Refinement and verification of test conditions for colony-forming unit assay in dog bone marrow cells with preservable colony specimens

The colony-forming unit assay reported by Kubota is a potentially useful method for toxicity evaluation since the method enables us to preserve the specimens. However, no study has yet reported on the utility of Kubota’s method in conducting the CFU assay in toxicity evaluation. Therefore, we examined the optimum test conditions for CFU assays by Kubota’s method using bone marrow cells from dogs, a widely used species in toxicity studies, to establish a useful hematopoietic toxicity evaluation system in vitro. We established test conditions for colony-forming unit granulocytes–macrophage assay and colony-forming unit erythroid assay. Optimum conditions for colony-forming unit granulocytes–macrophage assay were 20 % of colony-stimulating dog serum, 1?×?10⁵ cells/dish, and culture periods of 3 days for neutrophil colonies and 8 days for macrophage colonies. Appropriate conditions for colony-forming unit erythroid assay were 2 IU/ml recombinant human erythropoietin, 0.265–2.5?×?10⁴ cells/dish, and a culture period of 2–4 days. We also examined the response to bone marrow toxicants doxorubicin and azidothymidine in these systems. Both compounds reduced the numbers of colonies in a concentration-dependent manner in both assays. Taken together with these results, we confirmed that our method efficiently functioned as an evaluation system for bone marrow toxicity in vitro.     

The time course of leucocyte colony-formation in cultures of bone-marrow progenitor cells from rats and dogs

The colony-forming units granulocyte and macrophage (CFU-GM) assay, using either rat or dog haematopoietic progenitor cells, assesses the toxicity of new compounds. To identify the characteristics of colony formation in this system, a time-course study of CFU-GM assays using rat and dog bone marrow cells was tested. Neutrophil colonies, macrophage colonies and mixed colonies of neutrophils and macrophages were formed in soft agar medium. Neutrophil colonies reached their maximum number on days 3–4 and decreased markedly thereafter. Macrophage colonies reached their maximum number on days 7–8 and remained steady thereafter. Only a small number of mixed colonies of neutrophils and macrophages were formed beginning around day 4. There were no significant differences between rat and dog bone marrow cells in the occurrence of these maxima, or in any other growth phenomenon. This result suggests that to evaluate the influence of compounds on neutrophil colonies and macrophage colonies, observations should be made on days 4 and 8, respectively.     

Antigen-induced proliferation in vitro of bone marrow precursors of granulocytes and macrophages

The injection of polymerized flagellin into C57BL mice increased the number of granulocyte cluster-forming cells and macrophage colony-forming cells in the bone marrow. The addition of polymerized flagellin to agar cultures of bone marrow cells from normal or antigen-injected mice increased the number and growth rate of macrophage colonies developing when mouse serum was used as the source of colony stimulating factor (CSF). Polymerized flagellin had no potentiating effect on colony growth when urine or medium conditioned by bone marrow, spleen, lymph node or thymic cells were used as the source of CSF. Antigens may stimulate the proliferation of granulocyte and macrophage precursor cells by an indirect mechanism involving the production by bone marrow cells of CSF, or a similar growth promoting factor.     

Omental lymphoid organ as a source of macrophage colony stimulating activity in peritoneal cavity.

To test the hypothesis that the omental lymphoid organ (OLO) made by peritoneal milky spots is either a source of haemopoietic (macrophage) progenitors or growth factors we attempted to culture OLO cells in vitro in a variety of assay combinations. With the culture in vitro in semisolid agar it was found that OLO cells do not form granulocyte-macrophage or macrophage colonies in response to stimulants. However, when in the same assay marrow cells were used as the targets and OLO-related preparations as stimulants it was observed that marrow cells formed exclusively macrophage colonies. These marrow cells, in response to stimulants derived from other organs, produced granulocyte-macrophage, granulocyte and macrophage colonies. OLO-related preparations tested for macrophage-colony stimulating activity included partly purified medium conditioned by OLO cells derived from mice, either injected with endotoxin or not, and medium conditioned by OLO cells after 14 days, liquid culture in vitro. While these results were observed in Swiss mice, C3H/W mice, which are genetically endotoxin-unresponsive, failed to show this reaction. These data may suggest that the local production of macrophage-colony stimulating activity in the peritoneal cavity could be one physiological role for OLO. OLO is the first organ in adult mice identified to stimulate exclusively macrophage colony growth, and not granulocyte-macrophage or pure granulocyte colonies.     

