Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34 + cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34 + cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34 + cells on AGM-S3 cells. The expansion potential of CD34 + cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34 + cells were negative for CD38 and cryopreservable. Cultured JMML CD34 + CD38 − cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34 + cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Globin switching of erythrocytes during primate (human and monkey) hematopoietic development has been most thoroughly investigated not only as a model of tissue- and temporally specific transcriptional control but also as a tool for drug discovery against hemoglobinopathies. However, the regulatory mechanisms of globin switching in primates remains to be unresolved in primates mainly due to lack of available model system to date which reproduces the process of hematopoiesis to reflect accurately in vivo development. Recently primate ES cell lines were established, which are expected to serve as an experimental model for tissue growth and development, along with an efficacy and toxicity screening system for new drugs and a cell source for regeneration therapy. Among them, we previously demonstrated that the transition from primitive into definitive erythropoiesis was induced from primate ES cells by coculture with OP9 stromal cells. We also demonstrated that the VEGFR-2high CD34+ cells, emerging onto OP9 stromal layer after initial 6-day differentiation, contain the hemogenic progenitors.

The in vitro proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells in its entirety has not been well delineated because of a lack of an appropriate culture system that mimics the growth pattern in a living body. We applied a NOD/SCID mouse fetal thymus organ culture (FTOC) for leukemic cells from fresh (one case) and frozen (seven cases) bone marrow (BM) samples of children with T-ALL. Cell growth was observed in all seven samples in the culture, reaching a proliferational peak at 4 weeks, and it was calculated that the proliferation potential was 212-to 319-fold. The FTOC-derived T-ALL cells showed similarity to the original cells morphologically and immunophenotypically, still possessed clonalities and were able to regenerate overt leukemia in NOD/SCID mice. These FTOC-derived T-ALL cells differed from ordinary cell lines because they always need FTOC support. Thus, we established a new in vitro culture for T-ALL cells. A comparison of the original and FTOC-derived T-ALL cells revealed that the proportion of cells expressing IL-7R increased in all seven cases. Sorting and re-seeding of FTOC-derived IL-7R+ and IL-7R- cells into secondary FTOC resulted in a predominant generation of IL-7R+ cells from both fractions, while IL-7R- cells proliferated more potently than did IL-7R+ cells, suggesting that a pathway for the conversion of IL-7R- to IL-7R+ exists during the proliferation of T-ALL lymphoblasts. Addition of exogenous IL-7 or neutralization with anti-IL-7 antibody did not influence the growth pattern of T-ALL cells in FTOC. The current study provides a unique assay system for the exploration of the hierarchy within human T-lymphoid leukemic cells, and should facilitate the establishment of novel therapeutic modalities.

Human blood cells, except for erythrocytes and platelets, express CD81, a member of the transmembrane 4 superfamily (TM4SF). CD81 is also expressed on most of human immature hematopoietic cells, CD34+ cells, which are divided into three populations according to the expression of CD34 and CD81; CD34+CD81+, CD34+CD81(High) and CD34(Low)CD81+. Myeloid and lymphoid progenitors exist in the CD34+CD81+ population, and megakaryocytic progenitors are only in CD34(Low)CD81+ population. Erythroid and multipotential progenitors are shared by CD34+CD81+ and CD34(Low)CD81+ populations, but multipotential progenitors in the CD34+CD81+ population have already lost most of their myeloid potential. NK cells and mast cells can be generated from all three populations. Long-term repopulating (LTR) lymphohematopoietic stem cells are present in the CD34+CD81+ population. Based on these findings, we propose a model for the development of CD34+CD81+ lymphohematopoietic stem cells. Along the differentiation cascade from CD34+CD81+ lymphohematopoietic stem cells, there appear to be pathways to CD34(Low)CD81 + or CD34+CD81(High) cells, even if they are indirect. CD34(Low)CD81+ pathways define the loss of LTR ability, and lymphoid and myeloid potentials, whereas CD34+CD81(High) pathways represent the exclusive commitment to NK cells and mast cells.

