Takafumi Kimura

Thrombopoietin augments ex vivo expansion of human cord blood-derived hematopoietic progenitors in combination with stem cell factor and flt3 ligand

We studied the effects of stem cell factor (SCF) and flt3 ligand (FL) on the ex vivo expansion of human umbilical cord blood (CB)-derived CD34+ cells in combination with various cytokines, including interleukin (IL)-3, IL-6, IL-11, and c-Mpl ligand (thrombopoietin, TPO), in a short-term serum-free liquid suspension culture system. Among the two-factor combinations tested, SCF plus IL-3 most effectively expanded committed progenitor cells, including mixed colony-forming units (CFU-Mix). The expansion efficiency (EE) of FL for each progenitor was inferior to that of SCF in the presence of various cytokines, except TPO. IL-6 significantly increased the EE for granulocyte/macrophage colony-forming units (CFU-GM) obtained with SCF + IL-3 or FL + IL-3. Interestingly, TPO markedly augmented the EE for committed progenitors, including CFU-GM, erythroid burst-forming units (BFU-E), and CFU-Mix, in the presence of SCF + IL-3 or FL + IL-3. The combinations of SCF + IL-3 + TPO + IL-6 or IL-11 maximally stimulated the expansion of committed progenitors. The maximum EE for CFU-GM, BFU-E, and CFU-Mix was respectively 197-fold (day 14), 60-fold (day 7) and 51-fold (day 14). Other combinations of cytokines without IL-3 failed to expand effectively these committed progenitors. Our data demonstrate that it is possible to expand human CB-derived committed progenitors in vitro using SCF or FL with several other cytokines including TPO, and that IL-3 is the key cytokine promoting the expansion of human hematopoietic progenitors in the presence of SCF or FL.

Functional Differences between Subpopulations of Mobilized Peripheral Blood-Derived CD34+ Cells Expressing Different Levels of HLA-DR, CD33, CD38 and c-kit Antigens

We have investigated the functional characteristics of peripheral blood‐derived CD34+ cells mobilized by a combination of chemotherapy and G‐CSF (mobilized peripheral blood‐derived [MPB] CD34+ cells). In this study, subpopulations of MPB CD34+ cells have been directly compared in clonal cultures, long‐term cultures with bone marrow (BM) stromal cells, and single‐cell cultures. MPB CD34+ cells could be subdivided by expression levels of HLA‐DR (DR), CD38, CD33 and c‐kit antigens. The majority of MPB CD34+ cells expressed DR and CD38 antigens. In contrast, approximately 60% and 20% of the MPB CD34+ cells expressed CD33 and c‐kit antigens, respectively. Interestingly, MPB CD34+ cells can be subdivided into three fractions which express high, low or negative levels of c‐kit receptor. All types of committed progenitors were observed in populations of CD34+DR+, CD34+DR−, CD34+CD33−, CD34+CD38+ and CD34+ c‐kitlow cells. Colony forming unit‐granulocyte/macrophage was highly enriched in the population of CD34+CD33+ cells, whereas BFU‐E was highly enriched in the population of CD34+ c‐kithigh cells. In the population of CD34+CD38− cells, however, a few myeloid progenitors were detected. In addition, limiting dilution analyses clearly showed that the long‐term culture‐initiating cell (LTC‐IC) is enriched in the populations of CD34+DR−, CD34+CD33− and CD34+c‐kit− or low cells, but very few in CD34+ c‐kithigh cells, and that CD38 antigen is not a useful marker for the enrichment of LTC‐IC derived from MPB CD34+ cells. Moreover, single cell clone sorting experiments clearly demonstrated the functional differences between CD34+CD38+ and CD34+CD38− cells as well as CD34+ cells expressing different levels of c‐kit receptor. Our results suggest that an immunophenotype of LTC‐IC is different between BM‐, cord blood‐ and MPB‐derived CD34+ cells and that primitive and committed progenitors existing in these sources may be functionally different.

Human cord blood-derived primitive progenitors are enriched in CD34+c-kit- cells: correlation between long-term culture-initiating cells and telomerase expression.

