Normal Bone Marrow Stem Cells Research Articles

Expression of Leukemia-Associated Antigen, JL1, in Bone Marrow and Thymus

The identification of immunophenotypic markers with restricted expression has long been a critical issue in diagnostic and therapeutic advances for acute leukemias. We previously developed a monoclonal antibody against a new thymocyte surface antigen, JL1, and showed that JL1 is expressed in the majority of acute leukemia cases. In this study, using multiparameter flow cytometric analyses, we found that JL1 was uniquely expressed in subpopulations of normal bone marrow (BM) cells, implying the association of JL1 with the differentiation and maturation process. Although CD34(+) CD10(+) lymphoid precursors and some of maturing myeloid cells express JL1, neither CD34(+) CD38(-/lo) nor CD34(+) AC133(+) noncommitted pluripotent stem cells do. As for the myeloid precursors, CD34(+) CD33(+) cells do not express JL1. During lymphopoiesis, JL1 on the earliest lymphoid precursors disappear in the CD20(+) sIgM(+) stage of B-cell development or after CD1a down-regulation in thymocytes. Despite the highly restricted expression of JL1 in normal BM cells, most of the leukemias express JL1 irrespective of their immunophenotypes. These results indicate that JL1 is not only a novel differentiation antigen of hematopoietic cells, but also a leukemia-associated antigen. Therefore, we suggest that JL1 be a candidate molecule in acute leukemia for the diagnosis and immunotherapy that spares the normal BM stem cells.

Stem cells: What's happening?

Stem cell enthusiasts are probably aware that we are experiencing an exciting period of new development in the field of progenitor cells. This brief report focuses on selected topics which have been recently reported in two special interest group meetings in the U.K. The potential advantage of stem cells derived from human umbilical cord (HUC) blood over normal bone marrow (NBM) or peripheral blood stem cells (PBSC), as source products for reconstitution of the immune system is briefly mentioned, together with some potential applications of HUC blood in gene therapy.

Effect of Ultraviolet-B Light on Lymphocyte Activity at Doses at Which Normal Bone Marrow Stem Cells Are Preserved

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.

Expression and signal transduction of the FLT3 tyrosine kinase receptor.

FLT3 is a receptor tyrosine kinase of 130-55 kDa expressed on normal bone marrow stem and early progenitor cells and on leukemic blasts from patients with acute leukemias. The FLT3 ligand, FL, is a new cytokine which acts on hematopoietic progenitors in synergy with other cytokines. FLT3 transduces FL-mediated signal through interaction with a number of cytoplasmic substrates.

Resistance to lymphoid engraftment in lpr recipients of normal bone marrow: characterization of chimeric stem cell, monocyte and peripheral lymphoid lineages

Lethally irradiated MRL/lpr mice reconstituted with T cell-depleted bone marrow stem cells from the non-autoimmune strain A. Thy had been shown to develop a state of long-term split chimerism; erythrocytes were derived from the A. Thy donor, while peripheral lymphocytes were derived from the lpr recipient. In contrast, recipients of the non-lpr-congenic strain, MRL/+, were fully repopulated in both lineages by donor-derived hematopoietic cells. In order to more fully understand the mechanisms responsible for this type of split chimerism, we have investigated additional genetic and developmental parameters. We found that histocompatible normal B cell precursors engrafted C3H/HeJ and C57BL/6/+ mice much better than they engrafted the corresponding lpr congenic strains, indicating that resistance to lymphoid engraftment was not unique to the MRL background. Bone marrow cells and peritoneal macrophages were found to express the donor H-2 phenotype in both non-lpr and lpr recipients, limiting resistance to the lymphoid lineage. Moreover, we showed that normal bone marrow stem cells passaged in an lpr host environment were subsequently able to repopulate the B cell lineage of non-lpr secondary recipients, proving that prelymphoid stem cells were intact. Although lymph node cells from A. Thy----MRL/lpr chimeras were lpr-derived, they did not show the abnormal surface marker expression associated with the lpr phenotype, nor did they develop lymphoid hyperplasia or elevated autoantibody levels. However, A. Thy----MRL/lpr chimeras differed from normal mice in that their spleens were markedly deficient in IgM+ B cells.

