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-kit^low 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-kit^high 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-kit^low and CD34⁺c-kit^high 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-kit^low cells than for those derived from CD34⁺c-kit^high cells. It was recently reported that CD34⁺c-kit^low cells represent a more primitive population than CD34⁺c-kit^high cells. Taken together, these results suggest that IL-4 helps to recruit primitive progenitor cells in the presence of SCF.     

Generation and functional characterization of human dendritic cells derived from CD34+ cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34⁺ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34⁺ cells, compared to that of BM CD34⁺ precursors in response to GM-CSF and TNF-α with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34⁺ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34⁺ cells enhanced both the percentage of total CD1a⁺ cells and CD1a⁺CD14^? cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34⁺ DC precursors into PB and circulating CD34⁺ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediate-late-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5 μg per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Despite considerably increasing numbers of CD34⁺ PB MNC, the latter were not found to be in S/G₂M phase, whereas, among the CD34^? MNC, the proportion of cells in S/G₂M phase increased from <0.1% to 0.75 ± 0.4% (GM-CSF) and to 1.34 ± 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34⁺ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G₂M phase. The non-cycling status of PB and CB progenitor cells was confirmed by the analysis of CD34⁺ cells enriched from the two cell sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34⁺ cells were in S/G₂M phase. The fact that cycling CD34⁺ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized ‘stem cells’ which may require the BM micro-environment for adequate proliferation in vivo     

Generation of multinuclear tartrate-resistant acid phosphatase positive osteoclasts in liquid culture of purified human peripheral blood CD34+ progenitors

Circulating CD34⁺ cells were isolated from leukapheresis products collected from patients with ovarian cancer. CD34^? contaminating cells, identified immediately after immunoselection, ranged from 5% to 25% in five different experiments and were predominantly CD3⁺ T-lymphocytes (range 2–12%), CD3^?/CD16⁺/CD56⁺ natural killer cells (range 2–11%) and rare mature CD15⁺/CD11b⁺ granulocytes (range 1–2%). CD34⁺ cells were cultured in liquid medium in the presence of interleukin-3, granulocyte-macrophage colony stimulating factor, stem cell factor, granulocyte colony stimulating factor and a powerful proliferation with prevalent differentiation along the granulocytic/monocytic lineage was obtained. After 10 d of culture a small but consistent number of early multinucleated osteoclasts were identified with a frequency of one cell per 700 granulocytic/monocytic cells, as revealed by cytologic examination. This observation was confirmed by staining for tartrate-resistant acid phosphatase activity which revealed red multinucleated elements with a frequency comparable to that reported above. Conversely, no osteoclasts were observed in those cultures in which macrophage overgrowth was obtained by culturing CD34⁺ cells until day 35. These observations suggest that circulating progenitors have a multilineage potential in vitro and contribute to the clarification of osteoclast development in humans; additionally, they provide the basis for the future development of optimized osteoclast culture techniques in liquid medium and the basic culture system, to test the distinct activity of 1,25(OH)₂D₃, parathyroid hormone, interleukin-11 and of other cytokines on osteoclast development in humans.     

All-trans retinoic acid shows multiple effects on the survival, proliferation and differentiation of human fetal CD34+ haemopoietic progenitor cells

