Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoietic stem cells: comparison with interleukin-1 alpha

Currently available evidence suggests that in the steady state, the majority of hematopoietic stem cells are dormant in cell cycle and reside in the so-called G0 period. Studies in our laboratory indicated that once a stem cell leaves G0, its subsequent proliferation requires the presence of interleukin-3 (IL-3). Recently it was reported that interleukin-1 (IL-1) may stimulate stem cells to become sensitive to IL- 3. In a separate study, we observed that interleukin-6 (IL-6, also known as B cell stimulatory factor-2/interferon beta 2) possesses synergism with IL-3, shortening the G0 period of murine hematopoietic stem cells. We report here that human IL-6 and IL-3 act synergistically in support of the proliferation of progenitors for human blast cell colonies and that IL-1 alpha reveals no synergism with IL-3 when tested against purified human marrow progenitors. Panned My-10+ human marrow cells were plated in culture and on day 14 of incubation, either IL-3, IL-6, IL-1 alpha or a combination of these factors was added to the cultures. Blast cell colony formation was analyzed daily between days 18 and 32 of culture. IL-6 or IL-1 alpha alone failed to support blast cell colony formation. In the presence of IL-3 alone, blast cell colonies continued to emerge between days 21 and 27. When a combination of IL-3 and IL-6 was added, blast cell colonies developed earlier than in cultures with IL-3 alone and twice as many blast cell colonies were identified. IL-1 alpha failed to augment IL-3-dependent blast cell colony formation. Replating studies of the individual blast cell colonies revealed various types of single as well as multilineage colonies. These observations suggest that IL-6 shortens the G0 period of human hematopoietic stem cells and that the reported synergistic activities of IL-1 on primitive hematopoietic cells may be indirect.

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Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoietic stem cells: comparison with interleukin-1 alpha

Blood ◽

10.1182/blood.v71.6.1759.1759 ◽

1988 ◽

Vol 71 (6) ◽

pp. 1759-1763 ◽

Cited By ~ 182

Author(s):

AG Leary ◽

K Ikebuchi ◽

Y Hirai ◽

GG Wong ◽

YC Yang ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Interleukin 6 ◽

Colony Formation ◽

Interleukin 1 ◽

Blast Cell ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

The Individual ◽

Cell Colony

Abstract Currently available evidence suggests that in the steady state, the majority of hematopoietic stem cells are dormant in cell cycle and reside in the so-called G0 period. Studies in our laboratory indicated that once a stem cell leaves G0, its subsequent proliferation requires the presence of interleukin-3 (IL-3). Recently it was reported that interleukin-1 (IL-1) may stimulate stem cells to become sensitive to IL- 3. In a separate study, we observed that interleukin-6 (IL-6, also known as B cell stimulatory factor-2/interferon beta 2) possesses synergism with IL-3, shortening the G0 period of murine hematopoietic stem cells. We report here that human IL-6 and IL-3 act synergistically in support of the proliferation of progenitors for human blast cell colonies and that IL-1 alpha reveals no synergism with IL-3 when tested against purified human marrow progenitors. Panned My-10+ human marrow cells were plated in culture and on day 14 of incubation, either IL-3, IL-6, IL-1 alpha or a combination of these factors was added to the cultures. Blast cell colony formation was analyzed daily between days 18 and 32 of culture. IL-6 or IL-1 alpha alone failed to support blast cell colony formation. In the presence of IL-3 alone, blast cell colonies continued to emerge between days 21 and 27. When a combination of IL-3 and IL-6 was added, blast cell colonies developed earlier than in cultures with IL-3 alone and twice as many blast cell colonies were identified. IL-1 alpha failed to augment IL-3-dependent blast cell colony formation. Replating studies of the individual blast cell colonies revealed various types of single as well as multilineage colonies. These observations suggest that IL-6 shortens the G0 period of human hematopoietic stem cells and that the reported synergistic activities of IL-1 on primitive hematopoietic cells may be indirect.

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Leukemia inhibitory factor differentiation-inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells

Blood ◽

10.1182/blood.v75.10.1960.bloodjournal75101960 ◽

1990 ◽

Vol 75 (10) ◽

pp. 1960-1964 ◽

Cited By ~ 1

Author(s):

AG Leary ◽

GG Wong ◽

SC Clark ◽

AG Smith ◽

M Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Leukemia Inhibitory Factor ◽

Blast Cell ◽

Inhibitory Factor ◽

Target Cells ◽

Inhibiting Activity ◽

Hematopoietic Stem ◽

Cell Colony

Leukemia inhibitory factor (LIF)/differentiation-inhibiting activity (DIA)/human interleukin for DA cells (HILDA) is a cytokine with biologic activities involving a variety of different types of target cells. Here we have tested LIF/DIA for possible effects on the growth and differentiation of normal human hematopoietic cells in culture. As a single agent, LIF/DIA had no effect on colony formation by CD34- positive human bone marrow cells. However, LIF/DIA was as effective as either interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G- CSF) in the enhancement of IL-3-dependent colony formation of very primitive blast colony-forming cells. Studies using neutralizing antibodies against IL-6 or G-CSF demonstrated that this was not due to induction in culture of either of the other known synergistic factors for blast cell colony formation. A 1-day delay in the time course of appearance of blast cell colonies grown in the presence of LIF/DIA relative to those grown in the presence of IL-6 suggests that the different synergistic factors may operate through different mechanisms, although we cannot rule out that high doses of LIF/DIA might yield accelerated blast cell colony formation. Our findings provide evidence that LIF/DIA may play an important role, along with IL-6 and G-CSF, in the regulation of early hematopoietic stem cells.

