Effects of interleukin-6 on hematopoiesis in allogeneic and syngeneic bone marrow chimeras

Effects of interleukin-6 (IL-6) on hematopoietic progenitor cells were analyzed in murine bone marrow chimeras. When IL-6 was injected into syngeneic [C3H/He-->C3H/He] bone marrow chimeras from day 1 to day 12, the numbers of highly proliferative potential colony-forming units (CFU-HPP) or colony-forming units mix (CFU-Mix) in spleen cells and bone marrow cells increased on day 14 although there was a marked increase in spleen cells but not in bone marrow cells on day 21. The numbers of CFU-HPP increased in spleen cells from allogeneic [BALB/c-->C3H/He] bone marrow chimeras injected with IL-6 on days 14 and 21. In syngeneic bone marrow chimeras, the numbers of colony-forming units granulocyte/macrophage (CFU-GM) and burst colony-forming units (BFU-E) increased similarly to those of CFU-HPP and CFU-Mix on day 14. On day 21, these were mainly increased in spleen cells. In allogeneic bone marrow chimeras, IL-6 decreased the numbers of CFU-GM and BFU-Mix dose-dependently on day 14. Only 10 micrograms of IL-6 increased the numbers of CFU-GM and BFU-E on day 21. In our previous work, we showed that platelet counts increased on day 14 in syngeneic bone marrow chimeras injected with IL-6, whereas platelet and leukocyte counts increased on days 14 and 24 in allogeneic bone marrow chimeras injected with IL-6, correlating inversely with the numbers of hematopoietic progenitor cells. Overall, primitive hematopoietic progenitors (i.e., CFU-HPP and CFU-Mix) existed primarily in spleen cells of allogeneic bone marrow chimeras on day 14, whereas those in spleen cells of syngeneic bone marrow chimeras were found on day 21. These findings indicate that the effect of IL-6 on hematopoiesis in allogeneic bone marrow chimeras is completely different from that in syngeneic bone marrow chimeras, probably via graft-versus-host reaction (GVHR) but not GVH disease (GVHD).     

Female genital mutilation. Female circumcision. Who is at risk in the U.S.?

It is known that osteoblast precursor cells are found in the low-density mononuclear (LDMN) fraction of human bone marrow (BM) aspirates. The purpose of this study was to investigate whether CD34, a hematopoietic progenitor cell marker, is present on osteoblast progenitor cells. LDMN, CD34+, and CD34- cells were cultured under conditions that promote growth and differentiation of mineral-secreting osteoblasts in a limiting dilution manner. With LDMN cells, osteoblast progenitor cells were found at an average frequency of 1/36,000 cells. With CD34- cells, osteoblast progenitor frequency remained at an average of 1/33,000, similar to LDMN cells. With CD34+ selected cells, osteoblast progenitor frequency increased to an average of 1/5,000. This osteoblast progenitor frequency is maintained in sorted CD34+/CD38+ cells. The osteoblasts generated from CD34+ cells were morphologically normal, and expression of skeletal-specific alkaline phosphatase and osteonectin increased upon differentiation induced by dexamethasone (DEX) treatment. Ultrastructurally, these CD34+ cell-derived osteoblasts displayed osteoblast-specific features. Functionally, these CD34+ cell-derived osteoblasts differentiated with DEX treatment, increased the level of cyclic adenosine monophosphate in response to parathyroid hormone stimulation, increased the level of alkaline phosphatase activity, and increased mineral secretion. These results demonstrate that osteoblast progenitor cells are enriched in the CD34+ cell population from BM and that these progenitor cells can differentiate into functional osteoblasts in culture.     

Changes in the number of stem cells during mouse bone marrow cultivation on a sublayer of fibroblast-like cells

A determination was made of the number of colonies in the spleen of irradiated mice after administration to them of a culture of mouse bone marrow cells. Colony-forming units proved to persist in the culture only for a short time. Use of preliminarily grown underlayer of fibroblasts of bone marrow origin had no effect on the perservation and dynamics of the changes in the number oc colony-forming units.     