Summary. To evaluate the effect of all-trans retinoic acid (RA) on fetal haemopoiesis, we performed serum-free liquid and semisolid cultures using CD34⁺ cells purified from mid-trimester human fetal blood samples. RA, at both physiological (10^-n and 10^-12M) and pharmacological (10^-6 and l(r⁷M) concentrations, significantly (P<0.01) promoted the survival of fetal CD34⁺ cells in liquid cultures from day 3 onwards, by suppressing apoptosis induced by serum and growth factor deprivation. On the other hand, RA alone had no significant effect on the proliferation and differentiation of fetal haemopoietic progenitors. In the presence of optimal concentrations of recombinant interleukin-3 (IL-3), stem cell factor (SCF), granulocyte/ macrophage-colony stimulating factor (GM-CSF), and erythropoietin (Epo), low and high doses of RA induced striking differential effects on CD34⁺ cell proliferation in liquid cultures and colony formation in semisolid assays. In fact, 1CT^U M and 1CT¹²M RA were able to: (i) significantly (P<0.05) increase ³H-thymidine uptake by fetal CD34⁺ cells in liquid cultures, and (ii) variably promote the growth of pluripotent (CFU-GEMM, P<0.05), early (BFU-meg) and late (CFU-meg, P<0.01) megakaryocyte, granulocyte/macrophage (CFU-GM. P<001) and erythroid (BFU-E) progenitors in semisolid cultures. On the contrary, 10^-6 and 10^-7 M RA induced: (i) an overall inhibition (P<0.01) of CD34⁺ cell growth in liquid cultures; (ii) a marked suppression of BFU-E colony formation (P<0.01) at all Epo concentrations examined (0-002-4IU/ml); and (iii) a significant (P<0.()1) stimulation of CFU-GM with a shift from mixed granulocyte/ macrophage to pure granulocyte colonies, whereas it had little effect on the growth of CFU-GEMM, BFU-meg and CFU-meg. Our data, as a whole, demonstrate that RA has direct complex effects on the survival, growth and clonal expansion of fetal haemopoietic progenitor cells, mainly depending on the presence of recombinant cytokines, the type of progenitor and the concentrations of RA.     

Expansion of granulocyte colony-stimulating factor/chemotherapy-mobilized CD34+ hematopoietic progenitors: role of granulocyte-macrophage colony-stimulating factor/erythropoietin hybrid protein (MEN11303) and interleukin-15

Ex vivo stroma-free static liquid cultures of granulocyte colony-stimulating factor (G-CSF)/chemotherapy-mobilized CD34+ cells were established from patients with epithelial solid tumors. Different culture conditions were generated by adding G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), Flt3 ligand (Flt3), megakaryocyte growth and development factor (Peg-rHuMGDF), GM-CSF/erythropoietin (EPO) hybrid protein (MEN11303), and interleukin-15 (IL-15) to the basic stem cell factor (SCF) + interleukin-3 (IL-3) + EPO combination. This study showed that, among the nine different combinations tested in our 5% autologous plasma-containing cultures, only those containing IL-3/SCF/Flt3/MEN11303 and IL-3/SCF/Flt3/MEN11303/IL-15 significantly expanded colony-forming unit granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), long-term culture-initiating cells (LTC-IC), CD34+, and CD34+/CD38- cells after 14 days of culture. Particularly, the addition of IL-15 to IL-3/SCF/Flt3/MEN11303 combination produced a significant increase of LTC-IC, with an average 26-fold amplification as compared to input cells, without any detrimental effect on CFU-GM and BFU-E expansion. This combination also produced a statistically significant 3.6-fold expansion of primitive CD34+/CD38- cells. Moreover, this study confirms the previously described erythropoietic effect of MEN11303, which, in our experience, was the only factor capable of expanding BFU-E. Compared to equimolar concentrations of GM-CSF and EPO, MEN11303 hybrid protein showed a significantly higher capacity of expanding CFU-GM, BFU-E, LTC-IC, CD34+, and CD34+/CD38- cells when these cytokines were tested in combination with IL-3/SCF/Flt3. These cultures indicated that Peg-rHuMGDF addition to IL-3/SCF/EPO/Flt3 does not affect CFU-GM and BFU-E expansion but, unlike G-CSF or GM-CSF, it does not decrease the ability of Flt3 to expand primitive LTC-IC. These studies indicate that, starting from G-CSF/chemotherapy-mobilized CD34+ cells, concomitant expansion of primitive LTC-IC, CFU-GM, BFU-E, CD34+, and CD34+/CD38- cells is feasible in simple stroma-free static liquid cultures, provided IL-3/SCF/Flt3/MEN11303/IL-15 combination is used as expanding cocktail in the presence of 5% autologous plasma.     

