Blast Cell Colonies Research Articles

Leptin Stimulates the Proliferation of Murine Myelocytic and Primitive Hematopoietic Progenitor Cells

AbstractLeptin, the product of obese gene, was originally identified as a factor regulating body-weight homeostasis and energy balance. The present study has shown that leptin acts on murine hematopoiesis in vitro. In the culture of bone marrow cells (BMC) of normal mice, leptin induced only granulocyte-macrophage (GM) colony formation in a dose-dependent manner, and no other types of colonies were detected even in the presence of erythropoietin (Epo). Leptin also induced GM colony formation from BMC of db/db mutant mice whose leptin receptors were incomplete, but the responsiveness was significantly reduced. The effect of leptin on GM colony formation from BMC of normal mice was also observed in serum-free culture, and comparable with that of GM-colony–stimulating factor (CSF ). Although leptin alone supported few colonies from BMC of 5-fluorouracil (5-FU)–treated mice in serum-free culture, remarkable synergism between leptin and stem cell factor (SCF ) was obtained in the colony formation. The addition of leptin to SCF enhanced the SCF-dependent GM colony formation and induced the generation of a number of multilineage colonies in the presence of Epo. When lineage (Lin)−Sca-1+ cells sorted from BMC of 5-FU–treated mice were incubated in serum-free culture, leptin synergized with SCF in the formation of blast cell colonies, which efficiently produced secondary colonies including a large proportion of multilineage colonies in the replating experiment. In serum-free cultures of clone-sorted Lin−c-Kit+Sca-1+ and Lin−c-Kit+Sca-1− cells, although synergism of leptin and SCF was observed in the colony formation from both cells, leptin alone induced the colony formation from Lin−c-Kit+Sca-1−, but not Lin−c-Kit+Sca-1+ cells. These results have shown that leptin stimulates the proliferation of murine myelocytic progenitor cells and synergizes with SCF in the proliferation of primitive hematopoietic progenitors in vitro.

Potential for clinical use of viable pluripotent progenitor cells in blood bank stored human umbilical cord blood

There are indications that a close HLA match may not be necessary when human umbilical cord blood (HUCB) is used to effect a hematopoietic transplant. This was first suggested in 1972 and was further supported by the ability of HUCB to produce mouse survival following lethal irradiation. In China multiple units of HLA unmatched HUCB was utilized successfully in children to effect transplants. A recent publication indicated that newborn rodent blood cells can engraft in adult mice across the H-2 antigens. Furthermore, there recently have been successful transplants with 3 antigens mismatched HUCB. In this study HUCB was stored in polyolefin blood bank bags at 4 °C. The storage was similar to that used in routine blood banking. Clonogenic assays were performed at Day 1 and 21 utilizing various growth factors. Replating efficiency was determined on colonies obtained from cord blood that was stored (non-frozen) for 21 days. The functional ability of day 21 old HUCB was determined by its ability to produce survival of lethally irradiated mice. It was found that approximately two-thirds (62.5%) of single primary blast cell colonies in day 21 stored HUCB could generate various types of secondary colonies. In some instances the secondary colonies were counted as high as 42 total mean colonies per single primary colony. These blast cell colonies (CFU-BL) were able to form single and multi lineage colonies composed of virtually every hematopoietic cell types. Animal survival studies were utilized in an effort to determine possible functional ability and were successful in producing fifty-day survival in 54% of lethally irradiated SJL/J mice and 100% ALB/C mice. This study holds the potential of making HUCB available for purposes of marrow transplantation to all who need it. It could further remove most of the moral and ethical issues related to HUCB, reduce to a fraction the cost in providing stem cells for marrow transplantation and potentially allow HUCB to be handled by existing blood banks.

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.