Induction of serum colony-stimulating activity (CSA) following dimethylnitrosamine (DMN) exposure: Effects on macrophage differentiation

Dimethylnitrosamine (DMN) exposure in vivo affects hematopoiesis at the level of CFU-GM precursor cells. This effect on hematopoiesis has been shown to be an indirect consequence of DMN exposure; therefore, we examined serum from animals exposed to DMN in vivo for the presence of hematopoietic growth factor activity. Serum obtained from animals exposed to DMN in vivo supported colony formation in normal bone marrow stem cells whereas serum obtained from untreated or vehicle-exposed animals failed to support colony formation. Differential staining of the cells which arose following the in vitro culture of normal bone marrow cells with serum from DMN-exposed animals demonstrated the presence of cells of the monocytic and granulocytic lineages. Pre-treatment of serum from DMN-exposed animals with anti-CSF-1 antibodies prior to in vitro culture had no affect on either cell number, cell phenotype or colony-stimulating activity, suggesting the presence of GM-CSF. Administration of serum from DMN-exposed animals to naive recipient animals resulted in increased percentages of both blood-borne monocytes and neutrophils, mimicking the profile observed in DMN-exposed animals. Studies using oligonucleotide-directed PCR demonstrated the presence of GM-CSF transcripts in the livers obtained following DMN exposure. These results demonstrate the presence of a serum-borne activity(ies) following DMN exposure, most likely GM-CSF, which is capable not only of enhancing macrophage and granulocyte hematopoiesis but also of increasing the numbers of these two cell types in the blood.

Fractionation and dose rate effects in mice: a model for bone marrow transplantation in man.

This study was designed to compare several fractionation and dose rate schedules to optimize the therapeutic ratio for total body irradiation (TBI). C3H/HeJ mice were given TBI and the bone marrow survival fraction was calculated using the CFUS assay. Irradiation was given at two dose rates: low dose rate (LDR) at 5 cGy/min or high dose rate (HDR) at 80 cGy/min in single fraction and fractionated regimens. The fractionated regimens were given as either 120 cGy three times daily, 200 cGy twice daily, or 200 cGy daily. The Do was 80 cGy for the single fraction, HDR group and 85 for the LDR group. For the fractionated regimens, the apparent Do's ranged from 55-65 indicating no sparing effect of fractionation for the normal bone marrow stem cells. Indeed, the Do's were smaller suggesting an increased sensitivity to irradiation with fractionation. Low dose rate (LDR) and fractionation were also studied for their influence on normal tissue toxicity following upper half body irradiation (UHBI). All the fractionated regimens had higher LD50/30 and LD50/30-180 values than those achieved by single fraction LDR alone. There was no significant dose rate effect for LD50/30 when 120 or 200 cGy fractions were used. However, dose rate was important for LD50/30-180 with 200 cGy but not with 120 cGy fractions. These results demonstrate protection of non-hematopoietic tissues with fractionation and low dose rate without protecting hematopoietic stem cells and may have implications for human bone marrow transplantation.

The lpr gene is associated with resistance to engraftment by lymphoid but not erythroid stem cells from normal mice.

Abstract This study demonstrates cell lineage-specific resistance to engraftment involving lymphocytes but not erythrocytes by the spontaneously autoimmune MRL/lpr mouse strain. In these experiments, MRL/lpr mice were lethally irradiated (1000 R) and reconstituted with normal A-Thy bone marrow stem cells. Periodic analysis from 6 wk to 6 mo posttransplantation demonstrated that the T and B cells of these chimeras were derived from the MRL/lpr host. However, in the same A-Thy----MRL/lpr chimeras, erythrocyte repopulation was completely of A-Thy donor origin. In contrast, control MRL/+ (congenic mice that differ from MRL/lpr at the lpr locus and do not develop accelerated autoimmune disease) recipients were successfully repopulated in both the lymphoid and erythroid compartments by the A-Thy donor cells.