Functional,phenotypic and molecular characterization of cytokine low-responding circulating CD34+ haemopoietic progenitors

Circulating CD34⁺ cell populations characterized by a low rate (up to five) or high rate (more than five) of cell divisions were isolated from 8 d cultures in the presence of stem cell factor (SCF), interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), Flt3 ligand and Peg-rHu megakaryocyte growth and development factor (Peg-rHuMGDF) using the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and flow cytometric cell sorting. Phenotypic characterization of cells which had experienced up to five divisions (CFDA-SE^bright) showed a similar surface antigen expression to starting, freshly isolated CD34⁺ cells. Conversely, cells which experienced more than five divisions (CFDA-SE^dim) showed a differentiating behaviour, down-regulating CD34 antigen and acquiring differentiation markers. CFDA-SE^bright cells were significantly enriched in CD105 (endoglin) positive precursors as compared to both freshly isolated CD34⁺ and CFDA-SE^dim cells. Functional analysis indicated that CFDA-SE^bright had a 3-fold and 10-fold greater cumulative cloning efficiency as compared to freshly isolated CD34⁺ cells and CFDA-SE^dim cells, respectively. CFDA-SE^bright cells retained the vast majority of LTC-IC and showed a LTC-IC frequency 2.8-fold higher than that found in freshly isolated CD34⁺ cells. RT-PCR and Western blot analyses showed significantly higher bcl-2 RNA and protein levels in CFDA-SE^bright cells as compared to freshly isolated CD34⁺ and CFDA-SE^dim cells. This study indicates that cytokine low-responding circulating CD34⁺ cells (CFDA-SE^bright cells) represent a functionally, phenotypically and molecularly distinct multipotent progenitor population with biological properties associated with primitive precursors.     

GM-CSF promotes differentiation of a precursor cell of monocytes and Langerhans-type dendritic cells from CD34+ haemopoietic progenitor cells

Epithelia-associated dendritic cells (DC) including Langerhans cells in the skin (LC) are precursors of lymph node located interdigitating DC (iDC). CD1a⁺ LC are known to be derived from CD34⁺ haemopoietic progenitor cells (HPC); however, cells of an intermediate differentiation state that are CD34^? and CD1a^? have not been identified. Monitoring the differentiation pathway of HPC in the presence of GM-CSF + IL-4, we observed the emergence of a distinct LC precursor population that was CD33⁺ CD13⁺ CD4⁺ CD38⁺ CD44⁺ CD34^? CD14^? CD1a^?. The cells could be separated by FACS due to a unique CD44/CD38 expression pattern or by CD44 expression in conjunction with the SSC profile. It was found that they were similarly generated in the presence of GM-CSF alone and were detectable in culture for at least a week. Irrespective of being generated in the presence of GM-CSF + IL-4 or GM-CSF alone, CD44/SSC-sorted precursor cells matured to MHC class II compartments (MIIC) and Birbeck granules (BG) expressing LC, when subsequently cultured in the presence of GM-CSF + IL-4. When IL-4 was omitted, however, the same cells matured to phagocytically active adherent macrophages (MΦ). These culture conditions were associated with a > 4-fold increase in the concentration of IL-6 when compared to those used for LC differentiation. The identification of a distinct oligopotent precursor cell population that can deliberately be induced to give rise to BG⁺ MIIC⁺ CD1a⁺ CD14^? LC or to adherent CD14⁺ MΦ further substantiates the close relationship of monocytes and DC and may help to identify its in vivo equivalent.     

G-CSF-mobilized CD34+ peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34+ cells: role of G-CSF as survival factor

The mechanism of release of CD34⁺ cells into the peripheral blood (PB) after mobilization treatment with chemotherapy and/or growth factors is not clearly understood. Growth factors may induce increased proliferation and self renewal within the stem cell compartment. It is possible that they alter adhesion molecule profiles or other progenitor:stroma interactions, to allow release of these cells into the periphery. However, CD34⁺ cells are present in the PB under steady-state conditions, albeit in low number. Growth factors such as granulocyte colony-stimulating factor (G-CSF) may promote the survival of CD34⁺ cells in the PB by suppressing apoptosis. In order to test this hypothesis, we have quantitated apoptotic cells in the CD34⁺ fraction of peripheral blood stem cell (PBSC) collections, using two-colour flow cytometry, after staining with anti-CD34 antibody and the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD). 7AAD differentially stains live, apoptotic and dead cells, due to the altered accessibility of DNA in each subpopulation.
We have shown a significant reduction in the proportion of apoptotic cells in the CD34⁺ population mobilized by G-CSF compared to CD34⁺ cells in unstimulated PB, consistent with the theory that G-CSF is acting, at least in part, by suppressing apoptosis. In addition, we found that G-CSF mobilized CD34⁺ cells are less apoptotic than CD34⁺ cells of unstimulated normal bone marrow, indicating that, at the doses used, G-CSF is significantly altering the survival capacity of the mobilized cells.     