Complex Effects of Interleukin 6 on Clonogenic Blast Cell Growth in Acute Myeloblastic Leukemia

The present in vitro study shows how interleukin (IL)-6 modulates clonogenic blast cell growth in complex ways in acute myeloblastic leukemia when used either as a single factor or in different hematopoietic growth factor combinations. In the presence of IL-6, the colony numbers in culture assay decreased to 50 +/- 29% from the basal values (p < 0.001) in 10 cases and increased to 384 +/- 278% of the basal values (p < 0.01) in 5. The inhibitory effect of IL-6 on blast cell colony formation was retained when IL-6 was combined with granulocyte colony stimulating factor, but was lost if IL-6 was used in combination with mast cell growth factor, IL-3, granulocyte-macrophage colony stimulating factor, or IL-4. The stimulatory effect of IL-6 was diminished in the presence of granulocyte colony stimulating factor, but preserved in the presence of other growth factor combinations. IL-6 had a neutral effect on colony growth in 7 cases with acute myeloblastic leukemia. In these cases, however, IL-6 stimulated significantly clonogenic cell growth if combined with mast cell growth factor or granulocyte-macrophage colony stimulating factor.

Blast colony forming cell-binding capacity of bone marrow stroma from myelodysplastic patients.

A specific stroma function can be quantitatively assessed by counting the stroma-adherent blast cell colonies (CFU-BL) that are formed from normal plastic nonadherent mononuclear bone marrow cells (PNAMNC) after a short-term coincubation ("panning") with the preformed stromal layer. In order to obtain information of stroma function in myelodysplasia (MDS), the "CFU-BL-binding capacity" of stroma from normal bone marrow and from patients with MDS were compared. Stromal cell cultures were established from mononuclear bone marrow cells in microplate cultures cultured with or without 10(-6) M hydrocortisone. CFU-BL-binding capacity was studied by counting blast colonies seven days after panning, and the results were expressed as CFU-BL/10(3) PNAMNC. Normal marrow stromal layers bound CFU-BL only if they were cultured with hydrocortisone, while MDS stromal layers also bound CFU-BL in the absence of hydrocortisone. For further studies of the function of MDS stroma, the effect of growth factors (stem cell factor [SCF], G-CSF, interleukin 3 [IL-3] and their combinations) on CFU-BL binding by normal or MDS stroma has also been compared. Twenty-hour incubation of the stromal layers with a standard dose (100 ng/ml) of various hemopoietic growth factors (IL-3 alone or in combination with SCF, G-CSF alone or in combination with SCF) did not have any effect on CFU-BL binding by normal marrow stroma, but increased the CFU-BL binding by stromal layers from MDS bone marrow. These findings suggest that although stromal microenvironment in MDS is capable of supporting hemopoiesis, bone marrow stroma from MDS patients differs in some characteristics from the normal stroma.

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

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.

Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis.

We examined the role of the ligand for c-mpl. thrombopoietin (TPO). in murine early haemopoiesis. using a serum-free culture system. TPO in combination with the ligand for c-kit (SF) or interleukin-3 (IL-3) supported colony formation by marrow cells of 5-fluorouracil (5-FU)-treated mice whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL-3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL-3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL-3-dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL-3 to act on early multipotential haemopoietic progenitors.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Role of cytokines in leukemic type growth of myelodysplastic CD34+ cells