Kinetics of normal hemopoietic stem cells during leukemia growth before and after induction of a complete remission. Studies in a rat model for acute myelocytic leukemia (BNML)

During the invasion of leukemic cells of the rat acute myelocytic leukemia model BNML in the bone marrow, the number of normal bone marrow stem cells (CFU-S) decreased while simultaneously an increase of CFU-S in the leukemic spleen was observed. A small reduction in the tumor load by low dose cyclophosphamide treatment (10 mg/kg) caused a temporary (CFU-S recovery in the bone marrow. After a therapeutic dose of cyclophosphamide (100 mg/kg), the CFU-S numbers in femur and spleen decreased to low levels but they rapidly increased immediately thereafter. In the spleen, however, the CFU-S increase halted when femoral CFU-S numbers reached normal levels. Splenectomy following cyclophosphamide treatment revealed that the splenic CFU-S population does not play a role in regeneration of hemopoiesis. During the subsequent leukemia relapse, CFU-S in the femur decreased again while spleen CFU-S tended to rise. It is concluded that the bone marrow CFU-S, which survive both the leukemia and the remission-induction treatment, and not the migrated, extramedullary localized stem cells are the major source for the restoration of normal hemopoiesis.

The effect of T cells from patients with infectious mononucleosis on CFU-C GM proliferation: A preliminary report

The neutropenia occurring during infection is a poorly understood phenomenon. Immunologically-stimulated T lymphocytes, acting upon normal bone marrow stem cells, have been etiologically implicated in several disorders. Fifteen patients, ages 17 to 25 years, and diagnosed with infectious mononucleosis by positive heterophile titers, were studied. Peripheral blood T lymphocytes were separated using sheep red blood cell rosetting. They were then cocultured with normal bone marrow cells, in a concentration of 2 × 10 4 cells/ml, in methylcellulose containing 10% colony-stimulating activity. Normal BM was obtained from patients with nonmalignant hematologic disorders, or leukemia in remission. Bone marrow cells were cultured at a concentration of 1 × 10 5 or 5 × 10 5 cells/ml, alone (control) or with T lymphocytes. Plates were incubated at 37°C with 5% CO 2. Colonies were scored at 14 days. Inhibition of normal, bone marrow growth was observed at both concentrations, after addition of T lymphocytes to the culture system. Such suppression was significant (p < 0.05) for the lower concentration of normal bone marrow cells only. Variable and partial abrogation of effect was seen after overnight incubation of T lymphocytes, possibly due to loss of suppressor activity. There were insufficient numbers of tests with supernatant to allow computation of statistical significance. Correlation between T-cell ratios and suppressive effect has not been determined, although it is suspected that the responsible cells are within the T-suppressor fraction.

Cellular specificity of the cure for osteopetrosis: Isolation of and treatment with pluripotent hemopoietic stem cells

Osteopetrosis in the ia rat is the result of reduced bone resorption due to abnormal osteoclasts. Studies in the ia mutant have shown that mononuclear cells from normal littermates could cure the skeletal sclerosis and result in the formation of normal osteoclasts when transplanted into ia rats. Recent studies demonstrated that the Ficoll-Hypaque isolates of spleen, bone marrow, and newborn liver were effective in curing the skeletal disease. These results suggest that the cellular source of the cure is a pluripotent hemopoietic stem cell (PHSC). Goldschneider et al. (1980) demonstrated that stem cells from rat bone marrow relatively large mononuclear cells that are strongly positive for Thy 1.1 antigen. Splenic and bone marrow samples from normal rats were treated with a monoclonal antibody developed against the rat Thy 1.1 antigen and guinea pig complement. This procedure was cytotoxic for 47% of the mononuclear spleen cells and 74% of the mononuclear bone marrow cells. When the Thy 1.1 depleted samples were transplanted into ia littermates, they were ineffective in reversing the osteoclast and bone resorption defects. Using the parameters described by Goldschneider, and fluorescence-activated cell sorting (FACS), the PHSC were isolated from normal rat bone marrow. The isolated cells were assayed for colony forming units — spleen (CFU-S) and were found to contain approximately 65–70% PHSC. Two, 3, and 5-week-old ia rats were given an intraperitoneal injection of normal bone marrow stem cells (1.4 × 10 4 to 7.0 × 10 4 cells). The effects of treatment were evaluated by radiography, by measuring the size of the tibial marrow cavity, and by identifying active (phenotypically normal) osteoclasts at 3 weeks after transplantation. At all of the ages transplanted and at all of the cell numbers tested, the stem cell suspensions were effective in curing the osteopetrotic condition. These positive findings and the fact that the Thy 1.1 depleted cells were ineffective suggests that the PHSC is a cellular source of the cure in the ia mutation.

In vitro cytodestruction of leukemic cells in human bone marrow using a cocktail of monoclonal antibodies.

