Kohichiro Tsuji

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis

A critical issue for clinical utilization of human ES cells (hESCs) is whether they can generate terminally mature progenies with normal function. We recently developed a method for efficient production of hematopoietic progenitors from hESCs by coculture with murine fetal liver-derived stromal cells. Large numbers of hESCs-derived erythroid progenitors generated by the coculture enabled us to analyze the development of erythropoiesis at a clone level and investigate their function. The results showed that the globin expression in the erythroid cells in individual clones changed in a time-dependent manner. In particular, embryonic ε-globin-expressing erythroid cells from individual clones decreased, whereas adult-type β-globin-expressing cells increased to ≈100% in all clones we examined, indicating that the cells undergo definitive hematopoiesis. Enucleated erythrocytes also appeared among the clonal progeny. A comparison analysis showed that hESC-derived erythroid cells took a similar differentiation pathway to human cord blood CD34+ progenitor-derived cells when examined for the expression of glycophorin A, CD71 and CD81. Furthermore, these hESC-derived erythroid cells could function as oxygen carriers and had a sufficient glucose-6-phosphate dehydrogenase activity. The present study should provide an experimental model for exploring early development of human erythropoiesis and hemoglobin switching and may help in the discovery of drugs for hereditary diseases in erythrocyte development.

Control of B Cell Production by the Adaptor Protein Lnk: Definition of a Conserved Family of Signal-Modulating Proteins

Lnk is an SH2 domain-containing adaptor protein expressed preferentially in lymphocytes. To illuminate the importance of Lnk, we generated lnk(-/-) mice. Whereas T cell development was unaffected, pre-B and immature B cells accumulated in the spleens. In the bone marrow, B-lineage cells were proportionately increased, reflecting enhanced production of pro-B cells that resulted in part from hypersensitivity of precursors to SCF, the ligand for c-kit. Hence, Lnk ordinarily acts to regulate B cell production. Further characterization of lnk(-/-) mice also revealed that full-length Lnk is a 68 kDa protein containing a conserved proline-rich region and a PH domain. Lnk is a representative of a multigene adaptor protein family whose members act, by analogy with Lnk, to modulate intracellular signaling.

The Role of mel-18, a Mammalian Polycomb Group Gene, during IL-7–Dependent Proliferation of Lymphocyte Precursors

mel-18 is a mammalian homolog of Drosophila melanogaster Polycomb group genes. Mice lacking the mel-18 gene show a posterior transformation of the axial skeleton, severe combined immunodeficiency, and a food-passing disturbance in the lower intestine due to hypertrophy of the smooth muscle layer. In this study, the severe combined immunodeficiency observed in mel-18 mutant mice is correlated with the impaired mitotic response of lymphocyte precursors upon interleukin-7 stimulation. Strikingly, the axial skeleton and lymphoid phenotypes are identical in both mel-18 and bmi-1 mutants, indicating that the Mel-18 and Bmi-1 gene products might act in the same genetic cascade. These results suggest that mammalian Polycomb group gene products are involved in cell cycle progression in the immune system.

Extensive proliferation of mature connective-tissue type mast cells in vitro.

There are two phenotypically distinct subpopulations of mast cells in rodents: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). These populations differ in their location, cell size, staining characteristics, ultrastructure, mediator content and T-cell dependency1–3. Several investigators4–9 recently reported a further subclass of mast cells which arise when normal mouse haematopoietic cells are cultured with interleukin-3 (IL-3); IL-3 is an activity similar or identical to mast-cell growth factor, histamine-producing factor, or P-cell stimulating factor4–9. These cultured mast cells are in many ways similar to MMC; they stain with Alcian blue but not safranin, contain chondroitin sulphate E proteoglycan rather than heparin proteoglycan and have relatively low histamine content, as do MMC10–13. Although proliferation of MMC is known to be T-cell dependent in vivo and thought to be IL-3-dependent in vitro, the factors on which CTMC proliferation depends remain elusive. Here we show that mature CTMC purified from mouse peritoneal cells can proliferate in vitro in methylcellulose culture and maintain the appearance and function of CTMC. We also present evidence that mature CTMC cannot proliferate in the presence of pure IL-3 alone.