Gamma delta-T cell-receptor-positive lymphocytes inhibit human hematopoietic progenitor cell growth in HIV type 1-infected patients

In severe HIV infection, the majority of patients exhibit signs of hematopoietic deficiency including anemia, leukopenia, and thrombocytopenia. Besides other pathophysiological mechanisms, the disturbed helper/suppressor ratio of T-lymphocytes suggests that alterations within T cell subpopulations may have a suppressive effect on HIV-associated hematopoiesis. Since a delta TCS-1- and mostly CD-8-positive subpopulation of cytotoxic T-lymphocytes expressing the gamma delta-receptor is increased in peripheral blood and bone marrow of HIV-infected persons, it was the aim of this study to investigate the role of gamma delta-positive cells in HIV-associated bone marrow deficiency. The number of bone marrow-derived pluripotent colony-forming units (CFU-GEMM), burstforming units-erythrocyte (BFU-E), and colony-forming units-granulocyte-monocyte (CFU-GM) of HIV-1-positive patients was significantly (p < 0.05) increased after depletion of CD-8-positive, gamma delta-positive, and delta TCS-1-positive T-lymphocytes. In contrast, the depletion of these subpopulations had no stimulatory effect in healthy controls. Further experiments identified direct cellular contact between effector and hematopoietic progenitor cells and the production of interferon-gamma and tumor necrosis factor-alpha as the mechanisms mediating the suppressive effect of the delta TCS-1-positive cells in HIV-positive patients.     

Endotoxin-induced size change in bone marrow progenitors of granulocytes and macrophages

Injection of 5 mug endotoxin to adult C57BL mice caused a marked increase in the sedimentation velocity of granulocytic and macrophage progenitor (colony-forming) cells in the bone marrow. This change was maximal two days after injection and was not accompanied by corresponding changes in total marrow nucleated cell populations. The endotoxin-induced shift was not dependent on the presence of the thymus but did not occur in mice challenged after preinjection with endotoxin. No changes in buoyant density, cell cycle status, pattern of differentiation and responsiveness of granulocytic and macrophage progenitor cells were observed after the injection of endotoxin. The increased sedimentation velocity of progenitor cells appears to indicate an increase in cell volume but the mechanisms involved have not been identified.     

Gender-specific effects of prenatal chlordane exposure on myeloid cell development

Work previously reported by this laboratory indicated that prenatal chlordane exposure affected macrophage function in young adult mice. Because these macrophage effects were due to exposure during the development of the immune system, the possibility of a persistent effect on the development of myeloid stem and progenitor cells was considered. Female mice were treated with either 0 or 8 mg of chlordane per kilogram body weight daily for 18 days during pregnancy. Myeloid hemopoietic activity of bone marrow cells from 6-week-old offspring was evaluated for in vitro colony-forming units-in-culture in response to exogeneously added recombinant forms of the cytokines granulocyte/macrophage-colony stimulating factor, macrophage-CSF, and interleukin 3 (IL-3). There was a significant depression of the numbers of bone marrow colony forming units-granulocyte/macrophage (CFU-GM), CFU-IL-3, and CFU-macrophage (CFU-M) in only the female offspring. Male offspring consistently demonstrated no difference in the CFU-GM, CFU-IL-3, or CFU-M. Prenatal treatment with chlordane did not significantly affect the number of recoverable viable bone marrow cells in either male or female mice.     

Measurement of secondary colony formation after 5 weeks in long-term cultures in patients with myelodysplastic syndrome

Pancytopenia is a frequent manifestation of myelodysplastic syndromes (MDS). In the presence of an empty bone marrow, clinical distinction from aplastic anemia may be difficult. The hypoplastic marrow morphology seen in some cases of MDS raises questions about etiologic and pathophysiologic relationships between aplastic anemia and MDS. The goal of our study was to compare the degree of the hematopoietic failure in these diseases at the level of the most immature progenitor and stem cells that can be measured in vitro. In a systemic, prospective fashion, we have studied bone marrow (n = 45) and peripheral blood (n = 33) of patients with MDS for the number of long-term culture initiating cells (LTC-IC) in comparison to 17 normal controls and patients with new, untreated aplastic anemia (46 marrow; 62 blood samples). Due to the low numbers of cells available for the analysis, formal limiting dilution analysis could not be performed, instead secondary colony-forming cells (CFC) after 5 weeks of LTBMC were measured. As the number of these cells is proportional to the input number of LTC-IC, the number of secondary CFC per 10(6) mononuclear cells (MNC) initiating the LTBMC can be used as a measure of the content of immature stem cells in bone marrow and peripheral blood. The MDS group consisted of 34 RA, three RARS, eight RAEB and two RAEB-T patients with mean absolute neutrophil values of 1992, 1413, 1441, and 380 per mm3, respectively. The diagnosis was established based on bone marrow morphology and results of cytogenetic studies. In comparison to controls (147 +/- 38/10(6) MNC), significantly decreased numbers of bone marrow secondary CFC were found in MDS: in patients with RA and RARS, 21 +/- 7 secondary CFC per 10(6) bone marrow MNC (P < 0.001); patients with RAEB and RAEB-T: 39 +/- 12 CFC per 10(6) marrow MNC (P < 0.001). In all groups tested, the decrease in peripheral blood secondary CFC numbers was consistently less pronounced. In MDS patients with hypocellular bone marrow, secondary CFC were lower but not significantly different in comparison to MDS with hypercellular marrow (18 +/- 6 vs 35 +/- 11; NS; hypoplastic bone marrow also was not associated with significantly lower neutrophil counts). However, in 24% of patients with MDS, bone marrow secondary CFC were within the normal range, while in the aplastic anemia group only one of the patients showed secondary CFC number within normal range. Bone marrow and blood secondary CFC numbers in hypoplastic RA were significantly higher than those in severe aplastic anemia 919 +/- 5 in bone marrow, P < 0.01; 7 +/- 2 in blood, P < 0.05). This trend was even more pronounced in hypoplastic RA with chromosomal abnormalities. However, no significant differences were found between the secondary CFC numbers in hypoplastic RA and moderate aplastic anemia. We concluded that, although the deficiency in the stem cell compartment is less severe in MDS than in aplastic anemia, depletion of early hematopoietic cells is an essential part of the pathophysiology in both diseases.     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cells transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho+/++ (dull/bright) and Rho- (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho+/++ cell population contained > 99% of committed progenitor cells with in vitro colony-forming ability. The Rho- cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho- stem cells or purified Rho- stem cells in combination with large numbers of Rho+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho- stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Inhibition of hemopoiesis in vitro by neuroblastoma-derived gangliosides