Hematopoietic effects in mice exposed to deltamethrin and hydrocortisone

The effects of deltamethrin on bone marrow and spleen progenitor cell responsiveness to granulocyte and macrophage colony-stimulating factors were evaluated. Delthamethrin was tested in parallel with hydrocortisone to further investigate some similarity in the in vivo effects of both compounds observed in previous studies in our laboratory. In vivo effects were studied after the subcutaneous administration in mice of three 5 mg/kg injections of deltamethrin and a single 30 mg/kg injection of hydrocortisone to Balb/c mice. Soft agar colony formation, marrow andspleen cell counts, body, spleen and thymus weights were determined. Data obtaied in vivo indicate that deltamethrin and hydrocortisone reversibly icrease the formation of granulocyte an macrophage colonies in the narrow, but not in the spleen. No changes were observed in total and differential cell counts in the marrow and spleen and spleen weights. Treatment with both compounds, however, resulted in a dramaic reduction in thymus weights. Assays for endotoxin demonstrate that these effects were not due to the liberation of endotoxin. In vitro addition of 10⁻⁵, 10⁻² 10⁻⁷ M deltamethrin and hydrocortisone to marrow cultures from untreated mice resulted in different effects from those observed in vivo. Hydrocortisone increased granulocyte and reduced macrophage colonies, whereas deltamethrin was without in vitro effects. It is suggested that deltamethrin effects are due to an indirect action on the hypothalamic - pituitary axis leading to increased corticosteroid levels. The importance of biotransformation mechanims is also emphasized.     

Candida albicans can stimulate stromal cells resulting in enhanced granulopoiesis

Previously, we have reported that although unperturbed granulocyte colony-stimulating factor (GCSF)-deficient (G-CSF-/-) mice are neutropenic, when challenged with Candida albicans, they develop a profound neutrophilia. In an attempt to understand the basis of Candida-induced neutrophilia in G-CSF-deficient mice, we have modified the Dexter bone marrow culture system to produce an in vitro model that mimics emergency granulopoiesis in vivo. In this model, stromal cultures are overlaid with bone marrow cells in the presence or absence of heat-inactivated (HI) Candida. Irrespective of the genotype of mice used as a source of bone marrow-derived stromal cells, stimulation of these cultures with HI Candida led to a significantly greater recovery of cells compared to unstimulated stromal cultures. In addition, there was a marked increase in the number of colony-forming units granulocyte-macrophage (CFU-GM), as well as in the percentage of granulocytes in the population of nonadherent cells recovered from HI Candida-stimulated cultures. The conditioned medium generated from stromal cultures derived from either wild-type or G-CSF-/- mice exposed to HI Candida, when applied to bone marrow cells in a soft agar clonogenic assay stimulated M-, GM-, and G- type colonies. Interleukin-3 (IL-3) and GM-CSF could not be detected in the conditioned medium from either HI Candida stimulated or unstimulated stromal cultures. However, IL-6 was detected in the conditioned media from both wild-type and G-CSF-/- stromal cultures. Addition of anti-IL-6 antibody significantly impaired granulopoiesis in unstimulated and HI Candida-stimulated, wild type, and G-CSF-/- stromal cultures. Conditioned medium generated from G-CSF/IL-6-deficient stromal cells had the capacity to stimulate bone marrow cells to form colonies comprised of granulocytes and macrophages in soft agar clonogenic assay. This study demonstrates that stromal cells can be stimulated with HI Candida and gives an insight into Candida mediated granulopoiesis.     

In vitro transformation with avian myelocytomatosis virus strain CMII: characterization of the virus and its target cells

Avian myelocytomatosis virus strain CMII induced an in vitro transformation in cells from various hematopoietic tissues and could be quantitated by focus and soft agar colony assay techniques. The CMII-transformed bone marrow cells had a high proliferative capacity in comparison to uninfected controls. The cells closely resembled hematopoietic cells transformed by strain MC29 myelocytomatosis virus, but differed from avian myeloblastosis virus (AMV)-transformed cells. They were phagocytic, became adherent under certain conditions of culturing, and required colony-stimulating factor for colony formation in semisolid medium. These properties are characteristic for cells of the granulocyte/macrophage lineage of differentiation. In contrast to avian erythroblastosis virus, CMII effectively transformed macrophage cultures suggesting that the target cell belongs to the corresponding differentiation lineage. That it is not identical to normal granulocyte/macrophage colony-forming cells was demonstrated by cell separation experiments. In addition to hematopoietic cells, CMII induced a morphological transformation in chicken fibroblasts. CMII was found to consist of a mixture of a transforming component and an associated nontransforming virus of subgroup B or D. The transforming component is defective for replication and could be complemented by standard helper viruses of subgroups B, C, and D.     