Megakaryocyte progenitors derived from bone marrow or G-CSF-mobilized peripheral blood CD34+ cells show a distinct phenotype and responsiveness to interleukin-3 (IL-3) and PEG-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF)

In the present study we investigated the proliferative response of megakaryocyte progenitor cells (CFU-MK) derived from peripheral blood stem cell (PBSC) collections of patients with haematological malignancies and normal donors. Highly purified CD34⁺ cells and mononuclear cell fractions were assayed in the presence of recombinant interleukin-3 (IL-3) and pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), alone or in combination, and megakaryocyte colony formation was evaluated in the plasma clot. In comparison, steady-state bone marrow samples from normal donors were highly enriched in CD34⁺ cells and tested with the cytokines studied. Our results showed that IL-3 was able to stimulate CFU-MK colony formation from bone marrow and peripheral blood CD34⁺ cells. Similarly, PEG-rHuMGDF stimulated, in a dose–response manner, CD34⁺ cells from the bone marrow. However, normal mobilized peripheral blood CD34⁺ cells were not induced to generate CFU-MK colonies by PEG-rHuMGDF. The same lack of response was observed when patients peripheral blood CD34⁺ cells primed with chemotherapy plus G-CSF or with G-CSF alone were assessed. In contrast, PEG-rHuMGDF stimulated CFU-MK growth when mononuclear cells, either from the bone marrow or from mobilized peripheral blood, were grown in plasma clot. Moreover, we analysed by flow cytometry the expression of Mpl receptor on the cell membrane of normal mobilized peripheral blood and normal steady-state bone marrow CD34⁺ cells. Our results showed a reduced expression of Mpl receptor on mobilized peripheral blood progenitor cells in comparison with bone marrow cells.     

Different behaviour of fresh and cultured CD34+ cells during immunomagnetic separation

In-vitro expansion of human cord blood (CB) cells could enhance peripheral blood recovery and ensure long-term engraftment of larger recipients in the clinical transplant setting. Enrichment of CD34+ cells using the MiniMACS column has been evaluated for the preparation of CB CD34+ cells before and after expansion culture. Repurification of CD34+ cells after culture would assist accurate phenotypic and functional analysis. When fresh CB mononuclear cells (MNC) were separated, the MACS positive (CD34+) fraction (90.1% pure) contained a mean (+/- SD, n = 5) of 93.0 +/- 8.0% of the eluted CD34+ cells, 99.6 +/- 0.7% of the CFU-GM and all of the eluted long-term culture-initiating cells (LTC-IC). Cord blood CD34+ cells were then cultured for 14 d with IL-3, IL-6, SCF, G-CSF and GM-CSF, each at 10 ng/ml. The total cell expansion was 2490 +/- 200-fold and the CD34+ cell expansion was 49 +/- 17-fold. The percentage of CD34+ cells present after expansion culture was 1.2 +/- 0.85%. When these cells were repurified on the MiniMACS column, the MACS positive fraction only contained 40.3 +/- 13.4% of the eluted CD34+ cells which was enriched for the mature CD34+ CD38+ subset, 24.4 +/- 8.8% of the eluted CFU-GM and 79.5 +/- 11.0% of the LTC-IC. The remaining cells were eluted in the MACS negative fraction. In conclusion, repurification of cultured CD34+ cells does not yield a representative population and many progenitors are lost in the MACS negative fraction. This can give misleading phenotypic and functional data. Cell losses may be important in the clinical setting if cultured cells were repurified for purging.     

Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.

We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.     

In vitro growth of human fetal CD34+ cells in the presence of various combinations of recombinant cytokines under serum-free culture conditions

Summary. CD34⁺ cells were purified from midtrimester human fetal blood and adult bone marrow samples and seeded in serum-free fibrin-clot cultures in order to evaluate the number and the responsiveness to recombinant cytokines of pluripotent (CFU-GEMM), erythroid (BFU-E), megakaryocyte (BFU-meg and CFU-meg) and granulocyte/macrophage (CFU-GM) haemopoietic progenitor cells.
The number of the different haemopoietic progenitors/1 × 10³ CD34⁺ cells, except CFU-meg, was significantly higher in fetal blood than in adult bone marrow in cultures stimulated by any combination of cytokines including interleukin-3 (IL-3), granulocyte/macrophage colony stimulating factor (GM-CSF) or stem cell factor (SCF) plus erythropoietin (Epo). Nevertheless, whereas adult BFU-E showed a maximal growth in the presence of Epo plus IL-3 or Epo plus SCF, fetal BFU-E showed an optimal growth in the presence of Epo alone, the sensitivity of fetal BFU-E to suboptimal concentrations of Epo being approximately 10–15-fold higher than that of adult BFU-E. Addition of optimal concentrations of IL-3, GM-CSF or SCF, alone or in various combinations, to Epocontaining cultures induced a significant increase in both the number and size of fetal CFU-GEMM, and CFU-GM, and a parallel decrease of fetal BFU-E. Finally, SCF potently syner-gized with IL-3 in increasing the growth of both classes of fetal megakaryocyte progenitors, BFU-meg and CFU-meg.     

IL-12 indirectly enhances proliferation of 5-FU-treated human hematopoietic peripheral blood CD34+ cells

Interleukin-12 (IL-12) or natural killer cell stimulatory factor (NKSF), has multiple effects on T lymphocytes and natural killer cells. In this study, the effect of IL-12 on human hematopoiesis was studied by analyzing the growth of CD34⁺ peripheral blood stem cells (PBSC), in steady state. In the presence of Epo, IL-12 alone or in combination with IL-3 or SCF had no effect on the formation of colonies from CD34⁺ cells. In culture with Epo, G-CSF, and IL-3, the effect of Flt3-ligand (FL) on CD34⁺ PBSC was investigated in the presence or absence of IL-12. No additional effect of IL-12 was observed when combined with FL. We evaluated 5-FU-treated human CD34⁺ PBSC proliferation in cultures with Epo, G-CSF, and IL-3, in the presence or absence of IL-12. No cytokine combination enhanced colony formation from 5-FU-treated CD34⁺ cells. However, in cultures of 5-FU-treated human CD34⁺ cells, the most efficient combination was IL-3 + Epo + G-CSF + Accessory cells (CD34⁻). Furthermore, IL-12 enhanced this colony formation significantly. To investigate whether immature CD34⁺ cells were responsible for FL or SCF, 5-FU-treated human CD34⁺ cells were cultured with or without IL-12. Whereas no synergistic effect was observed in combination with IL-12, SCF alone significantly enhanced colony formation. However, the colony number was found to be smaller than with the potent combination of accessory cells in the presence of IL-12. These results indicate that accessory cells, lost in CD34⁺ cell purification, could be partly responsible for an IL-12 effect on immature human PBSC proliferation. J. Hematol. 58:183–188, 1998. © 1998 Wiley-Liss, Inc.     

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-kit^high 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-kit^{high or low} cells in the presence of Epo. Replating of E-Mix colonies derived from CD34⁺c-kit^high 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-kit^low cells which represent a more immature population than CD34⁺c-kit^high 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.     