The clonal growth of progenitor cells from myelodysplastic syndromes (MDS) can be subdivided into four growth patterns: (1) normal, (2) no growth or low plating efficiency, (3) low colony and high cluster number, and (4) normal or high colony number with a large number of clusters. The former two (1 and 2) can be referred to as nonleukemic patterns and latter two (3 and 4) as leukemic. In a search for a role for cytokines in leukemic-type growth of MDS progenitor cells, marrow CD34+ cells were purified up to 94% for 8 normal individuals and 88% for 12 MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). The purified CD34+ cells were cultured for 14 days with various combinations of cytokines, including recombinant human macrophage colony-stimulating factor (rM-CSF), granulocyte-CSF (rG-CSF), granulocyte-macrophage-CSF (rGM-CSF), interleukin-3 (rIL-3), and stem cell factor (SCF; a ligand for c-kit) in serum-free medium. The clonal growth of MDS CD34+ cells supported by a combination of all of the above cytokines was subdivided into the two patterns of leukemic or nonleukemic, and then the role of individual or combined cytokines in proliferation and differentiation of MDS CD34+ cells was analyzed in each group. Evidence we obtained showed that SCF plays a central role in the leukemic-type growth of MDS CD34+ cells and that G-CSF, GM-CSF; and/or IL-3 synergize with SCF to increase undifferentiated blast cell colonies and clusters over that seen in normal CD34+ cells. SCF is present in either normal or MDS plasma at a level of nanograms per milliliter, and this physiologic concentration of SCF can stimulate progenitor cells. This means that progenitor cells are continuously exposed to stimulation by SCF in vivo and that MDS leukemic cells have a growth advantage over normal blast cells. This depends, at least in part, on cytokines such as G-CSF, GM-CSF, IL-3, and SCF.

Isolation and Biological Characterization of Two Classes of Blast-Cell Colony-Forming Cells From Normal Murine Marrow

In this study, a primitive progenitor cell, the blast-cell colony-forming cell (BC-CFC), which is thought by some to be equivalent to the hematopoietic stem cell (HSC), those cells capable of long-term marrow repopulation, has been isolated from normal murine marrow. The cell separation method we employed has, as its final step, the purification of marrow cells based on their ability to take up (bright) or exclude (dull) the mitochondrial dye, Rhodamine (Rho)-123. Rho-bright and Rho-dull cells are enriched for multipotential progenitor cells or for HSC, respectively. It was found that Rho-bright cells contain more BC-CFC than Rho-dull cells (310 +/- 50 v 120 +/- 40 per 10(5) purified cells, respectively). However, the BC-CFC that copurified with the Rho-bright and the Rho-dull cells were different in terms of replating efficiency and response to interleukin-3 (IL-3) and stem cell factor (SCF). In fact, on replating, the blast-cell colonies cultured from the Rho-dull population give rise to many more secondary colonies and had a greater replating efficiency than those obtained from Rho-bright cells (replating efficiency: 29.0 +/- 6.3 v 19.5 +/- 3.7, respectively, P < .01). Furthermore, while the same numbers of blast-cell colonies were detected in culture of Rho-bright cells stimulated with IL-3 alone or in combination with SCF, blast-cell colonies were generated in cultures of Rho-dull cells only in the presence of both IL-3 and SCF. After 5 days in suspension culture stimulated with IL-3 and SCF, Rho-dull cells generated BC-CFC whose replating potential was similar to the replating potential of BC-CFC contained in the Rho-bright population. These results indicate that BC-CFC contained in the Rho-bright and -dull populations are qualitatively different. Because the Rho-dull population contains HSC, the results indicate that few, if any, BC-CFC are HSC.

Roles of Stem Cell Factor in theIn VitroGrowth of Blast Clonogenic Cells from Patients with Acute Myeloblastic Leukemia