The triterpenoid CDDO-Me promotes hematopoietic progenitor expansion and myelopoiesis in mice

The synthetic triterpenoid CDDO-Me has been shown to directly inhibit the growth of myeloid leukemias and lends itself to a wide array of therapeutic indications, including inflammatory conditions, because of its inhibition of NF-κB. We have previously demonstrated protection from acute graft-versus-host disease after CDDO-Me administration in an allogeneic bone marrow transplantation model. In the current study, we observed that CDDO-Me promoted myelopoiesis in both naive and transplanted mice. This effect was dose dependent, as high doses of CDDO-Me inhibited myeloid growth in vitro. All lineages (granulocyte macrophage colony-forming unit, BFU-E) were promoted by CDDO-Me. We then compared the effects with granulocyte colony-stimulating factor, a known inducer of myeloid expansion and mobilization from the bone marrow. Whereas both drugs induced terminal myeloid expansion in the spleen, peripheral blood, and bone marrow, granulocyte colony-stimulating factor only induced granulocyte macrophage colony-forming unit precursors in the spleen, while CDDO-Me increased these precursors in the spleen and bone marrow. After sublethal total-body irradiation, mice pretreated with CDDO-Me further displayed an accelerated recovery of myeloid progenitors and total nucleated cells in the spleen. A similar expansion of myeloid and myeloid progenitors was noted with CDDO-Me treatment after syngeneic bone marrow transplantation. Combined, these data suggest that CDDO-Me may be of use posttransplantation to accelerate myeloid recovery in addition to the prevention of graft-versus-host disease.     

Multiple hemopoietic lineages are found after stimulation of mouse bone marrow precursor cells with interleukin 3

When the murine T-lymphocyte clone L2 is stimulated with concanavalin A, it secretes at least two distinct factors that affect hemopoietic precursor cells, interleukin 3 (IL3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). IL3 accounts for approximately 10% of the colony-stimulating activity in L2-cell-conditioned medium. The IL3 secreted by L2 cells is similar antigenically to the IL3 secreted by WEHI-3 cells. Like the IL3 from WEHI-3 cells, IL3 secreted by L2 cells does not bind to DEAE Sephacel and can be separated from the L2-cell GM-CSF, which does bind to DEAE. By assessment of the functional, morphologic, surface phenotypic, and cytochemical characteristics of bone marrow cells 6 days after stimulation with IL3 in liquid culture, four hemopoietic lineages were found, including macrophage, neutrophilic granulocyte, megakaryocyte, and basophil/mast cell. In addition, when bone marrow cells were stimulated with IL3 in semisolid medium, several types of colonies were found, including mixed colonies containing macrophage, megakaryocyte, and granulocyte lineages.     

A colony assay system that detects B cell progenitors in fresh and cultured bone marrow

This report describes a colony assay system, based on methods used to grow myeloid precursors in semisolid medium, in which B cell progenitors can be grown. The formation of these B cell progenitor colonies is dependent upon soluble mediators from a stromal cell line known to support B lymphopoiesis. In initial experiments a double layer culture system was employed in which target cells in methylcellulose medium were separated from an adherent layer of S17 stromal cells by an agar interface. Target cells were harvested from Dexter type long-term bone marrow cultures at a time after transfer to the lymphoid Whitlock-Witte conditions, when myeloid progenitors were depleted and mature B cells had not yet appeared. On day 15 of culture a colony could be identified that contained several hundred tightly clustered lymphoid cells. There was a linear relationship between the number of cells plated and the number of colonies that developed. Identically appearing colonies were also observed in agar using fresh bone marrow cells as targets with either an underlayer of S17 cells or S17 conditioned medium to potentiate colony growth. Lymphoid colonies derived from fresh bone marrow appeared on days 6 and 14 of growth. A proportion of the cells from the fresh or cultured marrow derived colonies expressed the B220 antigen and cytoplasmic mu heavy chains, but surface IgM was never observed. Cell depletion experiments on antibody coated plates demonstrated the colony forming unit to be B220 antigen positive, surface IgM negative, and replating experiments indicated the colonies were lymphoid restricted in their differentiative potential.     