We studied the functional characteristics of subpopulations of cord blood-derived CD34+ cells expressing different levels of CD38 and c-kit antigens, using clonal cell culture and long-term culture with allogeneic bone marrow stromal cells or the MS-5 murine stromal cell line to assay long-term culture-initiating cells (LTC-IC) in each subpopulation. To investigate the capacity for replication, proliferation, and differentiation of each subpopulation of CD34+ cells, we also studied the correlation between LTC-IC and telomerase activity. After 5 weeks of coculture, LTC-IC accounted for one out of 32 CD34+CD38− cells and one out of 33 CD34+c-kit− cells. In contrast, the frequency of LTC-IC was low in their antigen-positive counterparts (one per 84 CD34+CD38+ cells, one per 90 CD34+c-kitlow cells, and very low among CD34+c-kithigh cells). It was noteworthy that some LTC-IC derived from CD34+CD38− as well as CD34+c-kit− cells generated colony-forming cells (CFCs) after up to 9 weeks of coculture. Telomerase activity was consistently low in CD34+CD38− and CD34+c-kit− cells compared to CD38+ or c-kithigh or low cells, suggesting that CD34+CD38− or c-kit− cells are likely to be more quiescent. These results suggest that the CD34+CD38− and CD34+c-kit− cell populations are primitive stem/progenitor cells, and that the telomerase activity of these cells correlates with their proliferative capacity as well as their stage of differentiation.

Simultaneous signalling through c-mpl, c-kit and CXCR4 enhances the proliferation and differentiation of human megakaryocyte progenitors: possible roles of the PI3-K, PKC and MAPK pathways

We assessed the effect of signalling through CXCR4 on the proliferation and differentiation of human megakaryocytic progenitor cells (CFU‐Meg) in the presence or absence of stem cell factor (SCF) and/or thrombopoietin (TPO), using peripheral blood‐derived CD34+IL‐6R− cells as a target. TPO alone induced a significant number of CFU‐Meg colonies. Although stromal cell‐derived factor‐1 (SDF‐1) or SCF alone did not support CFU‐Meg colony formation, these factors had a synergistic effect on CFU‐Meg colony formation in the presence of TPO. The combination of SDF‐1, SCF and TPO induced twice as many CFU‐Meg colonies as TPO alone. To investigate the mechanism of this synergistic action, we examined the effects of various protein kinase inhibitors on CFU‐Meg colony formation. LY294002 and GF109203X (inhibitors of PI3‐K and PKC respectively) completely or partially inhibited this synergistic action. In contrast, a MEK inhibitor (PD98059) did not inhibit CFU‐Meg colony formation. It significantly increased the higher ploidy classes (16N to 64N) of megakaryocytes supported by TPO, TPO + SCF, TPO + SDF‐1, and TPO + SCF + SDF‐1, whereas it abolished the effect of SDF‐1 on the increase of higher ploidy classes of megakaryocytes supported by TPO. These results suggest that MAPK may negatively or positively regulate the nuclear maturation of megakaryocytes, known as endomitosis. In the presence of PD98059, proplatelet formation (PPF) was significantly augmented, suggesting that the MAPK pathway may also inhibit the initiation of PPF. In conclusion, simultaneous activation of three signals through c‐mpl, c‐kit and CXCR4 can induce the in vitro proliferation and differentiation of CFU‐Meg, and SDF‐1 is a potentiator of human megakaryocytopoiesis.

Signal through gp130 Activated by Soluble Interleukin (IL)‐6 Receptor (R) and IL‐6 or IL‐6R/IL‐6 Fusion Protein Enhances Ex Vivo Expansion of Human Peripheral Blood‐Derived Hematopoietic Progenitors

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)‐6 receptor (R) and IL‐6 on the ex vivo expansion of human peripheral blood (PB)‐derived hematopoietic progenitor cells in a short‐term serum‐free liquid suspension culture system, using PB‐derived CD34+IL‐6R+/– cells as a target. In combination with stem cell factor (SCF), IL‐3, and sIL‐6R/IL‐6, the expansion efficiency (EE) for granulocyte/macrophage colony‐forming unit (CFU‐GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU‐E) and mixed colony‐forming unit (CFU‐Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL‐3 + sIL‐6R/IL‐6 + flt3 ligand (FL) most effectively expanded CFU‐GM and CFU‐Mix. The maximum EEs for CFU‐GM and CFU‐Mix were 208‐fold and 42‐fold, respectively. While the EE for BFU‐E was 70‐90‐fold in the presence of SCF + IL‐3 + sIL‐6R/IL‐6, FL significantly augmented the EE for CFU‐GM and CFU‐Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU‐Mix. Interestingly, in combination with IL‐3 and SCF, newly generated IL‐6R/IL‐6 fusion protein (FP) expanded PB‐derived BFU‐E and CFU‐Mix twice more effectively than a combination of sIL‐6R and IL‐6. These results demonstrated that human PB‐derived committed progenitors were effectively expanded in vitro using sIL‐6R/IL‐6 or FP, in combination with IL‐3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL‐6R and IL‐6.

