% hamster spleen conditioned medium (SCM). The clones generate varying numbers of "blast" cells, myeloid and macrophage elements, and occasional mastlike cells. Addition of increasing amounts of SCM produces a dose-dependent proliferation and differentiation of FD cells and also stimulates CFU-C but not BFU-E or CFU-GEMM colony formation. FD cells will also respond to FPCM, but concomitant addition of SCM and FPCM generates four times more cells than either CM alone. These results suggest that the hamster suspension cultures contain both stem cells and distinct regulatory cells that stimulate the growth and differentiation of myelopoiesis.     

Inhibition against CFU-C and CFU-E colony formation by soluble factor(s) derived from hairy cells

The inhibitory effect by hairy cell conditioned medium (HCCM) on the growth of granulocyte and erythrocyte colony forming cells was studied in vitro. The percent inhibition of CFU-C formation by HCCM from four hairy cell leukemia (HCL) patients ranged from 36% to 76%, while no inhibition was observed with conditioned medium (CM) obtained from three B-cell chronic lymphocytic leukemia (B-CLL) patients nor from two normal controls. HCCM inhibited specially the growth of rG-CSF responding stem cells. The hairy cell-derived colony inhibitory factor from HCCM was nondialyzable, fairly stable to heat treatment, and trypsin sensitive. Its maximal inhibitory activity against granulopoiesis was observed in the fractions of 5,000 to 6,000 daltons. Moreover HCCM inhibited CFU-E colony formation but not BFU-E. These results indicate that hairy cells produce a factor that inhibits granulopoiesis and erythropoiesis in vitro. This factor may play a role in neutropenia and anemia observed in HCL.     

Influence of murine mast cell growth factor (c-kit ligand) on colony formation by mouse marrow hematopoietic progenitor cells.

Purified natural and recombinant murine mast cell growth factor (MGF, a c-kit ligand) were evaluated alone and in combination with other cytokines for effects in vitro on colony formation by multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells from BDF1 mouse bone marrow. Both preparations stimulated Epo-dependent CFU-GEMM and enhanced Epo-dependent BFU-E colony numbers and size. MGF had some stimulating activity for CFU-GM. When used in combination with plateau concentrations of pokeweed mitogen mouse spleen cell conditioned medium or granulocyte-macrophage colony stimulating factor (CSF), MGF enhanced in greater than additive fashion colony formation by CFU-GM. MGF also enhanced the size of colonies formed, an enhancement greatest for colonies containing granulocytes and macrophages. MGF did not enhance Macrophage-CSF stimulated colony numbers or size. MGF seems to be an early acting cytokine with preferential effects on the growth of more immature hematopoietic progenitor cells.     

Adherent lymphokine-activated killer cells suppress autologous human normal bone marrow progenitors

We have generated a homogeneous population of recombinant interleukin-2 (rIL-2)-stimulated effector cells termed adherent lymphokine-activated killer cells (A-LAK) from peripheral blood mononuclear cells (PBMNC) of 14 normal individuals and tested the effect of A-LAK cells on autologous hematopoietic bone marrow (BM) progenitor growth. Enrichment of A-LAK from PBMNC depended on the propensity of A-LAK precursors to adhere to plastic and proliferate in the presence of rIL-2. The resultant population had the morphologic appearance of large granular lymphocytes, and the majority of cells (73% +/- 4%) expressed the CD56+/CD3- phenotype associated with rIL-2-stimulated natural killer (NK) cells. The A-LAK population had potent lytic activity in chromium release assays against both NK-sensitive (K562) and NK-resistant (Raji) targets. When BM mononuclear cells (BMMNC) were coincubated with autologous A-LAK and rIL-2 (1,000 U/mL) added at the start of culture, dose-dependent suppression of burst-forming unit-erythroid (BFU-E) and colony-forming unit mix (CFU-MIX) colony growth was observed at effector to target ratios (E:T) ranging from 0.25:1 to 5:1 (maximal suppression BFU-E = 85% +/- 6%; CFU-MIX = 95% +/- 3%). This suppression was rIL-2 dose-dependent, and no suppression was seen in the absence of rIL-2. Depletion of BM monocytes and T lymphocytes did not alter A-LAK suppression of progenitors coincubated with A-LAK cells. Addition of polyclonal neutralizing antibodies against both interferon-gamma (IFN-gamma) and tumor necrosis facto alpha (TNF-alpha) to the coincubation culture completely abrogated the suppressive effect of A-LAK on BFU-E and CFU-MIX colony growth while each neutralizing antibody used alone had intermediate effects. In contrast to coincubation studies, 36 hours of preincubation of A-LAK cells with autologous BM (E:T 2.2:1) and rIL-2 (1,000 U/mL) followed by plating of preincubated BM cells in hematopoietic progenitor culture produced significant suppression of day 14 BFU-E (47% +/- 5%), but spared the more primitive CFU-MIX (7% +/- 9%), suggesting a divergent effect of A-LAK cells on hematopoietic progenitors at different stages of differentiation. Addition of neutralizing antibodies against IFN-gamma and TNF-alpha in preincubation failed to abrogate the suppressive effect of A-LAK on BFU-E colony growth, suggesting that this suppression occurs by a different mechanism than that seen in coincubation studies. Previous studies have demonstrated that the A-LAK population has cytotoxic and proliferative advantages over other killer cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of erythroid differentiation factor (EDF) on proliferation and differentiation of human hematopoietic progenitors.