Characteristics of colony growth from normal human bone marrow

The number of granulocyte/macrophage colonies grown in vitro from bone marrow cells obtained from 90 rib segments and 30 ;normal' bone marrow aspirates was found to be highly variable. Considerable variation was also noted in the relationship between colony number and the number of cells cultured in both groups. The aspirate group was found to have a significantly greater ability to form colonies without the addition of colony-stimulating factor to the culture medium at low cell concentrations.     

骨髓基质细胞系BMSC1自发分泌多种细胞因子

骨髓基质细胞（ＢＭＳＣ）及其分泌的细胞因子在造血细胞分化发育过程中起重要作用。应用特异性细胞因子依赖株检测了自建的小鼠骨髓基质细胞株ＢＭＳＣ１的细胞因子分泌情况。实验说明ＢＭ－ＳＣ１细胞能自发分泌高水平的ＩＬ－６，中等水平的ＩＬ－７及较低水平的ＧＭ－ＣＳＦ，未检测到化学趋化因子。此细胞上清对骨髓造血干细胞有明显的促集落形成效应，所形成的集落以ＣＦＵ－ＧＭＭ及ＣＦＵ－ＧＭ为主，其促集落形成作用呈现剂量依赖关系。上述结果表明，骨髓基质细胞能够产生多种类型的细胞因子从而对于细胞骨髓内分化发育成Ｐｒｏ－Ｔ细胞可能起重要作用。     

Murine sarcoma viruses block corticosteroid-induced differentiation of bone marrow preadipocytes associated with long-term in vitro hemopoiesis.

A unique population of clonagenic bone marrow cells accumulates neutral fat and differentiates to adipocyte morphology in response to 10^?7M 17-hydroxy-corticosteroid. Differentiation of these adherent marrow preadipocytes is associated with maintenance of pluripotent hemopoietic stem cell and granulocyte-macrophage progenitor cell proliferation in bone marrow cultures in vitro. The present studies demonstrate that infection of preadipocytes with Harvey, Kirsten, or Moloney strains of murine sarcoma virus (MSV) but not the associated helper leukemia virus blocks corticosteroid-induced lipogenesis. This effect is observed both in whole marrow culture and in a new assay for the individual colony-forming unit adipocyte (CFUa). CFUa are single cells concentrated in light density fractions of an isopycnic density gradient, normally accumulate lipid, and grow to ≥50 cell colonies following suspension in 0.8% methylcellulose-containing medium with horse serum and hydrocortisone. An MSV-specific block of both CFUa formation and insulin-dependent differentiation of the 3T3-L1 embryo preadipocyte cell line is detected by 14 days. In contrast, MSV infection of granulocyte-macrophage progenitor cells in the same marrow does not block differentiation in response to colony-stimulating factor. These data indicate that cells of a specific phenotype can be identified within an organ culture as targets for the early biological effects of a particular class of RNA type-C viruses.     

Morphological pursuit of blood stem cells emerging from aplastic bone marrow of mouse after a single high dose of 5-FU injection

A single high dose of 5-fluorouracil (5-FU) was administered intravenously in mice to induce the aplastic bone marrow and to seize the morphological characteristics of blood stem cells. Time course observations of histological and cytological changes in bone marrow hematopoiesis and stem cell assay revealed that the day 2 bone marrow contained a number of quiescent blood stem cells. For advanced morphological studies of blood stem cells, the day 2 marrow cells were seeded in methylcellulose culture medium for stem cell assay. Blast cell colonies (BCC) which contained CFU-GEMM (colony-forming unit granulocyte, erythroid, monocyte/macrophage, megakaryocyte) were formed within 2 weeks. BCC-constituting cells (BCC-cell) were lifted and processed for morphological analyses. The BCC-cells were variable in size as well as morphology, and were divided into 3 sub-groups according to their size. Large-sized group (greater than 14 microns) consisted of large blastic cells with a tendency of differentiation into early granulocyte or monocyte/macrophage lineage. Medium-sized group (10-14 microns) consisted of more immature blastic cells which lacked a differentiation. Small-sized group (7-10 microns) consisted of two cell-types. One type (named 'transitional cell' or 'TC cell') had distinctive features as a dark nucleus and a narrow basophilic cytoplasm seemed to be the most immature cell type. The other (named 'lymphoid cell') was morphologically indistinguishable from the small lymphocyte. Blast cell colony-replating experiments disclosed that the proportion of small-sized cells in BCC-cells (about 4%) was equivalent to the incidence of GEMM colonies formed in replated culture (about 2-5%). Furthermore, immunohistochemical staining of wheat germ agglutinin (WGA), anti-Thy-1 antibody and anti-B cell antibody revealed that most of TC cells were positive for WGA suggesting of CFU-GEMM, and negative for anti-B cell antibody. Most of lymphoid cells were positive for anti-B cell antibody, and negative for WGA and anti-Thy-1 antibody, and were estimated to be true B-lymphocytes. Based on these results, it was reasonable to consider that a majority of TC cells were multipotent blood stem cells.     