Role of human interleukin-9 as a megakaryocyte potentiator in culture

OBJECTIVE This study investigated the effect of interleukin-9 (IL-9) on the proliferation and differentiation of human colony-forming unit megakaryocytic progenitor cells (CFU-Meg). MATERIALS AND METHODS Peripheral blood-derived CD34(+)IL-6R(-) cells were sorted and cultured in the presence of IL-9, erythropoietin (Epo), stem cell factor (SCF), and thrombopoietin (TPO) alone or in combination. The number of pure and mixed megakaryocyte colonies, the size of pure megakaryocyte colonies, the ploidy distribution of megakaryocytes, and proplatelet formation were investigated. RESULTS Apart from TPO, no single factor could support CFU-Meg-derived colony formation, but each two-factor combination among IL-9, Epo, and SCF supported a few CFU-Meg colonies. Interestingly, the combination of Epo+SCF+IL-9 induced four to six times as many CFU-Meg colonies as any of the two-factor combinations. Neutralizing monoclonal antibodies (mAbs) for IL-9 receptor and c-kit completely abolished this synergistic effect. In contrast, addition of neutralizing anti-c-Mpl or anti-CXCR4 Abs did not influence colony formation, indicating that this synergistic effect was independent of TPO or SDF-1. Moreover, the endogenous production of TPO by cultured CD34(+)IL-6R(-) cells in the presence of Epo+SCF+IL-9 was ruled out by reverse transcriptase polymerase chain reaction for TPO mRNA. Interestingly, the combination of TPO, Epo, SCF, and IL-9 supported the largest number of pure and mixed megakaryocyte colonies, suggesting that this combination of cytokines might recruit primitive megakaryocytic as well as multipotential progenitors. This combination also potently enhanced proplatelet formation compared with TPO alone or a combination of Epo, SCF, and IL-9. CONCLUSION This study demonstrated for the first time that human IL-9 can potentiate human megakaryocytopoiesis in the presence of Epo and/or SCF.

Human FLT3 ligand acts on myeloid as well as multipotential progenitors derived from purified CD34+ blood progenitors expressing different levels of c-kit protein

Abstract:  We studied the effect of human flt3/flk2 ligand (FL) on the proliferation and differentiation of purified CD34+ blood progenitors which express different levels of c‐kit protein in clonal cell culture in comparison with that of stem cell factor (SCF). FL alone did not support significant colony formation. However, FL significantly enhanced neutrophil colony (CFU–G) formation in the presence of granulocyte‐colony stimulating factor (G–CSF) by peripheral blood (PB)‐derived CD34+c‐kit− cells which contained a large number of CFU–G. In addition, FL could synergistically increase the number of CFU–G supported by a combination of interleukin (IL)‐3 and G–CSF, as did SCF. As we reported previously, SCF showed a significant burst‐promoting activity (BPA). In contrast, FL did not exhibit any BPA on PB‐derived CD34+c‐kithigh cells in which erythroid‐burst (BFU‐E) was highly enriched. However, FL could synergize with IL‐3 or GM–CSF in support of erythrocyte‐containing mixed (E‐Mix) colony by PB‐derived CD34+c‐kithigh or low cells in the presence of Epo. Replating of E‐Mix colonies derived from CD34+c‐kithigh cells supported by IL‐3+Epo+SCF yielded more secondary colonies than those supported by IL‐3+Epo or IL‐3+Epo+FL. When PB‐derived CD34+c‐kitlow cells which represent a more immature population than CD34+c‐kithigh cells were used as the target, number of secondary colonies supported by IL‐3+Epo, IL‐3+Epo+SCF or IL‐3+Epo+FL was comparable. However, the number of lineages expressed in the secondary culture was significantly larger in the primary culture containing IL‐3+Epo+FL than in that containing IL‐3+Epo. These results suggest that FL not only acts on neutrophilic progenitors, but also on more immature multipotential progenitors.

Haematopoietic action of flt3 ligand on cord blood‐derived CD34‐positive cells expressing different levels of flt3 or c‐kit tyrosine kinase receptor: comparison with stem cell factor