The effects of erythroid differentiation factor (EDF) on normal human hematopoietic progenitor cells were examined by bone marrow colony assay. Addition of EDF to the erythroid colony assay system enhanced erythroid burst-forming unit (BFU-E)-derived colony formation, and this effect disappeared on removal of adherent cells. Conditioned medium of EDF-treated monocytes also enhanced BFU-E colony formation, whereas conditioned medium of EDF-treated T cells did not. In contrast, EDF inhibited erythroid colony-forming unit (CFU-E) colony formation dose-dependently, although it had no effect on colony formation by myeloid cells. These data show that EDF has a specific effect on human hematopoietic progenitors of the erythroid lineage. The results also indicate that EDF enhanced BFU-E colony formation by stimulating adherent cells to produce factors with burst-promoting activity (BPA), but suppressed CFU-E colony formation by promoting differentiation of these cells.     

A role for calmodulin in the growth of human hematopoietic progenitor cells.

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.     

L-triiodothyronine augments erythropoietic growth factor release from peripheral blood and bone marrow leukocytes

To investigate cellular mechanisms involved in thyroid hormone stimulation of erythropoiesis, we studied the response of erythroid burst-forming unit (BFU-E) proliferation to L-triiodothyronine (L-T3) in a serum-free culture system. When added directly to culture, L-T3 stimulates erythroid burst formation by normal human bone marrow cells. In contrast, granulocyte-macrophage colony formation is unaffected. Enhancement of erythroid burst formation by L-T3 required the presence of nylon wool adherent and/or B-4 antigen-positive light-density marrow populations. Addition of other erythropoietic factors including platelet-derived growth factor and insulinlike growth factor II did not abrogate this apparent cellular requirement. Pulse exposure of marrow and peripheral blood mononuclear cells (greater than 95% lymphocytes) to L-T3 accelerates the release of a soluble factor that augments BFU-E proliferation into serum-free liquid culture medium. Time-course studies show that this factor appears in conditioned medium (CM) coincidentally with erythroid burst-promoting activity (BPA). Furthermore, incubation of CM with an antibody known to react with and adsorb BPA from solution removes the inducible mitogen. Biochemical analysis of CM prepared from unexposed and L-T3 pulse-exposed cells indicates that the rate of protein appearance is accelerated by L-T3 in a fashion that immediately precedes growth factor release and that several polypeptides are quantitatively increased. We conclude that unlike erythropoietin, which is mitogenic for progenitor cells directly, L-T3 enhances BFU-E proliferation indirectly by augmenting the release of soluble BPA-like molecules from accessory cells in culture.     

Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors.