The Effects of Methimazole on Haematopoiesis in Mice

Abstract

The effects of anti-thyroid drug, methimazole (MMI), on haematopoiesis in inbred C57BL/6 mice were studied. The in vitro proliferative response of bone marrow (BM) cells to interleukin-3 (IL-3) was significantly increased when mice were provided with 0.1% MMI in water (w/v) ad librium for 4 to 6 weeks. Using soft agar agar colony assay, the numbers of myeloid cell colonies were also significantly increased in mice treated with MMI. However, the proliferative response of BM cells to IL-3 was found to be greatly reduced 10 weeks after MMI treatment. In vitro studies showed that MMI alone at the concentrations of 500μM or above inhibited both the growth of normal BM cells in liquid culture and the formation of macrophage (M) - / granulocyte (G) - colonies in soft agar culture in a dose dependent manner. Direct cytotoxic effect of MMI (0 - 1250 μM) to normal BM cells was not observed. Results from this study suggested that MMI can modulate the development of myeloid haematopoietic cells.     

Effects of exogenous glucocorticoids on colony-forming activity of the bone marrow under cytotoxic exposure

Experiments on CBA/CaLac mice receiving a half-maximum tolerated dose of 5-fluorouracil demonstrated an inhibitory effect of physiological concentrations of dexamethasone (127×10⁻⁹M) on colony-forming activity of erythroid and granulomonocytic precursors in intact and regenerating bone marrow. Dexamethasone exhibited protective effects on granulocyte, macrophage, and fibroblast precursors during maximum myelosuppression. Various responses of hemopoietic progenitor cells to exogenous corticosteroidin vitro are probably determined by their functional state. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 412–414, April, 1999     

A simple, one-step clonal assay allows the sequential detection of committed (CFU-GM-like) progenitors and several subsets of primitive (HPP-CFC) murine progenitors.

Murine bone marrow (BM) cells were cultured in semisolid medium containing interleukin 3 (IL-3) and high doses of G-CSF. Colonies were counted twice, at day 7 and day 14, and the number of granulocyte/macrophage colony-forming units (CFU-GM) accurately estimated by the subtraction of day-14 from day-7 colonies, based on the principle that colonies detectable at day 7 and persisting beyond day 14 are generated by significantly more immature progenitors. The frequency of colonies relative to their size was determined and used to define subsets of high proliferative potential colony-forming cells (HPP-CFC). Two main groups of HPP-CFC were considered: those generating colonies of 0.6-1.8 mm of diameter or larger than 1.8 mm. The characterization of these groups showed that they correspond to different functional subsets of HPP-CFC. The replating ability of colonies was estimated. The percentage of clonogenic progenitors in the S phase of cell cycle was measured by cytosine arabinoside suicide assay. The sensitivity of colonies to 5-fluorouracil (5-FU) in vitro was determined and their survival after an in vivo treatment with 5-FU compared with that of colony-forming units in spleen (CFU-S). This technique allowed identification of: A) CFU-GM; B) relatively mature HPP-CFC, probably corresponding to CFU-S day12; C) more primitive HPP-CFC, relatively resistant to 5-FU in vivo and closely corresponding to CFU-S day 14, and D) very primitive HPP-CFC, resistant to 5-FU in vitro. This simple, rapid, and versatile method allows the detection of a broad range of hematopoietic progenitors in murine BM, from committed progenitors to largely quiescent, primitive stem cells, as well as the evaluation of the progenitors' self-renewal and proliferative potential.     