We compared the effect of human flt3 ligand (FL) and stem cell factor (SCF) on cord blood (CB)‐derived CD34+ cells expressing different levels of flt3 or c‐kit tyrosine kinase (TK) receptor in clonal cell culture. The c‐kit receptor was expressed by 58.5±16.7% of CB CD34+ cells (n = 19), in which c‐kithigh, c‐kitlow and c‐kit‐ cell populations could be identified. In contrast, the flt3 receptor (FR) was weakly expressed on 58.6±8.3% (n = 9) of CB CD34+ cells. FL+erythropoietin (Epo) failed to support erythroid burst (BFU–E) formation by any subpopulation of CD34+ cells. However, SCF+Epo supported BFU–E and erythrocyte‐containing mixed (CFU–mix) colony formation from all subpopulations. Interestingly, FL markedly augmented CFU–mix colony formation supported by interleukin (IL)‐ 3+Epo when CD34+c‐kitlow or CD34+FR+ cells were used as the target. On the other hand, SCF significantly enhanced CFU‐mix colony formation supported by IL‐3+Epo when CD34+c‐kithigh or low and CD34+FR+ cells were used. The replating potential of CFU–mix supported by IL‐3 + Epo + FL was greater when CD34+c‐kitlow or CD34+FR+ cells were used. When the CD34+c‐kitlow cells were used, the number of lineages expressed in secondary cultures of CFU–mix colonies derived from primary cultures containing IL‐3 + Epo+FL or SCF was significantly larger than when the primary cultures contained IL‐3+Epo. Furthermore, the number of long‐term culture‐initiating cells found in CD34+FR+ cells was larger than that in FR‐ cells. CB‐derived CD34+c‐kitlow cells represent a less mature population than c‐kithigh cells, as reported previously. Therefore, these results indicate that both FL and SCF can act on primitive multipotential progenitors. However, it is still uncertain whether CB‐derived CD34+FR+ cells are less mature than CD34+FR‐ cells.

Interleukin 6 receptor expression by human cord blood- or peripheral blood-derived primitive haematopoietic progenitors implies acquisition of different functional properties

The significance of interleukin 6 receptor (IL‐6R) expression by cord blood (CB)‐ and peripheral blood (PB)‐derived primitive haematopoietic progenitors was investigated. IL‐6R was preferentially expressed by PB‐derived myeloid progenitors. Most PB‐derived erythroid bursts (BFU‐E) and mixed colony‐forming cells (CFU‐Mix) did not express this receptor. However, CB‐derived primitive progenitor cells possessed multipotentiality, irrespective of IL‐6R expression. Interestingly, the long‐term culture‐initiating cell (LTC‐IC) population was enriched in PB‐derived CD34+ IL‐6R+ cells, but the extended LTC‐IC (ELTC‐IC) population, which represents a less mature class of haematopoietic progenitors, seemed to be equally distributed in the IL‐6R+ and IL‐6R− cell populations. In contrast, the number of LTC‐ICs and ELTC‐ICs was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. It is noteworthy that the number of LTC‐ICs and ELTC‐ICs in CB‐derived CD34+ cells was markedly higher than that in PB‐derived CD34+ cells regardless of IL‐6R expression. Telomerase activity was consistently lower in PB‐derived CD34+ IL‐6R− cells than in CD34+ IL‐6R+ cells. In contrast, telomerase activity was similar in CB‐derived CD34+ IL‐6R+ or IL‐6R− cells. The pattern of telomerase induction upon cytokine stimulation differed between CB‐ and PB‐derived CD34+ IL‐6R+ or IL‐6R− cells. However, overall telomerase activity per dish was well correlated with the proliferative potential of both cell populations, suggesting that induction of telomerase plays an important role in the escape from replicative senescence of primitive haematopoietic progenitors. Collectively, these results suggest that CB‐derived primitive progenitors are less mature than PB‐derived progenitors and that the expression of IL‐6R by primitive haematopoietic progenitors may have different implications for PB‐ and CB‐derived CD34+ cells.

High Incidence of Chemotherapy-Induced Acral Erythema in Female Patients with non-Hodgkin's Lymphoma Treated with the Vacop-B Regimen

Seven patients, all females out of 29 with non-Hodgkins lymphoma (NHL) (16 males and 13 females) treated with the VACOP-B regimen utilizing granulocyte-colony-stimulating factor (G-CSF) support developed chemotherapy-induced acral erythema (CAE). In contrast, none of 32 patients with NHL who were treated with CHOP, MACOP-B, or biweekly CHOP regimens without G-CSF developed CAE. Total dose intensities of VACOP-B regimen were higher than those of the three other regimens. However, no significant difference in dose intensities of each drug in the patients treated with the VACOP-B regimen was found between male and female patients and between female patients with or without CAE. The cause of the high incidence of CAE (7/13) in the female patients treated with VACOP-B regimen remains unknown. However, female sex hormones may increase susceptibility to CAE. Since the occurrence of CAE interrupts intensive chemotherapy and reduces the cure rate, high risk patients for CAE should be carefully monitored for early symptoms and signs of CAE and should be treated early and appropriately.