We investigated the effects of stem cell factor (SCF) on the growth of blast clonogenic cells from 27 patients with acute myeloblastic leukemia (AML) and 3 patients with chronic myelocytic leukemia in myeloid crisis. SCF alone showed a significant stimulatory activity in 15 of 30 patients (50%). A marked reduction in the number of blast cell colonies supported by SCF alone was noted by the addition of neutralizing antibody (Ab) against granulocyte-macrophage colony-stimulating factor (GM-CSF). Ab against interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) also moderately reduced the number of colonies, whereas Ab against granulocyte CSF (G-CSF) failed to do so. All four Ab together completely abolished the growth in 5 of 6 patients tested. c-kit antisense oligonucleotides reduced the colony formation supported by IL-3 or G-CSF or, in the absence of growth factor, in only 2 of 10 patients tested. SCF caused stimulation by acting synergistically with G-CSF, GM-CSF, IL-3, IL-6, IL-9, IL-11, and IL-12 in 20 of 27 (74%), 17 of 27 (63%), 14 of 28 (50%), 9 of 28 (32%), 1 of 15 (7%), 3 of 28 (11%), and 2 of 15 (13%) patients, respectively. Thus, SCF alone or in combination with some other factor stimulated the growth in 27 of 30 (90%) patients. Of 3 nonresponders, 2 were AML, M3 at presentation. G-CSF at the optimal concentration increased the sensitivity of blasts to SCF. Taken together, SCF acting in combination with other factors, but not alone, stimulates the growth of blast clonogenic cells. GM-CSF, IL-6, and TNF-alpha may be produced endogenously, whereas G-CSF and SCF may be supplied exogenously. Autocrine regulation of the growth of blasts seems to increase the responsiveness of the cells to any of these factors, allowing them to achieve a highly active growth state.

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.

Porcine Brain Microvascular Endothelial Cells Support the In VitroExpansion of Human Primitive Hematopoietic Bone Marrow ProgenitorCells With a High Replating Potential: Requirement for Cell-to-CellInteractions and Colony-Stimulating Factors

Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long-term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).

Gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells.

gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

The self-renewal process of murine hemopoietic stem cells supported by interleukin-3 and the synergistic factors and the probability of its occurrence.

By use of the in vitro murine blast cell colony (Bl) assay system, Bl-constituting cells supported by interleukin-3 (IL-3), IL-3 + interleukin-6 (IL-6), IL-3 + granulocyte colony-stimulating factor (G-CSF) and IL-3 + interleukin-1 (IL-1) were replated and the frequencies of secondary (2nd) granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colony and Bl progenitors were examined. According to the statistical method reported by Nakahata et al, the p values for the hemopoietic stem cells to self-renew were calculated and all cytokine groups produced similar p values ranging between 0.576 and 0.596. Further studies using IL-3-supported and IL-3 + G-CSF-supported Bl showed that the 2nd Bl progenitors could be produced even when there were more than 150 primary Bl-constituting cells per colony in the case of IL-3 + G-CSF, but no 2nd Bl progenitors were found in the case of IL-3. Their appearance was limited within the smaller primary Bl when supported by IL-3. Again there was a difference in the net product number of 2nd Bl progenitors, that is, the addition of G-CSF to the primary culture could produce around double the number of 2nd Bl progenitors. These data led us to hypothesize that synergistic factors could not modify the self-renewal probability, but maintained the self-renewal process for a longer period, in other words, for several cellular divisions.

All-Trans Retinoic Acid Directly Inhibits Granulocyte Colony-Stimulating Factor-Induced Proliferation of CD34+ Human Hematopoietic Progenitor Cells

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte-macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)-induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.

All-trans retinoic acid directly inhibits granulocyte colony- stimulating factor-induced proliferation of CD34+ human hematopoietic progenitor cells

In this study we examine the effects of retinoids on purified CD34+ human hematopoietic progenitor cells. All-trans retinoic acid inhibited granulocyte colony-stimulating factor (G-CSF)-induced proliferation of CD34+ cells in short-term liquid cultures in a dose-dependent fashion with maximal inhibition of 72% at a concentration of retinoic acid of 1 mumol/L. Although no significant effects were observed on granulocyte- macrophage CSF (GM-CSF)--interleukin-3--or stem cell factor (SCF)- induced proliferation, the combinations of G-CSF and each of these cytokines were all inhibited. Moreover, retinol (3 mumol/L) and chylomicron remnant retinyl esters (0.1 mumol/L) in concentrations normally found in human plasma also had inhibitory effects. Single-cell experiments showed that the effects of retinoic acid were directly mediated. Retinoids also significantly inhibited G-CSF-induced colony formation in semisolid medium, with 88% inhibition observed at a concentration of retinoic acid of 1 mumol/L. However, we did not observe any effects of retinoic acid on G-CSF-induced differentiation as assessed by morphology and flowcytometry. Similar to previous findings using total bone marrow mononuclear cells, we observed a stimulation of GM-CSF-induced colony formation after 14 days. We also observed a stimulatory effect of low doses of retinoic acid (30 nmol/L) on blast-cell colony formation on stromal cell layers. Taken together, the data indicate that vitamin A present in human plasma has inhibitory as well as stimulatory effects on myelopoiesis.

