Ryuhei Tanaka

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.

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.

Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38- cells expressing Flk2/Flt3.

Summary. In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34+ haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34+ cells and their immature subpopulations, CD34+CD33– and CD34+CD38– cells. Methycellulose clonal culture of sorted CD34+Flk2/Flt3+ and CD34+Flk2/Flt3– cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 × 104 lineage marker‐negative (Lin–)CD34+Flk2/Flt3– cells were transplanted into non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45+ cells in bone marrow 11–12 weeks after the transplantation. In contrast, all recipients transplanted with 1 × 104 Lin–CD34+Flk2/Flt3+ cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin–CD34+CD38–Flk2/Flt3+ cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long‐term‐repopulating haematopoietic stem cells.

Characterization of peripheral blood progenitor cells (PBPC) mobilized by filgrastim (rHuG-CSF) in normal volunteers: dose-effect relationship for filgrastim with the character of mobilized PBPC.

Filgrastim (rHuG‐CSF)‐mobilized peripheral blood progenitor cells (PBPC) in healthy Japanese volunteers were characterized in detail using two clonal cell culture systems and double‐colour flow cytometry to detect multilineage colony‐forming cells and subsets of CD34+ cells. The kinetics of PBPC during the administration of filgrastim was studied, and possible differences in the character of progenitor cells relative to given doses of filgrastim were investigated. Filgrastim was administered subcutaneously to normal volunteers for 7 d at doses of 100, 200 or 400 μg/m2 (10 per cohort). Treatment with 100 or 200 μg/m2 filgrastim was well tolerated; however, the 400 μg/m2 dose level was not completed because of bone pain and myalgia. The treatment strikingly mobilized various types of progenitor cells, including highly proliferative megakaryocytic colony‐forming cells. The number of progenitor cells peaked on days 5 and 6. The fold increase of circulating progenitor cells from the baseline value in the volunteers treated with 200 μg/m2 filgrastim was more pronounced than in those treated with 100 μg/m2. Treatment with 200 μg/m2 also released the less mature progenitor cells (i.e. mixed colony‐forming cells, CD34+/33− cells, and CD34+/HLA‐DR− cells) into circulation better than the 100 μg/m2 dose. These results suggest that daily subcutaneous injection with 200 μg/m2 filgrastim for 5 d will effectively mobilize, both qualitatively and quantitatively, PBPC in healthy donors.

Thrombopoietin-independent effect of interferon-γ on the proliferation of human megakaryocyte progenitors

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon‐γ receptor (IFN‐γR). To clarify the precise functional roles of IFN‐γR in human CD34+ cells, we examined the effect of IFN‐γ alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum‐free clonal culture. Surprisingly, IFN‐γ alone supported only megakaryocyte (MK) colonies in a dose‐dependent manner with a plateau level at 1000 U/ml of IFN‐γ. IFN‐γ at 1000 U/ml induced 10 ± 1.2 MK colonies from 1 × 103 CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)‐3, stem cell factor (SCF) or IL‐6 alone induced 22 ± 4.0, 22 ± 4.2, 4 ± 0.6 and 0 MK colonies, respectively. The addition of anti‐IFN‐γ monoclonal antibody (mAb) to the IFN‐γ culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO‐dependent production of MK colonies. In contrast, although anti‐TPO polyclonal Ab almost completely blocked TPO‐dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN‐γ, suggesting that the observed effect of IFN‐γ on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN‐γ to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL‐3‐dependent MK colony formation. Additionally, IFN‐γ induced the increase of DNA content of cultured glycoprotein IIb/IIIa‐positive megakaryocytes. These results suggest that IFN‐γ may have regulatory roles in human megakaryocytopoiesis.

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells.

Although granulocyte colony‐stimulating factor (G‐CSF) has been reported to act on cells of neutrophilic lineage, the administration of G‐CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G‐CSF (G‐CSFR) on human bone marrow and G‐CSF‐mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G‐CSFR+ and CD34+G‐CSFR− cells using methylcellulose clonal culture. Flow cytometric analysis showed that G‐CSFR was expressed on 14.9 ± 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G‐CSFR+ cells produced only myeloid colonies, whereas CD34+G‐CSFR− cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G‐CSFR− cells generated CD34+G‐CSFR+ myeloid progenitors. In G‐CSF‐mobilized peripheral blood, CD34+ cells contained 10.8 ± 5.8% of G‐CSFR+ cells, most of which were also myeloid progenitors, although CD34+G‐CSFR− cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G‐CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G‐CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G‐CSF in humans.