We investigated the in vitro hematopoietic stimulatory activity of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) on bone marrow progenitor populations in 17 normal individuals. In serum-free cultures LIF/HILDA did not induce colony growth. In serum containing media, LIF/HILDA stimulated the growth of colony forming unit (CFU)-MIX and CFU-EO in a dose-dependent fashion and resulted in an increased CFU-MIX and burst forming unit-erythrocytes (BFU-E) colony size. Similar stimulatory effects were seen on a highly purified hematopoietic progenitor population obtained after immunomagnetic depletion of mature myeloid precursors and lymphoid cells. Addition of LIF/HILDA to cultures containing maximally stimulatory concentrations of recombinant human interleukin-3 (rhuIL3), rhuIL3 + rhuIL6, or rhu granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) in serum containing media significantly increased the number of CFU-MIX and eosinophil colonies and increased size and cluster number of CFU-MIX and BFU-E. Depletion of accessory T lymphocytes or monocytes from bone marrow progenitors did not alter the response of hematopoietic precursors to LIF/HILDA. A similar increased colony growth was seen when LIF/HILDA was added to cultures of positively selected CD34/HLA-DR+ or CD34+/HLA-DR- bone marrow hematopoietic progenitor cells stimulated with maximally stimulatory concentrations of rhuIL3 + rhuIL6. LIF/HILDA is a novel cytokine capable of stimulating growth and proliferation of multi-lineage, erythroid, and eosinophil colonies in the presence of serum. LIF/HILDA exerts its activity by direct interaction with highly purified immature bone marrow progenitor cells, has an additive effect when used with other cytokines known to stimulate primitive hematopoietic precursors, and does not require accessory cells.     

Lack of normal CFU-C/suppressor activity in population of alloantigen-stimulated T cells from patients with chronic myelogenous leukemia

Alloantigen-stimulated T cells from patients with Ph1-positive chronic myelogenous leukemia (CML) and normal individuals were obtained by one-way mixed lymphocyte cultures (MLC). Effects of alloantigen-stimulated T cells and MLC supernatants on colony formation were studied in vitro. Alloantigen-stimulated T cells were cocultured with normal allogeneic bone marrow cells (target cells) grown for CFU-C and BFU-E formation. Target cells were also cultured with MLC supernatants. Alloantigen-stimulated T cells from normal controls significantly suppressed CFU-C formation in the presence of human placental conditioned medium (HPCM), while those from CML patients failed to reduce it. MLC supernatants from CML patients were significantly less suppressive on CFU-C formation than those from normal controls. In contrast, not only normal and CML alloantigen-stimulated T cells but also their MLC supernatants increased BFU-E formation in a similar manner. Furthermore, MLC supernatants from normal controls as well as CML patients stimulated the growth of CFU-C from target cells in the absence of HPCM. These results suggest that CML T cells lack the ability to release soluble inhibitors of granulopoiesis, although the production of colony-stimulating factors and burst-promoting activities was equivalent to that of normal T cells after alloantigen stimulation in vitro. The failure of CML T cells to suppress granulopoiesis may contribute to the preferential proliferation of granulocytes in the pathophysiology of CML.     

Regulation of early human hematopoietic (BFU-E and CFU-GEMM) progenitor cells in vitro by interleukin 1-induced fibroblast-conditioned medium

Stimulators of human erythroid burst-forming units (BFU-E) and multipotential colony-forming cells (CFU-GEMM) can be produced by a number of different cell types. A product of human peripheral blood monocytes, interleukin 1 (IL-1), was evaluated for its ability to stimulate fibroblast cultures to produce stimulators of human bone marrow BFU-E and CFU-GEMM colony formation. BFU-E and CFU-GEMM colony formation was evaluated using low-density, nonadherent low-density, and T lymphocyte-depleted nonadherent low-density human bone marrow cells cultured in the presence of a source of pure human erythropoietin. Both human monocyte conditioned medium (MCM) and human recombinant IL-1 (hrIL-1) induced fibroblasts to produce stimulators of BFU-E and CFU- GEMM in a dose-dependent fashion with maximal colony formation occurring when fibroblasts were stimulated by 25% MCM or 140 ng/mL ROO6B hrIL-1, or 1.25 to 5 ng/mL ROO6T hrIL-1. Preincubation of MCM and hrIL-1 with an antibody to IL-1 inactivated the ability of MCM and hrIL- 1 to induce the release of erythroid and multipotential colony stimulating activity from fibroblasts. These results suggest that monocyte-derived IL-1 is involved in regulating the production of humoral stimulators of early human hematopoietic progenitors.     