Decreased retinoblastoma protein expression in acute myeloblastic leukaemia is associated with the autonomous proliferation of clonogenic blasts

We have previously shown that blasts from some patients with acute myeloblastic leukaemia (AML) grow autonomously in vitro and that this growth pattern is related to the production of autocrine growth factors including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 beta. However, another potential mechanism of autonomous growth involves the deletion of growth inhibitory molecules. One such inhibitory protein is the product of the retinoblastoma (Rb) gene which is expressed in normal haemopoietic cells and functions in cell cycle control and as a transcriptional repressor. Deletion of the Rb gene has been reported in various types of malignancy. We have examined the expression of the Rb gene product in blasts from 39 patients with AML by Western analysis. Using an antibody, Rb (Ab-2) which recognizes the C-terminal region, 11/39 samples (28%) failed to express Rb protein. The results of the Western blot analysis were confirmed by flow cytometry using a second antibody, Rb (Ab-1), against residues 300-380 of the Rb protein. Samples from seven of the 11 Rb-negative cases were studied for the expression of Rb mRNA; Rb mRNA was absent in four and three cases were positive. Analysis of the growth characteristics of these cells showed that blasts from 24/39 cases (62%) had partially or totally autonomous growth in a blast cell colony assay. Of these 24 samples with autonomous growth, 10 (41%) were Rb protein negative; in contrast, of the 15 cases with non-autocrine growth only one was Rb protein negative (7%, P < 0.05). Also, in Rb positive blasts, suppression of Rb protein production using an antisense oligonucleotide significantly increased proliferation of clonogenic AML blasts, confirming that the Rb protein acts as a negative regulator of growth in AML blasts. Our data suggest that deletion of Rb protein expression is frequently found in AML and is associated with the acquisition of autocrine growth characteristics, possibly as the consequence of derepression of genes involved in growth control and cytokine production.

Interleukin-4--a dual regulatory factor in hematopoiesis.

Interleukin-4/B-cell stimulatory factor-1 (IL-4/BSF-1) is a unique cytokine which may have multiple regulatory functions in vitro and in vivo. It has been shown to produce diverse effects on hematopoietic progenitors and can act on the proliferation and differentiation of committed as well as primitive hematopoietic progenitors. It acts synergistically with G-CSF to support neutrophil colony formation. In contrast, it inhibits IL-3-dependent colony formation and macrophage colony formation supported by IL-3 plus M-CSF, GM-CSF, or by M-CSF alone. It also suppresses pure and mixed megakaryocyte colony formation supported by IL-3 in the presence of Erythropoietin (EPO). IL-4, however, supports multipotential blast cell colony formation. And there are apparent differences between the functions of IL-4 and IL-3. IL-4 is able to exert its hematopoietic actions directly and indirectly. For example, it enhances the release of GM-CSF or G-CSF from T-lymphocytes and TNF-alpha from monocytes. Since IL-4 receptors have been shown to be expressed on bone marrow stromal cells as well as hematopoietic cells, it may also act in the bone marrow microenvironment. These results suggest that IL-4 is an intermediate-acting, lineage-non-specific factor just like IL-3 or GM-CSF. Complex interactions between many cytokines including IL-4 may act in the regulation of normal as well as pathological hematopoiesis.