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c-kitis expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker-negative, c-kit-positive (Lin–c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin–c-kit+Sca-1+cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 × 103of Linc+Sca-1– cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin–c-kit+Sca-1+cells are primitive hematopoietic stem cells and that Sca-1– cells do not contain stem cells that reconstitute hematopoiesis. Lin–c-kit+Sca-1+cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin– c-kit+Sca-1– cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+to c-kit+Sca-1– increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kitis found both on the primitive hematopoietic stem cells and progenitors, Sca-1+cells are more primitive and respond better than Sca-1– cells to a combination of hematopoietic factors, including SCF and stromal cells.

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 × 103blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 × 103blast cells) donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.

To study the relationship between hematopoietic factors and their responsive hematopoietic progenitors in the differentiation process, both purified factors and enriched progenitors are required. We isolated total CD34+cells, CD34+, CD33+cells, and CD34+, CD33–cells individually from normal human bone marrow cells by fluorescence-activated cell sorter (FACS), and examined the effects of granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3), and IL-5 on in vitro colony formation of these cells. CD34+,CD33+cells formed granulocyte colonies in the presence of G-CSF. Both CD34,CD33+cells and CD34+,CD33–cells formed granulocyte/macrophage colonies in the presence of IL-3. Eosinophil (Eo) colonies were only formed by CD34+,CD33–cells in response to IL-3, but scarcely formed by CD34+cells in the presence of IL-5. We performed the two-step cultures consisting of the primary liquid culture for 6 days and the secondary methylcellulose culture, and serially examined changes in phenotypes of the cells cultured in the primary culture. CD34–,CD33+cells derived from CD34+,CD33+cells by preincubation with G-CSF or IL-3 formed Eo colonies in the presence of IL-5 but not IL-3. CD34–,CD33+cells derived from CD34+, CD33–cells by preincubation with IL-3 also formed Eo colonies by support of IL-5 as well as IL-3. Both CD34+cells gradually lost the CD34 antigen by day 6 of incubation with G-CSF or IL-3. Loss of this antigen was well-correlated with aquisition of susceptibility to IL-5. It was concluded that G-CSF supported the neutrophil differentiation of committed colony-forming cells, IL-3 supported that of both committed and multipotent colony-forming cells. G-CSF and IL-3 also supported the early stage of E. differentiation; IL-5 supported the late stage of that.

CD4+cells comprise approximately 3% to 6% of murine bone marrow (BM) cells. The majority are CD4dull+, but there are two distinct sub populations: CD4 brightly positive Gr-1−cells (CD4hiGr-1-) and CD4+Gr-1 +cells (CD4loGr-1lo). CD4hiGr-1−cells are considered to be mature T cells by cell surface antigen expression and morphology. CD4loGr-1locells, which comprise approximately 0.6% of the BM cells, express small amount of B220 and Thy1 antigens. Interestingly, colony-forming units (CFU)-spleen and CFU-C are not enriched in this population. However, when injected into lethally irradiated mice, CD4loGr-1locells were shown to differentiate into T-cell, B-cell, and myelo-monocyte lineages when assayed 26 weeks after transplantation. Furthermore, donor-derived CD4loGr-1locells were present in the recipients’ BM at least 16 weeks after transplantation. These observations suggest that murine CD4loGr-1locells in BM have self-renewal capability and retain the ability to differentiate into at least three lineages in long-term hematopoiesis.

To clarify the phenotypes of various classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with CD34 and a newly developed mouse antihuman c-kitproto-oncogene product (KIT) monoclonal antibody (MoAb). We characterized three cell fractions in CD34+cells that express KITlowand KIThighcells in addition to KIT–cells. A clonogenic assay showed that most granulocyte-macrophage colony-forming cells (GM-CFC) were present in CD34+KIThighpopulations, whereas erythroid burst-forming cells (BFU-E) were detected mainly in the CD34+KITlowpopulation. CD34+-KIT–fraction contained a small number of BFU-E. Morphologic analysis showed that blast-like cells were more enriched in the CD34+KITlowfraction. KITlowcells contained CD34+CD38–cells that were considered to be very primitive progenitor cells, as determined by a replating assay. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell functions by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At week 2, more CFC recovered from the culture in the fraction initiated with a CD34+KIThighpopulation. However, more LTC-IC were present during weeks 5 to 9 in the CD34+KITlowpopulation. These results indicate that primitive progenitors are more enriched in the KITlowpopulation and that the KIThighpopulation contains many GM-committed progenitor cells. We also showed that anti-KIT MoAb inhibited the ability of CD34+cells to generate CFC on the stromal layer in the LTC system. This suppressive effect was more evident in the generation of BFU-E by CD34+KITlowcells. Moreover, we confirmed that CD34+KIThighcells emerged from CD34+KITlowcells during coculture with allogeneic stromal cells or from liquid culture in the presence of stem cell factor (SCF), interleukin-6, and erythropoietin. These results emphasize the pivotal role of the KIT and SCF interaction in hematopoiesis and indicate that KITlowcells are more primitive than KIThighcells.