Hemopoiesis is disturbed in bone marrow-involving cancers like leukemia and neuroblastoma. Shedding of gangliosides by tumor cells may contribute to this tumor-induced bone marrow suppression. We studied in vitro the inhibitory effects of murine neuroblastoma cells (Neuro-2a and C1300) and their gangliosides on hemopoiesis using normal murine hemopoietic progenitor colony-forming assays. Transwell cultured neuroblastoma cells showed a dose-dependent inhibition on hemopoiesis, indicating that a soluble factor was responsible for this effect. Furthermore, the supernatant of Neuro-2a cultured cells inhibited hemopoietic proliferation and differentiation. To determine whether the inhibitory effect was indeed due to shed gangliosides and not, for instance, caused by cytokines, the effect of DL-threo-1 -phenyl-2-decanoylamino-3-morpholino-1-propanol (DL-PDMP) on Neuro-2a cells was studied. DL-PDMP is a potent inhibitor of glucosylceramide synthase, resulting in inhibition of the synthesis and shedding of gangliosides. The initially observed inhibitory effect of supernatant of Neuro-2a cells was abrogated by culturing these cells for 3 days in the presence of 10 microM DL-PDMP. Moreover, gangliosides isolated from Neuro-2a cell membranes inhibited hemopoietic growth. To determine whether the described phenomena in vitro are a reflection of bone marrow suppression occurring in vivo, gangliosides isolated from plasma of neuroblastoma patients were tested for their effects on human hemopoietic progenitor colony-forming assays. These human neuroblastoma-derived gangliosides inhibited normal erythropoiesis (colony-forming unit-erythroid/burst-forming unit-erythroid) and myelopoiesis (colony-forming unit-granulocyte/macrophage) to a higher extent compared with gangliosides isolated from control plasma. Altogether these results suggest that gangliosides shed by neuroblastoma cells inhibit hemopoiesis and may contribute to the observed bone marrow depression in neuroblastoma patients.     

Retroviral transfer of a bacterial alkyltransferase gene (ada) into human bone marrow cells protects against O6-benzylguanine plus 1, 3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity

The antitumor activity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is limited by the O6-alkylguanine-DNA alkyltransferase (ATase) in tumor cells and by delayed myelosuppression. Inactivation of neoplastic ATase by O6-benzylguanine (BG) improves the therapeutic index for BCNU. We have demonstrated previously that BG + BCNU-induced myelosuppression in mice is reduced by expression of the BG-resistant ATase ada in murine bone marrow. We have now generated an amphotropic retrovirus containing the ada gene and tested the effectiveness of ada expression in preventing BG + BCNU cytotoxicity in human hematopoietic progenitor cells. A retroviral producer clone with a biological titer of 6.5 x 10(4) colony-forming units/ml and 4.4 pmol ATase/mg protein was used for transduction of bone marrow. Cocultivation of these ada producer cells with progenitor cells from six normal individuals resulted in 1.9-3. 9-fold protection against BG + BCNU-induced cytotoxicity in committed progenitor cell assays. Furthermore, this cytoprotective effect was associated with a high transduction efficiency (40%) and a 2-fold increase of ATase activity in the surviving committed progenitor cell colonies. These data provide a basis for testing the clinical effectiveness of retroviral ada gene transfer into hematopoietic cells to increase the therapeutic index of BG + BCNU.     

