Increased DR antigen expression by committed hemopoietic progenitor cells but not primitive progenitor cells in chronic myeloid leukemia

We used a complement-dependent cytotoxic assay to study the expression of DR antigens by hemopoietic progenitor cells derived from normal marrow and from the blood of patients with chronic phase chronic myeloid leukemia (CML). Almost all normal and CML day-12 granulocyte-macrophage colony-forming cells (d12 GM-CFC) and erythroid burst-forming units (BFU-E) expressed DR antigens. A primitive progenitor cell, the "blast colony-forming cell" (Bl-CFC), also expressed DR antigens whether derived from normal marrow or CML blood. The expression of DR antigen by normal and CML Bl-CFC was similar but the density of DR antigens on CML d12 GM-CFC and BFU-E was much greater than that of their counterparts from normal marrow. The similarity in DR antigen density on normal and CML Bl-CFC means that purging of CML marrow using a DR-specific antibody would not select in favor of normal cells and thus would not be useful in an autograft setting.     

Long-term hemopoietic repopulation by Thy-1lo, Lin-, Ly6A/E+ cells.

Murine bone marrow Thy-1lo, Lin-, Ly6A/E+ cells have been isolated by fluorescence-activated cell sorting (FACS). This population contained up to 27% in vitro colony-forming cells (CFC) when stimulated by interleukin 3 (IL-3) and 5% day-12 spleen colony-forming units (CFU-S) (uncorrected for seeding efficiency). As few as 100 cells were able to reconstitute the myeloid and lymphoid compartments of lethally irradiated recipients for periods of up to 72 weeks. Over the range of 30-560 transplanted cells, three patterns of engraftment were observed; the proportion of donor cells increased gradually to values of > 90%, gradually declined, or remained static over the 72-week observation period. When the donor cell percentage in the peripheral blood exceeded 60%, both myeloid (neutrophils and monocytes) and lymphoid (T- and B-lymphocytes) cells were of donor origin. Primary animals containing > 60% donor cells in their peripheral blood were able to engraft secondary recipients with donor cells of both myeloid and lymphoid lineages. Primary animals with < 20% donor cells in their peripheral blood contained bone marrow cells that were only able to produce lymphoid cells in the peripheral blood of secondary recipients. With > 90% of animals, including both primary and secondary recipients, there were no large fluctuations in the proportion of donor myeloid or lymphoid cells in the peripheral blood. Where changes occurred, with three exceptions, these were gradual. These data suggest that the Thy-1lo, Lin-, Ly6A/E+ population is heterogeneous and contains in vitro CFC, day-12 CFU-S, and long-term repopulating cells for both the myeloid and lymphoid lineages and cells capable of long-term repopulation of only the lymphoid lineage.     

Changes in cell surface antigen expression during hemopoietic differentiation

Human bone marrow cells were separated on a fluorescence activated cell sorter (FACS) according to their binding of a series of monoclonal antibodies; the positive and negative fractions were cloned for erythroid burst and colony-forming units (BFU-E and CFU-E) and myeloid colony-forming units (CFU-GM), and cytocentrifuge slides were prepared for microscopy of maturing precursors. The pattern of antigen expression on hemopoietic progenitor and precursor populations has been established using antibodies defining blood group (A, I/i), HLA- associated (*A, B, C, DR, DC1), lineage specific, and transferrin receptor antigens. Like monomorphic HLA-DR, the antigen defined by monoclonal antibody OKT10 is expressed on the earliest progenitors and lost during differentiation, suggesting a role in interactions regulating the differentiation of these cells. The HLA-linked DC1 determinant, in contrast to HLA-DR, is not expressed at a detectable level on progenitor cells. Although a lineage-specific early antigen has not been identified, the transferrin receptor is expressed on the majority of erythroid progenitors, but only weakly on myeloid progenitors, and may provide an approach to isolating erythroid progenitors. These and earlier studies with monoclonal antibodies against HLA-DR and glycophorin now provide a detailed "map" of antigen expression during hemopoietic differentiation.     