The proto-oncogene c-kitencodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kitmolecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kitmolecule (approximately 5%) were B220(B)−, TER119(erythroid)−, Thy1negative-low, and WGA+. A small number of Mac-1(macro-phage)+or Gr-1(granulocyte)+cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)−c-kit+cells were rhodamine-123lowand this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin−WGA+c-kit+cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin−c-kit+cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 μg ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kitmolecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.

Natural killer (NK) cells can be cultured in interleukin-2 (IL-2)–containing medium from selected human bone marrow (BM) cells obtained after the elimination of mature T and NK cells. To isolate and characterize IL-2—responsive NK progenitors in the selected BM cells, we investigated the expression of IL-2 receptors (IL-2R) on these cells. Neither CD25 (IL-2Rα) nor IL-2Rβ antigen was observed on the selected BM cells before culture. However, CD25+cells without detectable levels of IL-2Rβ antigen appeared 24 hours after culture in IL-2-containing medium. Cells were sorted from each fraction of the selected BM cells 24 hours after culture after staining with anti-CD33, anti-CD34, and anti-CD25 monoclonal antibodies. The generation of NK cells (CD3–CD56+cells) and NK activity were observed only from the CD33–/CD34–/CD25+cell fraction after culture in IL-2—containing medium. The frequency of IL-2–responsive NK progenitors relative to the fraction was 1/231 (95% confidence range, 1/156 to 1/289), which corresponded to the frequency relative to the total number of selected BM cells when the frequency relative to the CD33–/CD34–/CD25+cell fraction was converted according to the percentage of these cells in the total number of selected BM cells. These results indicated that IL-2—responsive NK progenitors were enriched in the CD33–/CD34–/CD25+cell fraction.

We confirmed that murine hematopoietic stem cells express the c-kitmolecule but not lympho-hematopoietic lineage markers. These lineage marker-negative c-kit-positive (Lin–c-kit+) cells were further divided according to the uptake of rhodamine-123 (Rh-123). Approximately 1.000 Lin-c-kit+rhodamine-123dullcells, which contained 4.0 ±1.3 and 12.5 ±1.9 day 8 and day 12 spleen colony-forming units (CFU-S), respectively, rescued the 100% of lethally irradiated mice. One third of these cells formed colonies in the presence of interleukin-3 plus erythropoietin. The time course of the hematopoietic reconstitution of this primitive hematopoietic stem cell fraction was investigated by using Ly-5 congenic mice. Although myeloid cells and B lymphocytes were detected in the peripheral blood 2 to 3 weeks after transplantation, T lymphocytes were not detected until 4 weeks after transplantation. It is generally assumed that myeloid cells and B lymphocytes grow in the bone marrow and that T lymphocytes must pass through the thymus. For the first 2 to 3 weeks after transplantation, donor-type T lymphocytes were not dominant in the thymus, and most donor type cells were CD4/CD8 double-negative or double-positive (including CD4lowand CD8low). Four weeks after transplantation, donor-type T lymphocytes were dominant and the ratio of CD4/CD8 cells had recovered to the normal pattern. However, significant numbers of T lymphocytes were detected in the peripheral blood at this stage. Sequential analysis of hematopoietic reconstitution from primitive stem cells demonstrates that myeloid and B-lymphoid lineages occurred earlier than that of the T-lymphoid lineages.

Advances in fluorescence-activated cell sorter technology have brought about multicolor analysis of cell phenotypes. To clarify the phenotypes of human hematopoietic stem cells (HSCs), we initially prepared novel antibodies against CD34 and labeled one of them (4A1) with allophycocyanin (APC). With this, we analyzed the phenotypes of CD34+HSCs and showed that primitive HSCs or CD34+CD33-cells expressed adhesion molecules such as CD43, CD44, CD11a, CD11c, CD18, and leukocyte adhesion molecule (LAM-1). The more primitive hematopoietic cells or CD34+CD38-cells also expressed CD11a and CD18 with an incidence of 20% to 30%. To clarify the role of adhesion molecules in HSCs, we examined the colony forming capacity after long-term culture with allogeneic irradiated stromal layers. Among CD34+CD33-cells, CD18+cells gave rise to colony-forming cells (CFCs) on stromal layers, but reached a maximum at week 2, after which the number of generated CFCs decreased. On the other hand, CD18-cells generated less CFCs than CD18+cells at 2 to 3 weeks, but increased after 4 weeks of culture. When CD18 or CD11a antibody was added to a coculture system of CD34+CD33-cells with stromal layers, the number of generated CFCs decreased significantly compared with the no antibody control. Leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) was expressed on some populations of hematopoietic cells and contributed to the proliferation by interacting with stromal cells. However, more primitive cells capable of reconstituting hematopoiesis did not express LFA-1. These data provide a rationale for the administration of anti-LFA-1 antibody after bone marrow transplantation for reducing the graft failure.