Juvenile chronic myelogenous leukemia: characterization of the disease using cell cultures

To characterize juvenile chronic myelogenous leukemia (JCML), the proliferative properties of bone marrow (BM) and peripheral blood (PB) cells from nine patients were studied using assays for CFU-C and CFU- GEMM and liquid cultures. All specimens showed two reproducible abnormalities: impaired growth of normal hematopoietic progenitors and excessive proliferation of monocyte-macrophage colonies in the absence of exogenous colony-stimulating activity (CSA). Cytogenetic studies in one patient indicated that the CFU-C were malignant because BM cells at diagnosis and monocyte-macrophage colonies showed an abnormal karyotype, whereas PB lymphocytes did not. In contrast to JCML, PB from six adults with Philadelphia (Ph1) chromosome-positive chronic myelogenous leukemia (Ph1 + CML) yielded CSA-dependent CFU-C colonies which were composed of granulocytes, macrophages, or both, as well as exuberant growth of BFU-E colonies. Co-cultures of JCML BM adherent or nonadherent cells with normal BM resulted in suppression of normal hematopoietic colony formation. Supernatant from JCML adherent cells in liquid culture or plasma from newly diagnosed untreated JCML patients also suppressed control BM colony growth in a dose-dependent manner. These findings confirm that JCML is a malignant disorder of monocytic lineage and suggest that the mechanism of hematopoietic failure in JCML is mediated by an inhibitory monokine secreted by malignant JCML cells.     

Study of early hematopoietic precursors in human cord blood.

Human cord blood is a source of transplantable stem cells. These stem cells express the antigen CD34, are resistant to treatment with 4-hydroperoxycyclophosphamide (CD34+/4-HCres), and do not give rise to colonies when plated in clonogenic assays. We studied the number of CD34+ cells present in cord blood and developed a two-step assay for early precursors (pre-colony-forming units, pre-CFU) capable of giving rise to committed progenitors. In this assay CD34+/4-HCres cord blood cells were cultured in suspension with different growth factors. After 7 days in suspension the remaining cells were plated in clonogenic assays, for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and mixed lineage colony-forming units (CFU-MIX), in the presence of pure factors or a combination of recombinant human (rh) interleukin 3 (IL-3) and medium conditioned by the PU34 primate cell line. Pre-CFU for all precursors were identified. These pre-CFU developed into committed progenitors in response to rhIL-3. The combinations of rhIL-3 plus rh interleukin 1 (IL-1) or rhIL-3 plus rh interleukin 6 (IL-6) did not enhance recovery of progenitors. The developing CFU-GM were responsive to rh granulocyte-macrophage colony-stimulating factor (GM-CSF) and rh granulocyte colony-stimulating factor (G-CSF) but much less so to rhIL-3. BFU-E and CFU-MIX developed in suspension but could only be detected when cells were replated in the presence of a combination of rhIL-3 and PU34 but not rhIL-3 alone. This assay may be useful in evaluating the number of early hematopoietic precursors present in cord blood samples and in defining growth factor combinations that could enhance hematopoietic recovery after cord blood stem cell transplants.     

K562 cells produce and respond to human erythroid-potentiating activity

Human erythroid-potentiating activity (EPA) is a 28,000 mol wt glycoprotein that stimulates the growth of erythroid progenitors in vitro and enhances colony formation by the K562 human erythroleukemia cell line. EPA has potent protease inhibitory activity, and is also referred to as tissue inhibitor of metalloproteinases (TIMP). We observed that colony formation by K562 cells in semi-solid medium containing reduced fetal calf serum (FCS) is not directly proportional to the number of cells plated, suggesting production of autostimulatory factors by K562 cells. Using radioimmunoprecipitation and a bioassay for EPA, medium conditioned by K562 cells was found to contain high levels of biologically active EPA; Northern hybridization analysis confirmed the expression of EPA mRNA. Radiolabeled EPA was used to identify cell surface receptors on K562 cells. Together, these results suggest that EPA may act as an autocrine growth factor for K562 cells.     