Therapy for human leukemia is currently limited by toxicity of chemoradiotherapy to normal bone marrow stem cells. This limitation can be circumvented by the prior removal and subsequent reinfusion of autologous bone marrow [1, 2]. In order for successful engraftment to occur, reinfused bone marrow must be pruged of leukemic cells ex vivo while the viability of stem cells remains intact. Monoclonal antibodies have the potential of providing this selective destructive effect.

A recommendation for high-dose Ara-C interval treatment based on studies in a slow-growing leukemia model (BNML).

The arabinoside cytosine (Ara-C) dose-response relationship was investigated in a slow-growing rat leukemia (BNML) which is a realistic model for human acute myelocytic leukemia (AML). For both the tumor load reduction determined as leukemic colony-forming units spleen (LCFU-s) and the normal stem cell toxicity determined as colony forming units spleen (CFU-s), a plateau curve was observed. Maximum LCFU-s reduction was observed after high-dose Ara-C (200 mg/kg), while the greatest CFU-s reduction occurred following a ten fold lower dose. For the high Ara-C dose, several methods of administration were compared: rapid intravenous (IV), intramuscular, subcutaneous, intraperitoneal, and continuous infusion. The most effective tumor load reduction was obtained by rapid IV interval treatment. In addition, rapid IV interval administration of Ara-C was less toxic to normal bone marrow stem cells than continuous infusion of the drug. The results of these studies may justify the application of high-dose Ara-C interval treatment in human AML.

Organization of haemopoietic stem cells: the generation-age hypothesis.

This paper proposes that the previous division history of each stem cell is one determinant of the functional organization of the haemopoietic stem cell population. Stem cells from a lineage of stem cells which have generated many stem cells (older stem cells) are used in the animal to form blood before stem cells which have generated few stem cells (younger stem cells). The stem cell generating capacity of a lineage of stem cells is finite. After a given number of generations a stem cell is lost to the stem cell compartment by forming two committed precursors of the cell lines. Its part in blood formation is taken by the next oldest stem cell. We have called this proposal the generation-age hypothesis. Experimental evidence in support of the proposal is presented. We stripped away older stem cells from normal bone marrow and 13 day foetal liver with phase-specific drugs and revealed a younger population of stem cells whose capacity for stem cell generation was three- to four-fold greater than that of the average normal, untreated population. We aged normal stem cells by continuous irradiation and serial retransplantation and found that their stem cell generative capacity had declined eight-fold. We measured the stem cell generative capacity of stem cells in the bloodstream. It was a half to a quarter that of normal bone marrow stem cells and we found a subpopulation of circulating stem cells whose capacity for stem cell generation was an eighth to a fortieth that of normal femoral stem cells. This subpopulation was identified by its failure to express the brain-associated antigen which was present on 75% of normal femoral stem cells but was not found on their progeny, the committed precursors of granulocytes.

Radiation Modifying Effect of the Free Radical Norpseudopelletierene-N-Oxyl on Normal Bone Marrow Stem Cells in Vitro and in Vivo

The sensitization of anoxic bone marrow stem cells irradiated in vitro in the presence of the free radical norpseudopelletierene-N-oxyl (NPPN) has been demonstrated. No sensitization, however, was ...

Factors determining cell killing by chemotherapeutic agents in vivo—I. : Cyclophosphamide

The effect of various chemotherapeutic agents in vivo on the survival of mouse haemopoietic spleen colony-forming cells and osteosarcoma cells is compared and a detailed study is made of the dose-effect curve of cyclophosphamide for the haemopoietic stem cells in normal-mouse bone marrow and in rapidly repopulating spleen. Different parts of the dose-effect curve could be attributed to different mechanisms and it was evident that cyclophosphamide is more effective against rapidly proliferating cells. After elimination of an artefact caused by the assay technique it could be shown that the greater activity versus rapidly proliferating cells is not caused by a higher sensitivity when the cells are exposed in a particular phase of the mitotic cycle, as is the case for instance with vinblastine. The hypothesis is advanced that DNA alkylation by cyclophosphamide or its metabolites may take place in any part of the cell cycle, but that cell killing will occur only if the lesion has not been repaired before DNA synthesis takes place. Finally the results show that only part of the difference in activity of cyclophosphamide versus normal bone marrow stem cells and tumour cells may be ascribed to differences in the rate of cell proliferation; other discriminating mechanisms must be involved.