Duality in physiological time: Euclidean and fractal

We have previously reported that bone marrow progenitors in dogs, specifically granulocyte-macrophage colony-forming units (GM-CFU), increase developing airway hyperresponsiveness after inhalation of the allergen Ascaris suum. In the present study, we evaluated whether this increased marrow hemopoietic activity can be stimulated by a factor in serum after allergen challenge. Serum samples taken from dogs prior to and 20 min, 2 h, and 24 h after Ascaris or diluent challenge were added to bone marrow cells aspirated prior to challenge, and GM-CFU measured. A second bone marrow aspirate was performed 24 h after challenge. Nonadherent mononuclear bone marrow cells were incubated for 8 days in the presence of the serum and recombinant canine hemopoietic cytokines (stem cell factor, granulocyte colony-stimulating factor, GM colony-stimulating factor). Eight dogs that developed (airway responders) and eight dogs that did not develop (airway nonresponders) allergen-induced airway hyperresponsiveness were studied. Allergen inhalation increased bone marrow GM-CFU in response to all three growth media in vitro for the airway responder (P < 0.05) but not airway nonresponder dogs. The 24-h serum, taken from the airway responder but not the airway nonresponder dogs, produced a similar increase in granulocyte progenitors when added to the bone marrow taken before allergen inhalation (P < 0.05). These findings demonstrate that bone marrow-derived granulocyte progenitors are upregulated by a factor that can be shown to be present in serum 24 h after allergen challenge in dogs that develop allergen-induced airway hyperresponsiveness. Whether in vivo stimulation of bone marrow inflammatory cell production is necessary for the development of allergen-induced airway hyperresponsiveness remains to be proven.     

Growth-supporting activities of fibronectin on hematopoietic stem/progenitor cells in vitro and in vivo: structural requirement for fibronectin activities of CS1 and cell-binding domains

Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34(+)/VLA4(bright)/VLA5(dull) hematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor-deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34(+) cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34(+) cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.     

Factors affecting progenitor cell yields using three tandem leukaphereses in previously treated malignancies

BACKGROUND: Mobilized peripheral blood progenitor cells (PBPCs) have increasingly been used to replace autologous bone marrow to allow faster hematopoietic reconstitution after myeloablative therapy in various malignancies. There is a paucity of data concerning factors that affect the total yield of three tandem leukaphereses. METHODS: Factors affecting the yield of PBPCs were analyzed in a series of 121 consecutive patients including 36 with non-Hodgkin's lymphoma, two with Hodgkin's disease, four with multiple myeloma, 44 with acute leukemia, 20 with breast cancer and 15 with other solid tumors. PBPCs were mobilized using granulocyte-colony-stimulating factor (G-CSF) alone (group I, n = 15), or after conventional-dose (group II, n = 70) or high-dose (group III, n = 36) chemotherapy followed by G-CSF. The total yield of three tandem PBPC collections for each patient was assessed by the number of mononuclear cells (MNCs), CD34+ cells and colony-forming units of granulocyte macrophages (CFU-GM). The factors evaluated included age, sex, diagnosis, history of marrow involvement, previous radiotherapy, the number of prior chemotherapy cycles and mobilization method. The two -sample t-test and logistic regression analysis were performed for univariate and multivariate analysis, respectively. RESULTS: With univariate analysis, a diagnosis of acute leukemia, positive history of bone marrow involvement, more chemotherapy cycles and mobilization with high-dose chemotherapy adversely affected the yields of CD34+ cells. By multivariate analysis, Group II had higher yields of MNCs (p = 0.039), CFU-GM (p = 0.002) and CD34+ cells (p = 0.011) than Group III. Fewer cycles of prior chemotherapy is the common favorable factor for the yields of both CD34+ cells (p = 0.016) and CFU-GM (p = 0.017). CONCLUSIONS: The number of prior chemotherapy cycles adversely affects progenitor cell yield. Conventional-dose chemotherapy followed by G-CSF seems to be the mobilization methods of choice for heavily pretreated cancer patients with limited bone marrow reserve. PBPCs should be harvested early, when the tumor burden is less, to avoid cumulative marrow toxicity from chemotherapy.     