Defective response to thrombopoietin and impaired expression of c‐mpl mRNA of bone marrow cells in congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon disorder in newborns and infants, characterized by isolated thrombocytopenia and megakaryocytopenia in the first year without physical anomalies. The defect of thrombopoiesis is not well understood. Recently, thrombopoietin (TPO), the ligand for the c‐mpl receptor, was cloned. Accumulating evidence from in vitro and in vivo studies indicate that TPO plays a key role in the regulation of megakaryocytopoiesis. In this study we examined the effect of TPO on megakaryocyte colony formation from a patient with CAMT using a plasma‐containing methylcellulose clonal culture. The in vitro results demonstrated a defective response to TPO in megakaryocyte colony formation from bone marrow mononuclear cells (MNC) of the patient, although interleukin‐3 (IL‐3) but not stem cell factor (SCF) induced only a small number of megakaryocyte colonies. These findings indicated that thrombocytopenia in CAMT could not be corrected by administration of TPO in vitro. Additionally, clonal cultures containing SCF, IL‐3, IL‐6 and erythropoietin showed decreased numbers of erythroid and myelocytic progenitors in the bone marrow of the patient. The serum TPO level measured by enzyme‐linked immunosorbent assay was significantly higher than that in healthy controls. By PCR, marrow MNC from healthy children and from a patient with essential thrombocytosis expressed c‐mpl mRNA, whereas no c‐mpl mRNA was detected in marrow MNC from the patient with CAMT. There was no difference in the CD34 expression and c‐kit mRNA between the CAMT patient and healthy children. The results of this study suggest that the pathophysiology in CAMT may be a defective response to TPO in haemopoietic cells through impaired expression of c‐mpl mRNA.

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long‐term culture system using the murine bone marrow stromal cells MS‐5 to support the growth of progenitor B cells with CD34–, CD10+, CD19+, and cytoplasmic μ chain (Cμ)‐negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 103 CB CD34+ cells/well were seeded on MS‐5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony‐stimulating factor (rhG‐CSF), but not rhIL‐7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS‐5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a Cμ+ phenotype, and a small number of cells had immunoglobulin μ chain on their cell surface in the presence of both rhSCF and rhG‐CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS‐5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG‐CSF, and that a species‐cross‐reactive membrane‐bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS‐5 stromal cells. Further studies are required to investigate the mechanism how rhG‐CSF acts on progenitor B cells to allow their proliferation and differentiation.

Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture

We examined the effects of recombinant human thrombopoietin (TPO, c‐Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum‐free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34+ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)‐3, IL‐6, stem cell factor, erythropoietin, granulocyte‐macrophage colony‐stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte‐macrophage (GM) colonies, erythroid bursts (E), granulocyte‐erythrocyte‐macrophage‐megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone‐sorted CD34+ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum‐free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long‐term culture‐initiating cells. These effects were completely abrogated by anti‐TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.

Hematopoietic Capability of CD34^+ Cord Blood Cells: A Comparison With CD34^+ Adult Bone Marrow Cells

The characteristics of hematopoietic progenitor and stem cell (HPC/HSC) populations in mammals vary according to their ontogenic stage. In humans, HPC/HSCs from umbilical cord blood (CB) are increasingly used as an alternative to HPC/HSCs from adult bone marrow (BM) for the treatment of various hematologic disorders. How the hematopoietic activity of progenitor and stem cells in CB differs from that in adult BM remains unclear, however. We compared CD34+ cells, a hematopoietic cell population, in CB with those in adult BM using phenotypic subpopulations analyzed by flow cytometry, the colony-forming activity in methylcellulose clonal cultures, and the repopulating ability of these cells in NOD/Shi-scid (NOD/SCID) mice. Although the proportion of CD34+ cells was higher in adult BM than in CB mononuclear cells, the more immature subpopulations, CD34+CD33- and CD34+CD38- cells, were present in higher proportions in CD34+ CB cells. Clonal culture assay showed that more multipotential progenitors were present in CD34+ CB cells. When transplanted into NOD/SCID mice, CD34+ adult BM cells could not reconstitute human hematopoiesis in recipient BM, but CD34+ CB cells achieved a high level of engraftment, indicating that CD34+ CB cells possess a greater repopulating ability. These results demonstrated that human hematopoiesis changes with development from fetus to adult. Furthermore, CD34+ CB cells contained a greater number of primitive hematopoietic cells, including HSCs, than did adult BM, suggesting the usefulness of CD34+ CB cells not only as a graft for therapeutic HSC transplantation but also as a target cell population for ex vivo expansion of transplantable HSCs and for gene transfer in gene therapy.

Novel Method for Efficient Production of Multipotential Hematopoietic Progenitors from Human Embryonic Stem Cells

We propose a novel method for the efficient production of hematopoietic progenitors from human embryonic stem cells (hESC) via coculture with murine fetal liver-derived stromal cells, in which embryonic hematopoiesis dramatically expands at midgestation. We generated various hematopoietic progenitors in coculture, and this hematopoietic activity was concentrated in cobblestone-like cells derived from differentiated hESC. The cobblestone-like cells mostly expressed CD34 and retained an endothelial cell potential. They also contained hematopoietic colony-forming cells, especially erythroid and multilineage colony-forming cells at high frequency. The multipotential hematopoietic progenitors abundant among the cobblestone-like cells produced almost all types of mature blood cells, including adult-type β-globin-expressing erythrocytes and tryptase/chymase double-positive mast cells. These progenitors showed neither the immature properties of ESC nor the potential to differentiate into endoderm and ectoderm at a clonal level. The coculture system developed for hESC can provide a novel source of hematopoietic and blood cells for applications in cellular therapy and drug screening.