In vitro interactions of PGE and cAMP with murine and human erythroid precursors

Addition of prostaglandins of the E series (PGE1, PGE2) in methylcellulose cultures of murine marrow results in a dose-dependent inhibition of the cloning efficiency of both BFU-E and CFU-C. However, CFU-E growth is unaffected. The inhibitory action of PGE is progressively overcome by increasing amounts of colony-stimulating factor (CSF), and with some limitations, also of erythropoietin (Ep). Addition of PGF2 alpha' associated or not with indomethacin, does not exert any significant effect on these hemopoietic precursors. In an attempt to unvail the mechanism(s) underlying these phenomena, dibutyryl-cyclic AMP (db-cAMP), theophylline (an inhibitor of phosphodiesterase), or theophylline + PGE were plated at various concentrations. Both db-cAMP and theophylline induce an inhibitory influence on both BFU-E and CFU-C growth, which mimicks that by PGEs; additionally, theophylline potentiates the inhibitory action of PGE1. In all these studies, the CFU-E number was not significantly modified. PGE action on BFU-E proliferation is clearly species-dependent, since PGE1 addition to human marrow methylcellulose cultures induces a significant enhancement of the number of both BFU-E and CFU-E derived colonies. This action was abolished upon removal of adherent cells, thus suggesting that PGE1 evokes a release of factor(s) enhancing human erythroid colony growth by adherent cells.     

Enhancement of the action of colony stimulating factor (CSF) by soluble component(s) of erythrocytes in mouse bone marrow cells cultures.

Rat erythrocytes, separated from other blood cells by SE-cellulose chromatography, were lysed by exposure to hypotonic solution, dialyzed and ultracentrifuged. The supernatant contained a substance which enhanced the activity of colony stimulating factor (CSF) in soft agar cultures of granulocyte-macrophage progenitor cells (CFU-C) from normal mouse bone marrow. The growth-enhancing effect of the erythrocyte factor was observed when mouse L-cell conditioned medium was used as the CSF source and also when serum from endotoxin-treated mice or from mice undergoing graft-versus-host reaction was used as the stimulant. The erythrocyte factor effect was also detected by 3H-thymidine uptake of bone marrow cells in liquid cultures. These results suggest that the effect of the erythrocyte factor on CSF is not restricted to CSF from a specific source nor to semi-solid agar cultures.     

Consequences of extremely high doses of irradiation on bone marrow stromal cells and the release of hematopoietic growth factors

Although hematopoietic growth factors have previously been difficult to demonstrate in long-term murine bone marrow cultures, it is possible to demonstrate release of growth factors from adherent cells of these long-term cultures following modest doses of irradiation. The present studies were undertaken to determine the maximally tolerated dose of irradiation for growth factor-producing stromal cells and to characterize the growth factor activities. It was discovered that stromal cells could survive extremely high doses of irradiation (500 Gy) and continue to elaborate hematopoietic growth factors. Using escalating doses of irradiation, a dose-dependent increment in detectable hematopoietic growth factors was detectable in unconcentrated conditioned medium. Conditioned medium from long-term cultures exposed to 500 Gy stimulated both fresh murine bone marrow cells (15 +/- 2 to 81 +/- 5 CFU-C/5 X 10(4) target cells) and the interleukin-3/GM-CSF-responsive cell line FDC-P1. In the CFU-C assay, this activity appeared to be predominantly monocyte/macrophage differentiating activity (M-CSF), based upon colony morphology. However, following stimulation of these irradiated stromal cells with endotoxin, there was a significant increase in FDC-P1 growth-promoting activity, and in the CFU-C assay there was increased production of granulocyte, megakaryocyte, and blast-cell type colonies indicating the increased release of a multilineage growth factor. The stromal cells surviving these extremely high doses of irradiation represent a heterogeneous population as demonstrated by morphologic, histochemical, and functional characterization. The two predominant cell populations included a macrophage-like cell and a large flat cell previously referred to as a "blanket" cell.     

Hemopoietic precursor cells in human peripheral blood.

Human peripheral blood contains two types of stem cells that differentiate along the granulocytic pathway. They are separable by their ability to form colonies in agar in vitro (CFU-C) and in plasma clots in diffusion chambers in vivo (CFU-DG). Kinetic studies suggest that CFU-DG represents an intermediate between the still hypothetical human pluripotent stem cell and CFU-C.