Differential activity of saporin 6 on normal and leukemic hemopoietic cells

The antiproliferative effect of saporin 6 (SO6), a ribosome-inactivating protein (RIP) purified from the seeds of Saponaria officinalis has been tested on three leukemic cell lines (K562, U937, and HL60), human normal bone marrow, and peripheral blood hemopoietic progenitor cells from normal subjects. In leukemic cell lines, SO6 appeared much more effective against erythrocytic than against monocytic and promyelocytic leukemic cells, as shown by protein synthesis assays carried out after up to 72 h of culture. Among the normal hemopoietic progenitor cells, erythroid burst-forming units were the most affected, with results similar to those observed in the erythroid leukemic cell line, both in treated and in pretreated cultures, with strong damage after 24 h of exposure to SO6. On the other hand, granulocyte-macrophage colony-forming units (CFU-GM) from bone marrow were significantly more affected than the myeloid leukemic cell lines after permanent treatment with the inhibitor, the damage being significantly lower after an exposure of 24 h. CFU-GM from peripheral blood and megakaryocyte CFU showed an intermediate sensitivity after 24 h of exposure to SO6, similar to that of the other normal precursors after permanent treatment with the drug.     

Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule.

We explored the expression of a lymphocyte homing-associated cell adhesion molecule (H-CAM, CD44) on hematopoietic progenitors. We demonstrate that immature myeloid and erythroid leukemic cell lines stain intensely with monoclonal antibodies Hermes-1 and Hermes-3, which define distinct epitopes on lymphocyte surface H-CAM, a glycoprotein involved in lymphocyte interactions with endothelial cells. Using fluorescence-activated cell sorting (FACS), human marrow cells were fractionated into Hermeshi, Hermesmed, and Hermeslo populations according to the expression of both the Hermes-1 and Hermes-3 epitopes. Granulocyte-macrophage colony-forming unit and erythroid burst-forming unit precursors were found predominantly in the brightly positive fractions. Two-color FACS analysis confirmed that the My10 (CD34) positive populations of cells in bone marrow, which contain most of the progenitor cell activity, are brightly positive for Hermes-1. Finally, we demonstrate that among bone marrow cells, the highest levels of H-CAM are expressed on myeloid and erythroid progenitors as well as mature granulocytes and lymphocytes. Thus we provide evidence that molecules related or identical to the H-CAM homing receptor are expressed on marrow progenitor cells. H-CAM may contribute to progenitor cell interactions with marrow endothelial and stromal cell elements important to the maintenance and regulation of hematopoiesis.     

Expression of intercellular adhesion molecule-1 (CD54) on hematopoietic progenitors.

The distribution of intercellular adhesion molecule-1 (ICAM-1), a ligand for lymphocyte function antigen-1, on hematopoietic tissue was determined using the anti-ICAM-1 monoclonal antibody CL203.4 with flow cytometry and short-term semi-solid hematopoietic progenitor cultures. After timed incubation in media with fetal bovine serum, 29% of erythroid burst-forming units (BFU-E), 24% of erythroid colony-forming units (CFU-E), and 52% of granulocyte-macrophage colony-forming units (CFU-GM) bone marrow progenitors expressed ICAM-1. This finding, which is consistent with the detection of ICAM-1 on acute non-lymphoblastic leukemic blasts, is at variance with recent reports. ICAM-1 was also detected on bone marrow blasts, proerythroblasts, promyelocytes, and cells of monocyte/macrophage lineage, but was not detected on erythroblasts, normoblasts, neutrophilic myelocytes, metamyelocytes, bands, or on most lymphocytes. These results indicate that maturation of cells of the erythroid and myeloid lineage is associated with loss of ICAM-1. The distribution of ICAM-1 on bone marrow progenitors, early precursor cells, and accessory cells in conjunction with the function of this molecule in cell-cell interactions suggests that ICAM-1 may play a role in the cell-cell and cell-stromal interactions that regulate hematopoiesis.     

Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TÜ 67)

Summary Anti-TÜ 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TÜ 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TÜ 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TÜ 67 antibody.Supported by the Deutsche Forschungsgemeinschaft (He 1380-2/1)     

The effect of a prolonged in vitro exposure to 1-beta-D arabinofuranosylcytosine and deoxycytidine on the survival of normal (CFU-GM) and leukemic (L-CFU) human myeloid progenitor cells in suspension culture

We examined the effect of a 96-h exposure to 1-beta-D arabinofuranosylcytosine (Ara-C) and deoxycytidine (dCyd) in medium lacking an exogenous source of colony-stimulating activity (phytohemagglutinin-stimulated leukocyte-conditioned medium, PHA-LCM) on the survival of normal human committed myeloid progenitor cells (day-7 and day-14 granulocyte-macrophage colony-forming units [CFU-GM] as well as leukemic progenitors (leukemic colony-forming units, L-CFU) derived from myeloblasts obtained from 13 patients with acute nonlymphocytic leukemia (ANLL). Coadministration of Ara-C (20-50 microM) in conjunction with dCyd (50-100 microM) permitted the survival of an average of 9%-57% of day-7 CFU-GM and 32%-65% day-14 CFU-GM, depending upon the relative concentrations of dCyd and Ara-C. In contrast, exposure of leukemic myeloblasts to identical regimens resulted in considerably greater reductions in L-CFU survival, which in general exceeded 90% and in some cases was 100%. In addition, exposure of leukemic myeloblasts to Ara-C and dCyd for 96 h in culture medium lacking PHA-LCM eliminated the secondary plating efficiency (PE2) of leukemic colonies in 11 of 13 samples assayed and reduced values dramatically in the remaining 2. Substantial preservation of CFU-GM formation was also noted when normal bone marrow samples depleted of T cells and marrows obtained from two patients with ANLL in remission were assayed. These studies suggest that in contrast to certain normal committed myeloid progenitor cells, leukemic progenitors, particularly those with self-renewal capacity, are highly vulnerable to a prolonged exposure to Ara-C and dCyd in the absence of an exogenous source of colony-stimulating activity. They also raise the possibility that a combined regimen utilizing chemotherapeutic agents in conjunction with modifications of long-term culture techniques may represent a novel approach to the ex vivo purging of leukemic cells from bone marrow in conjunction with autologous bone marrow transplantation.     

Analysis of human hemopoietic progenitor cells for the expression of glycoprotein IIIa

Human hemopoietic progenitor cells were examined for the expression of glycoprotein IIIa (GPIIIa). This protein, which forms the beta-subunit of the GPIIb/IIIa receptor for cytoadhesive proteins as well as the beta-subunit of the vitronectin receptor, represents the most sensitive cell surface marker so far identified for the megakaryocytic lineage. Bone marrow cells were fractionated by a discontinuous Percoll gradient to separate cells that form megakaryocytic colonies in culture (1.05 greater than rho less than 1.077 g/ml). Density centrifugation was followed by indirect immunopanning to select for an enriched population of progenitor cells depleted of most of the mature cells of the myeloid, lymphoid, and erythroid lineages. This cell suspension was labeled with antibodies directed against determinants of GPIIIa and analyzed using a fluorescence-activated cell sorter (FACS). Fractions of cells were sorted and analyzed for the ability to form hemopoietic colonies in culture. Our study demonstrated that megakaryocytic progenitor cells (CFU-M) as well as granulocyte-macrophage colony-forming units (CFU-C), erythroid colony-forming units (BFU-E), and mixed lineage colony-forming units (CFU-GEMM) express HLA-DR antigens but lack GPIIIa. Therefore GPIIIa represents a marker that is not present on hemopoietic progenitor cells, but is expressed on the progenies of CFU-M. In view of the importance of GPIIIa as a component of receptors for cytoadhesive proteins, this finding may help to elucidate the adhesive interactions between early hemopoietic cells and bone marrow interstitium.     

Effect of lenalidomide therapy on hematopoiesis of patients with myelodysplastic syndrome associated with chromosome 5q deletion

Background

Lenalidomide improves erythropoiesis in patients with low/intermediate-1 risk myelodysplastic syndrome and interstitial deletion of the long arm of chromosome 5 [del(5q)]. The aim of this study was to explore the effect of lenalidomide treatment on the reserves and functional characteristics of bone marrow hematopoietic progenitor/precursor cells, bone marrow stromal cells and peripheral blood lymphocytes in patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q).