Osteopontin expression by osteoclast and osteoblast progenitors in the murine bone marrow: demonstration of its requirement for osteoclastogenesis and its increase after ovariectomy

Osteoclast development requires cell-to-cell contact between hematopoietic osteoclast progenitors and bone marrow stromal/osteoblastic support cells. Based on this, we hypothesized that osteopontin, an adhesion protein produced by osteoclasts and osteoblasts, plays a role in osteoclastogenesis. Using in situ hybridization, we demonstrate that cells expressing the osteopontin messenger RNA (mRNA) appear after 3 days of culturing murine bone marrow cells. The number of these cells increases thereafter, reaching a peak on day 5. In the same cultures, cells expressing alkaline phosphatase (AP) or tartrate resistant acid phosphatase (TRAP), phenotypic markers for osteoblastic and osteoclast-like cells, respectively, appeared subsequent to the appearance of the osteopontin-positive cells. By means of a combination of in situ hybridization and histostaining, it was shown that the osteopontin mRNA was localized in 30-50% of the AP-positive or the TRAP-positive, as well as in nonspecific esterase (NSE)-positive, cells. The number of cells expressing both the osteopontin mRNA and either one of the three phenotypic markers was significantly increased in bone marrow cultures from estrogen-deficient mice, as compared with controls. Conversely, the number of all three populations of double positive cells was decreased in cultures treated with a specific antimouse rabbit osteopontin antibody or an RGD peptide. These findings indicate that osteopontin is expressed during the early stages of the differentiation of osteoclast and osteoblast progenitors in the bone marrow and that its cell adhesion properties are required for osteoclastogenesis.     

Ovariectomy-induced bone loss and the hematopoietic system

To investigate the relationship of the hematopoietic system to the loss of bone due to ovarian hormone deficiency, we examined the effects of ovariectomy and estrogen administration on the thymus, spleen and the bone marrow, and on the proliferation of marrow progenitors of osteoclasts. We also assessed the effects of daily administration of interleukin-1 receptor antagonist (IL-1ra) on bone loss due to ovarian hormone deficiency. Ovariectomy resulted in decreased cancellous bone volume, increased trabecular osteoblast and osteoclast numbers, and increased serum alkaline phosphatase levels that were prevented by 17 beta-estradiol treatment. Thymus weight, spleen weight, thymus and spleen lymphocytes, and bone marrow monocytes and lymphocytes also increased significantly following ovariectomy, and the increases were suppressed by 17 beta-estradiol. Ovariectomy, in addition, caused a 4-fold increase in the number of tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells formed in cultures of marrow cells and the increase was partially inhibited by 17 beta-estradiol. IL-1ra administration did not prevent the bone loss due to ovariectomy. Our findings indicate that ovariectomy-induced bone loss in the rat is accompanied by marked changes in the hematopoietic system, and that these changes are modulated by estrogen administration. In spite of the negative finding with IL-1ra, the nature of the involvement of the hematopoietic system in the pathogenesis of bone loss due to ovarian hormone deficiency merits continued exploration.     

The role of blood stem cells in hematopoietic cell renewal

It has been the purpose of this keynote address to review available evidence for the notion that the stem and progenitor cells circulating in the peripheral blood play a decisive role in the homeostasis of blood cell formation distributed throughout dozens of bone marrow units in the skeleton. Furthermore, if this notion is correct, one could speculate that the quantity and quality of stem and progenitor cells in the blood should reflect the functional state of the hematopoietic stem cell system throughout the skeletal bone marrow and provide a new tool for the evaluation of alteration in blood cell production. On this basis, the following questions are considered: A) What do we know about the quality and quantity of blood stem cells in steady-state conditions? B) In what way do blood stem cells respond to perturbations of the "steady-state" of blood cell formation? C) Which role do blood stem cells play during hemopoietic development assuming that the establishment of bone marrow hemopoiesis requires the "seeding" of blood stem cells into an appropriate cellular environment? D) What is the role of blood stem cells in hemopoietic regeneration after partial body irradiation with a small volume of marrow (and hence stem cells) protected? and E) What are the mechanisms and/or kinetics of hemopoietic recovery if stem cells introduced into the circulation were collected from exogenous (autologous or allogeneic) sources? In this review presentation, experimental work of our group and of other members of the scientific community is summarized. It becomes obvious that blood stem and progenitor cells play a key role in hematopoietic homeostasis. Furthermore, their physiology and pathophysiology deserve rigorous experimental studies in order to develop a novel tool in the diagnosis and prognosis of neoplastic and non-neoplastic disorders of blood cell formation.     

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.