The early process of T-cell development prior to thymic colonization has been poorly investigated because of the lack of a sensitive assay. We have developed a two-step in vitro culture system by combining a clonal culture with a fetal thymus organ culture (FTOC) and analysed the early development of T cells from lymphohaematopoietic progenitors. Cells of immature colonies derived from bone marrow cells of 5-fluorouracil (5FU)-treated mice using various combinations of early acting cytokines were transferred into a FTOC. All the combinations of stem cell factor (SCF), interleukin (IL)-3 and IL-6 capable of inducing colony formation supported T-cell generation. IL-11 and the Flt3 ligand possessed T-lineage promotional effects similar to IL-6 and SCF respectively. However, there were some quantitative differences in the final T-cell yield among cytokine combinations. Thus, the commitment towards T lineage in lymphohaematopoietic progenitors may be an event determined intrinsically rather than induced by specific stimuli, but there may be a hierarchy between the activity of cytokines in further development. Furthermore, we examined the T-lineage potential of individual colonies derived from Lin(-)c-Kit(+)Sca-1(+) cells clone-sorted from post-5FU marrow cells. No colonies that contained only myelocytic progenitors showed T-lineage potential, but 23.3% of colonies with a haematopoietic multipotentiality did. Therefore, the divergence of the T lineage from other lineages such as myeloid potential may occur at an early stage of the hierarchy of haematopoiesis. The proposed method should prove valuable for exploring the molecular and cellular changes that occur during early T-cell development before thymic colonization.

We found that the stromal cell-derived factor-1/pre-B cell growth-stimulating factor receptor, CXC chemokine receptor 4 (CXCR4), is expressed on human CD34+ bone marrow (BM) cells. Stringently FACS-sorted CD34+CXCR4+ BM cells completely lack myeloid, erythroid, megakaryocytic, and mixed colony-forming potential (myeloid progenitors), but give rise to B and T lymphoid progenitors, whereas CD34+CXCR4- BM cells can generate colonies formed by myeloid progenitors and can also develop into these lymphoid progenitors. Therefore, expression of CXCR4 on CD34+ BM cells can allow lymphoid progenitors to be discriminated from myeloid progenitors. Because CD34+CXCR4+ cells are differentiated from CD34+CXCR4- cells, multipotential progenitors located in the BM are likely to be negative for CXCR4 expression. CXCR4 seems to be expressed earlier than the IL-7R and terminal deoxynucleotidyl transferase during early lymphohemopoiesis. These results suggest that the expression of CXCR4 on CD34+ BM cells is one of the phenotypic alterations for committed lymphoid progenitors.

We recently established a human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent cell line (HML) from colony‐constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All‐trans‐RA, 13‐cis‐RA and 9‐cis‐RA at 10−8 mol/l to 10−5 mol/l inhibited the GM‐CSF‐dependent cell growth. Some of the RA‐treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein‐positive cells. However, eosinophil‐derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL‐5 alone could not stimulate cell growth, the addition of IL‐5 to the cultures containing stem cell factor + all‐trans‐RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.

We investigated the effects of interferon gamma (IFN-gamma) on the growth of murine hematopoietic progenitors. IFN-gamma inhibited granulocyte colony-stimulating factor (G-CSF)- and interleukin-3 (IL-3)-dependent colony growth by granulocyte-macrophage (GM) progenitors derived from the bone marrow cells of normal mice. However, the number of IL-3-dependent GM colonies formed by the bone marrow cells of 5-fluorouracil (5-FU)-treated mice was not influenced by the addition of IFN-gamma. Replating experiments suggested that IFN-gamma suppressed GM colony growth directly and that it exerted an inhibitory effect on the proliferation, but not on the commitment, of GM progenitors. In contrast, IFN-gamma failed to suppress colony growth by mast cell progenitors. Erythroid and megakaryocytic progenitors exhibited different responses to IFN-gamma depending on mouse strains. These results suggest that potent negative regulators are not always inhibitors of hematopoietic progenitors.