Design and Methods

We evaluated the number and clonogenic potential of bone marrow erythroid/myeloid/megakaryocytic progenitor cells using clonogenic assays, the apoptotic characteristics and adhesion molecule expression of CD34⁺ cells by flow cytometry, the hematopoiesis-supporting capacity of bone marrow stromal cells using long-term bone marrow cultures and the number and activation status of peripheral blood lymphocytes in ten patients with low/intermediate-1 risk myelodysplastic syndrome with del(5q) receiving lenalidomide.

Results

Compared to baseline, lenalidomide treatment significantly decreased the proportion of bone marrow CD34+ cells, increased the proportion of CD36⁺/GlycoA⁺ and CD36⁻/GlycoA⁺ erythroid cells and the percentage of apoptotic cells within these cell compartments. Treatment significantly improved the clonogenic potential of bone marrow erythroid, myeloid, megakaryocytic colony-forming cells and increased the proportion of CD34⁺ cells expressing the adhesion molecules CD11a, CD49d, CD54, CXCR4 and the SLAM antigen CD48. The hematopoiesis-supporting capacity of bone marrow stroma improved significantly following treatment, as demonstrated by the number of colony-forming cells and the level of stromal-derived factor-1α and intercellular adhesion molecule-1 in long-term bone marrow culture supernatants. Lenalidomide treatment also increased the proportion of activated peripheral blood T lymphocytes.

Conclusions

The beneficial effect of lenalidomide in patients with lower risk myelodysplastic syndrome with del(5q) is associated with significant increases in the proportion of bone marrow erythroid precursor cells and in the frequency of clonogenic progenitor cells, a substantial improvement in the hematopoiesis-supporting potential of bone marrow stroma and significant alterations in the adhesion profile of bone marrow CD34⁺ cells.     

Comparison of in vitro inhibition of etoposide (VP16) on leukemic and normal myeloid, erythroid clonogenic cells

We have compared in various clonogenic assays the in vitro sensitivity to etoposide (VP16) of 1) human leukemic precursors (leukemia colony-forming units; L-CFU), 2) normal erythroid progenitors (erythroid burst-forming units; BFU-E, and 3) normal committed myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM and more primitive hemopoietic precursors (PPC) that adhere to cultured marrow stromal cells. Bone marrow samples were obtained from 15 normal subjects and 16 leukemic patients: 9 in the acute phase of acute nonlymphoblastic leukemia (ANLL) and 7 in complete remission. VP16 was tested at concentrations ranging from 10(-8) to 10(-3) M. The median recoveries at 10(-3) M VP16 were respectively 0%, 0.5%, 0%, and 0% for leukemic progenitors, CFU-GM from leukemic patients in complete remission, normal CFU-GM, and BFU-E, and 23% for PPC. This indicates that CFU-GM, BFU-E, and L-CFU are highly sensitive to VP16, whereas PPC, more primitive myeloid precursors, are spared. These results suggest that VP16 may be used as an "ex vivo" purging agent for autologous bone marrow.     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

Autoimmune pancytopenia: Lymphocyte inhibition of autologous but not allogenic bone marrow growth in vitro

A patient with autoimmune renal failure, cavitary lung lesions and arthritis experienced pancytopenia while prednisone therapy was being tapered. Utilizing semisolid culture techniques, a population of nonadherent peripheral blood mononuclear cells was demonstrated, which inhibited autologous but not allogeneic bone marrow erythroid colony-forming units (CFU-E) and myeloid colony-forming units (CFU-c) in vitro. No inhibition of CFU-E or CFU-c colony formation was seen when patient's serum or immunoglobulin G (IgG) was added to cultures. Reinstitution of prednisone therapy resulted in normalization of peripheral blood counts, which was accompanied by the loss of the hemopoietic inhibitor cell activity in the patient's peripheral blood. These results demonstrate the need for testing autologous marrow samples when looking for possible immunemediated inhibition of hematopoiesis.     