Role of glycoprotein 130 and c-Kit signaling in proliferation and differentiation of human hematopoietic progenitor cells

Abstract Glycoprotein (gp) 130, a receptor component for interleukin 6 (IL-6), can associate with a soluble IL-6 receptor (sIL-6R) – IL-6 complex. To examine the role of gp130 signaling in human hematopoietic progenitor-cell proliferation and differentiation, we studied the effects of the sIL-6R – IL-6 complex in combination with other cytokines on human CD34+ cells in clonal and suspension cultures. The sIL-6R – IL-6 complex, but not sIL-6R or IL-6 alone, in the presence of stem-cell factor (SCF) produced dramatic increases in the populations of various cell lineages, including erythroid cells and various hematopoietic progenitors, in suspension culture. Significant numbers of colonies of (particularly) multilineage and blast cells were generated in methylcellulose culture supplemented with a combination of sIL-6R – IL-6 complex and SCF. Addition of anti-gp130 monoclonal antibodies (MAbs) and anti-IL-6R MAbs to the above-mentioned cultures dose-dependently inhibited the generation of cells of various lineages and of progenitor cells in suspension culture and completely blocked multilineage colony production in methylcellulose culture; an anti-erythropoietin antibody did not cause inhibition. These findings demonstrate that both proliferation and differentiation of hematopoietic progenitor cells can be induced through gp130 and c-Kit signaling, indicating that progenitor cells are responsive to the sIL-6R – IL-6 complex, even though they do not express IL-6R. Together with previous studies showing that detectable levels of sIL-6R, IL-6, and SCF are present in human serum, these results suggest that gp130 signaling may play an important role in human hematopoiesis in vivo.

Prompt and durable hematopoietic reconstitution by unrelated cord blood transplantation in a child with Fanconi anemia

We describe here the case of an 8-year-old girl with Fanconi anemia (FA) whose hematopoiesis was successfully restored by unrelated umbilical cord blood (UCB) transplantation. The patient became resistant to androgen therapy, and developed intracranial hemorrhage and dyserythropoiesis. Her hematopoietic recovery after the transplantation was excellent and a complete chimerism has been durably maintained. UCB should be considered as a stem cell source for transplantation when a patient with FA does not have an HLA-identical unaffected sibling donor. Bone Marrow Transplantation (2001) 27, 767–769.

A possible change in doubling time of haemopoietic progenitor cells with stem cell development.

We separated haemopoietic progenitors derived from marrow cells of 5‐fluorouracil (5‐FU)‐treated mice into three groups, based on the stages of stem cell development and studied doubling time, using a serum‐free clonal culture system. Stage I progenitors were those present in primary marrow cells from 5‐FU‐treated mice. Stages II and III progenitors were early and late progenies in culture of stage I progenitors, respectively. The morphological analysis of colonies derived from stage I, II and III progenitors demonstrated an association of progression of stages with loss of multipotentiality. The doubling time of haemopoietic progenitors was estimated by sequential analysis of colony formation and studies of growth fraction. The time required for haemopoietic progenitors to double shortened as their stage of development progressed. Alteration in one doubling time of haemopoietic progenitors at progressive stages of stem cell development was seen in cultures supported by various combinations of growth factors, including interleukin‐3 (IL‐3), IL‐11, and steel factor (SF). Cell‐cycle analysis suggested that reduction of the doubling time of haemopoietic progenitors is probably due to a decrease in the time spent in the G1 phase of the cell cycle. Our results suggest that in early haemopoiesis the doubling time of haemopoietic progenitors may change with stem cell development.

Cytokine requirement for the development of T-lymphoid lineage potential in clonal lymphohaematopoietic progenitors in vitro: Development of T Cells from Lymphohaematopoietic Progenitors

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.