Spontaneous alloreactivity of natural killer (NK) and lymphokine-activated killer (LAK) cells from athymic rats against normal haemic cells. NK cells stimulate syngeneic but inhibit allogeneic haemopoiesis.

We wanted to re-examine the hypotheses that natural killer (NK) cells preferentially react with immature cells, and that they are not directed against major histocompatibility complex (MHC) gene products. Rat marrow cells could be separated according to maturity on a four-step discontinuous density gradient of Percoll. Almost all the immature bone marrow cells with progenitor activity, as measured in vivo in a diffusion chamber assay or in vitro in a granulocyte/macrophage colony-forming assay, resided within the lighter density cell fraction (density approximately 1.065). The higher density cells (density approximately 1.082) contained mainly the more mature, non-proliferative cells within the granulocyte series. NK and lymphokine-activated killer (LAK) cells from athymic rats, being devoid of T cells, efficiently killed low- as well as high-density bone marrow cells from a fully allogeneic and a MHC congenic rat strain, while little or no killing was observed against syngeneic bone marrow cell fractions. LAK cells also effectively inhibited granulocyte/macrophage colony formation from allogeneic bone marrow precursors in vitro, while stimulating colony formation from syngeneic bone marrow cells. The NK-mediated killing of allogeneic bone marrow cells was effectively inhibited by NK-sensitive tumour cells, while there was much less inhibition of the killing of tumour cells by allogeneic bone marrow cells. We conclude that NK cells recognize MHC incompatibilities on both immature and mature allogeneic bone marrow cells through recognition systems not related to T-cell receptors, and that allospecific killing can explain the contrasting effect of NK cells on allogeneic and syngeneic haematopoiesis.     

The Effects of Methimazole on Haematopoiesis in Mice

The effects of anti-thyroid drug, methimazole (MMI), on haematopoiesis in inbred C57BL/6 mice were studied. The in vitro proliferative response of bone marrow (BM) cells to interleukin-3 (IL-3) was significantly increased when mice were provided with 0.1% MMI in water (w/v) ad librium for 4 to 6 weeks. Using soft agar agar colony assay, the numbers of myeloid cell colonies were also significantly increased in mice treated with MMI. However, the proliferative response of BM cells to IL-3 was found to be greatly reduced 10 weeks after MMI treatment. In vitro studies showed that MMI alone at the concentrations of 500μM or above inhibited both the growth of normal BM cells in liquid culture and the formation of macrophage (M) - / granulocyte (G) - colonies in soft agar culture in a dose dependent manner. Direct cytotoxic effect of MMI (0 - 1250 μM) to normal BM cells was not observed. Results from this study suggested that MMI can modulate the development of myeloid haematopoietic cells.     

Suppressive effects of human herpesvirus 6 on in vitro colony formation of hematopoietic progenitor cells

Human herpesvirus 6 (HHV-6) has been reported to be involved in bone marrow failure after bone marrow transplantation (BMT). To elucidate the role of HHV-6 in the marrow failure, we examined the comparative effect of two variants of HHV-6 (HHV-6A and HHV-6B) and human herpesvirus 7 (HHV-7) on in vitro colony formation of hematopoietic progenitor cells in methylcellulose semi-solid media. Progenitor cells prepared from cord blood mononuclear cells (CBMNCs) were infected with one of these viruses at various multiplicity of infection (MOI), and were subjected to methylcellulose colony assay. Formation of both granulocyte/macrophage (CFU-GM) and erythroid (BFU-E) colonies was MOI-dependently suppressed after infection with the Z29 strain of HHV-6B. Although HHV-6A suppressed the formation of BFU-E colonies as efficiently as HHV-6B, the former did not exhibit significant suppressive effect on the formation of CFU-GM colonies at an MOI 1. HHV-7 had no effect on hematopoietic colony formation at all. Based on frequent positivity of viral DNA in single colonies obtained from HHV-6-infected progenitor cells by polymerase chain reaction and in situ hybridization, direct effects of HHV-6 on the hematopoietic progenitor cells are suggested as the cause of the suppression rather than indirect effects via accessory cells of the bone marrow. J. Med. Virol. 52:406–412, 1997. © 1997 Wiley-Liss, Inc.