Hideaki Sakabe

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.

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-11 (IL-11) enhances clonal proliferation of acute myelogenous leukemia cells with strong expression of the IL-11 receptor α chain and signal transducing gp130

We examined the effect of recombinant human interleukin (IL)-11 alone or in combination with various colony-stimulating factors (CSFs), including IL-3, granulocyte/macrophage (GM)-CSF, granulocyte (G)-CSF, stem cell factor (SCF), flt3 ligand (FL), and thrombopoietin (TPO), on colony formation by leukemic progenitor cells (L-CFU) obtained from 33 patients with acute myelogenous leukemia (AML). Leukemic colony formation was found in approximately 70 to 80% of the patients in the presence of at least one of the above CSFs. Although IL-11 alone did not support L-CFU, the growth of these progenitors in the presence of other cytokines was enhanced by IL-11 in 16 out of 33 patients and it showed a synergistic action with G-CSF in 12 of them. This synergistic action occurred in seven out of nine M5 patients (French–American–British (FAB) class- ification). A single cell clone-sorting experiment clearly demonstrated that this synergistic effect was operative at the single progenitor cell level. The number of leukemic cells proliferating in the presence of G-CSF+IL-11 was significantly higher than in the presence of G-CSF alone, suggesting that IL-11 recruited dormant leukemic progenitors into the cell cycle. Flow cytometric analysis revealed that all types of AML blast cells (M0∼M6) ubiquitously expressed gp130, although the level of expression was significantly higher in M5 cells. In contrast, expression of the IL-11 receptor α chain (IL-11Rα) varied between FAB types. Blast cells obtained from M1, M3 and M5 patients showed higher levels of expression, with M5 cells showing the strongest expression. Interestingly, the leukemic progenitor cells for which proliferation was synergistically enhanced by IL-11 had significantly higher expression of both IL-11Rα and gp130. These results suggest that administration of IL-11 in vivo may stimulate the proliferation of leukemic progenitor cells, particularly M5 cells, in the presence of G-CSF, and that the responsiveness of L-CFU to IL-11 may be predicted by a simple receptor assay.

Action of human interleukin-4 and stem cell factor on erythroid and mixed colony formation by peripheral blood-derived CD34+c-kithigh or CD34+c-kitlow cells

We studied the interaction of interleukin (IL)‐4 and other burst‐promoting activity (BPA) factors, such as IL‐3, granulocyte/macrophage colony‐stimulating factor (GM‐CSF), IL‐9 and stem cell factor (SCF), on erythroid burst‐forming unit (BFU‐E) and erythrocyte‐containing mixed (CFU‐Mix) colony formation in serum‐free culture. IL‐4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL‐4 showed weak but significant BPA when peripheral blood (PB)‐derived CD34+c‐kitlow cells were used as the target population. The BPA of IL‐4 was much weaker than that of IL‐3, which exerted the most potent activity, as previously reported. When CD34+c‐kithigh cells were used as the target, four factors known to have BPA, IL‐3, GM‐CSF, IL‐9 and SCF, could express BPA. In contrast, IL‐4 alone failed to support erythroid burst formation. Interestingly, IL‐4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte‐containing mixed colonies from CD34+c‐kitlow and CD34+c‐kithigh cells. Delayed addition of SCF + Epo or IL‐4+Epo to the cultures initiated with either IL‐4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU‐E and CFU‐Mix progenitors. In contrast, the survival effect of IL‐4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34+c‐kitlow cells than for those derived from CD34+c‐kithigh cells. It was recently reported that CD34+c‐kitlow cells represent a more primitive population than CD34+c‐kithigh cells. Taken together, these results suggest that IL‐4 helps to recruit primitive progenitor cells in the presence of SCF.