The tetrapeptide AcSDKP, an inhibitor of the cell-cycle status for normal human hematopoietic progenitors, has no effect on leukemic cells.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) inhibits the entry into DNA synthesis of murine spleen colony-forming units (CFU-S) and protects these cells during chemotherapy. This synthetic peptide also inhibits the growth of normal human marrow progenitors granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) and decreases their percentage in DNA synthesis at nanomolar concentration. In view of its clinical application as a marrow protector, we have investigated its effects on malignant cells. Studies were carried out on HL-60 cells and on fresh leukemic cells from patients with either chronic myeloid leukemia (CML) or acute myeloid leukemia (AML). Results showed that AcSDKP, whatever the doses used, did not modify the proliferation of both HL-60 cells and AML cells even when enhanced by stimulating factors such as interleukin 3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, no change in the number and the percentage in S-phase of both HL-60 clonogenic cells and CML progenitors was observed. Our data clearly demonstrate that the tetrapeptide AcSDKP was ineffective on leukemic cells and therefore by acting selectively on normal progenitors represents a potent therapeutical agent for the protection of normal bone marrow progenitors during chemotherapy.     

Immunophenotypic demonstration of two natural killer surface markers, H25 and H366, on fresh human leukemic cells

Using a modified alkaline-phosphatase/antialkaline-phosphatase method for phenotyping fresh human leukemias, we could demonstrate peripheral blood and bone marrow-derived blast cells to specifically react with two monoclonal antibodies (MoAbs), H25 and H366, previously shown to recognize natural killer cells, activated T lymphocytes and a proportion of normal hematopoietic precursor cells. MoAbs H25 and H366 were found to identify the majority of leukemic cells in patients presenting with T-ALL, LGL leukemia, pre-B-ALL, CML, and AML, respectively.     

Anti-K562 cell monoclonal antibodies recognize hematopoietic progenitors.

The K562 leukemia cell has properties of self-renewal and pluripotency similar to those of the hematopoietic stem cell. Monoclonal antibodies to K562 cells have been produced by using hybridoma technology. By radioimmunoassay, some anti-K562 cell antibodies also bind to erythrocyte antigens or peripheral blood mononuclear cells; others are more specific for K562 cells. Antibody binding to hematopoietic progenitors was assayed by using the ability of these cells to form colonies in vitro. After exposure of human bone marrow cells to anti-K562 antibodies and complement, myeloid or erythroid colony formation was inhibited. Some of the inhibitory antibodies showed little binding to mature blood cells by radioimmunoassay, immunofluorescence, and complement cytotoxicity, suggesting that they recognize antigens specific to undifferentiated cells. With the fluorescence-activated cell sorter, one inhibitory antibody was shown to stain only 3% of bone marrow cells. Inhibitory anti-K562 antibodies also bind to myelogenous leukemia cells and virus-transformed lymphocytes. Thus, these antibodies appear to recognize antigens shared by normal hematopoietic progenitors, leukemic cells, and transformed lymphocytes.     

Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.     

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).     

Effects of juzen-taiho-toh (TJ-48), a traditional Oriental medicine, on hematopoietic recovery from radiation injury in mice

Effects of Juzen-taiho-toh (TJ-48) on the recovery of hemopoietic systems from radiation injury are analyzed. Female C57BL/6N mice (6-8 weeks old) were irradiated at doses of 1, 2, 3, 5, or 7 Gy from a 60Co source. After irradiation, the mice were given TJ-48 (1.25 g in 100 ml drinking water). Seven days after irradiation, the mice were sacrificed, and bone marrow (both femurs), thymus, spleen, and peripheral blood counts were made. The bone marrow cells were used for fibroblast colony-forming unit (CFU-f), spleen colony-forming unit (CFU-S), granulocyte-macrophage colony-forming unit (CFU-GM), erythroid colony-forming unit (CFU-E), and erythroid burst-forming unit (BFU-E) assays. No difference was observed between the experimental and control groups except for CFU-S counts. In the assay for day-14 CFU-S, the mice injected with TJ-48-treated bone marrow cells showed better general condition (including increased body weight) and heavier spleens with larger and more numerous colonies. The effect of TJ-48 does not seem to be elicited via the hemopoietic microenvironment, because mice that had been given TJ-48 before irradiation at 8 Gy and then injected with syngeneic bone marrow cells did not show enhanced day-14 CFU-S counts. These results suggest that TJ-48 manifests a radioprotective effect by increasing the number and size of day-14 CFU-S.