The purification of CD34+ cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors

This study was performed to assess the methods which gave maximal recovery of purified CD34/45⁺ cells from a cord blood specimen and optimal growth of progenitors cultured from the purified cells. Cord blood samples were separated using Percoll gradients (either one (1.080) or two successive (1.080 and 1.068) gradient(s)) and commercially available devices for CD34⁺ cell isolation (affinity columns as manufactured by CellPro Inc. or immunomagnetic separation procedure as devised by Baxter Inc.). ‘CellPro’ or ‘Baxter’ techniques gave similar results in terms of nucleated, CD34/45⁺ and progenitor cell concentration; however, the yield of CD34/45⁺ cells in the CD34⁺ enriched fraction was significantly higher when using the ‘CellPro’ technique. We also found significantly higher numbers of CD34/45⁺ cells in the CD34⁺ enriched final fraction when using only one, 1.080, Percoll density gradient in the first separation step. Using one density separation step followed by the ‘CellPro’ technique, we obtained an average of 3×10⁶ purified CD34/45⁺ cells from samples containing 8.5×10⁸ nucleated cells. Granulomonocytic progenitors (CFU-GM) and mixed progenitors (CFU-GEMM) cells from light-density and purified CD34/45⁺ cell fractions were evaluated. We found that 20–30% of the light-density cells and the purified CD34/45⁺ cells, yielded a granulomonocytic colony in serum free medium in the presence of interleukins 3 and 6, erythropoietin, granulomonocytic and granulocytic colony-stimulating factors and stem cell factor. The addition of tumour-necrosis factor α to the cocktail significantly improved the growth of CFU-GEMM allowing 10% of the purified CD34/45⁺ cells to yield a mixed colony, which confirms the role of this cytokine on CD34⁺ cells from cord blood. This study provides an improved method for the recovery of CD34/45⁺ purified cells and their colony formation. These methods may serve as a basis for studies on CD34/45⁺ cell amplification and gene transfer.     

CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells

Malignant cells in haemopoietic autografts can contribute to post-transplant relapse. Engraftment of myeloma patients with CD34⁺ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7–4.5 logs. Residual tumour cells detected in CD34⁺ selected cells may be due to selection impurity or the existence of malignant CD34⁺ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34⁺ progenitors selected with immunomagnetic beads and highly purified CD34⁺ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections. Using allele-specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi-quantitative ASO-PCR was capable of detecting one malignant cell in 10⁴–10⁵ normal white blood cells. Selection of CD34⁺ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two-colour flow-cytometry we observed CD34^?, BB4⁺ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of >99% pure CD34⁺ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO-PCR detected no malignant cells in >99.9% pure CD34⁺ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34⁺ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34-negative cells.     

STEM CELL FACTOR AND THE REGULATION OF DENDRITIC CELL PRODUCTION FROM CD34+ PROGENITORS IN BONE MARROW AND CORD BLOOD

Dendritic cells (DC) are essential for the presentation of antigen in primary immune responses and they develop from CD34⁺ cells in the bone marrow. Although both granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor (TNF) are known to stimulate the development of mature DC from their progenitor (CFU-DL), the function of stem cell factor (SCF) in this pathway remains to be determined. The interactions of SCF with GM-CSF, TNF, interleukin-3 (IL-3) and macrophage colony stimulating factor (M-CSF) in promoting CFU-DL development have now been studied in serum-free cultures of unfractionated as well as progenitor enriched cells from either bone marrow or cord blood. Although SCF alone is without effect on colony formation, it enhances both the numbers and size of DC colonies generated in vitro by GM-CSF and TNF. It acts directly on progenitors and in the presence of GM-CSF can also induce suboptimal DC growth even in the absence of TNF. SCF appears to recruit very early progenitors of a high proliferative potential with the capacity to differentiate into erythroid and myeloid as well as dendritic cell progeny. In combination with other cytokines it may therefore be useful for the ex vivo generation of large numbers of DC for clinical purposes.     

Malignancy: Insulin-like Growth Factor-1 (IGF-1) is a Costimulator of the Expansion of Lineage Committed Cells Derived from Peripheral Blood Mobilized CD34+ Cells in Multiple Myeloma Patients

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte - colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high) /CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high) /CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.     

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages

We generated red blood cells (RBC) from cord blood (CB) CD34⁺ cells using a four-phase culture system. We first cultured CB CD34⁺ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34⁺ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34⁺ cells from a different donor using the same co-culture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 × 10¹² RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 × 10¹³ RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.