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Leukemia inhibitory factor differentiation-inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells

Blood ◽

10.1182/blood.v75.10.1960.1960 ◽

1990 ◽

Vol 75 (10) ◽

pp. 1960-1964 ◽

Cited By ~ 95

Author(s):

AG Leary ◽

GG Wong ◽

SC Clark ◽

AG Smith ◽

M Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Leukemia Inhibitory Factor ◽

Blast Cell ◽

Inhibitory Factor ◽

Target Cells ◽

Inhibiting Activity ◽

Hematopoietic Stem ◽

Cell Colony

Abstract Leukemia inhibitory factor (LIF)/differentiation-inhibiting activity (DIA)/human interleukin for DA cells (HILDA) is a cytokine with biologic activities involving a variety of different types of target cells. Here we have tested LIF/DIA for possible effects on the growth and differentiation of normal human hematopoietic cells in culture. As a single agent, LIF/DIA had no effect on colony formation by CD34- positive human bone marrow cells. However, LIF/DIA was as effective as either interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G- CSF) in the enhancement of IL-3-dependent colony formation of very primitive blast colony-forming cells. Studies using neutralizing antibodies against IL-6 or G-CSF demonstrated that this was not due to induction in culture of either of the other known synergistic factors for blast cell colony formation. A 1-day delay in the time course of appearance of blast cell colonies grown in the presence of LIF/DIA relative to those grown in the presence of IL-6 suggests that the different synergistic factors may operate through different mechanisms, although we cannot rule out that high doses of LIF/DIA might yield accelerated blast cell colony formation. Our findings provide evidence that LIF/DIA may play an important role, along with IL-6 and G-CSF, in the regulation of early hematopoietic stem cells.

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Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.93.9.4040 ◽

1996 ◽

Vol 93 (9) ◽

pp. 4040-4044 ◽

Cited By ~ 153

Author(s):

Y. Yonemura ◽

H. Ku ◽

F. Hirayama ◽

L. M. Souza ◽

M. Ogawa

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Interleukin 1 ◽

Hematopoietic Stem ◽

Interleukin 3

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Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells

Blood ◽

10.1182/blood.v86.6.2123.bloodjournal8662123 ◽

1995 ◽

Vol 86 (6) ◽

pp. 2123-2129 ◽

Cited By ~ 30

Author(s):

AC Berardi ◽

A Wang ◽

J Abraham ◽

DT Scadden

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Hematopoietic Stem Cells ◽

Fibroblast Growth Factor ◽

Colony Formation ◽

Fibroblast Growth ◽

Colony Stimulating Factor ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Stimulating Factor

Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony- stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell.

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CD34+/CD33- cells reselected from macrophage inflammatory protein 1 alpha+interleukin-3--supplemented "stroma-noncontact" cultures are highly enriched for long-term bone marrow culture initiating cells

Blood ◽

10.1182/blood.v84.5.1442.1442 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1442-1449 ◽

Cited By ~ 23

Author(s):

CM Verfaillie ◽

JS Miller

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Antigen Expression ◽

Hematopoietic Stem ◽

Interleukin 3 ◽

Inflammatory Protein ◽

Hla Dr ◽

Macrophage Inflammatory Protein

Abstract Human hematopoietic stem cells are thought to express the CD34 stem cell antigen, low numbers of HLA-DR and Thy1 antigens, but no lineage commitment antigens, CD38, or CD45RA antigens. However, fluorescence- activated cell sorted CD34+ subpopulations contain not more than 1% to 5% primitive progenitors capable of initiating and sustaining growth in long-term bone marrow culture initiating cells (LTBMC-ICs). We have recently shown that culture of fresh human marrow CD34+/HLA-DR- cells separated from a stromal layer by a microporous membrane (“stroma- noncontact” culture) results in the maintenance of 40% of LTBMC-ICs. We hypothesized that reselection of CD34+ subpopulations still present after several weeks in stroma-noncontact cultures may result in the selection of cells more highly enriched for human LTBMC-ICs. Fresh marrow CD34+/HLA-DR- cells were cultured for 2 to 3 weeks in stroma- noncontact cultures. Cultured progeny was then sorted on the basis of CD34, HLA-DR, or CD33 antigen expression, and sorted cells evaluated for the presence of LTBMC-ICs by limiting dilution analysis. We show that (1) LTBMC-ICs are four times more frequent in cultured CD34+/HLA- DR- cells (4.6% +/- 1.7%) than in cultured CD34+/HLA-DR- cells (1.3% +/- 0.4%). This suggests that HLA-DR antigen expression may depend on the activation status of primitive cells rather than their lineage commitment. We then sorted cultured cells on the basis of the myeloid commitment antigen, CD33. (2) These studies show that cultured CD34+/CD33- cells contain 4% to 8% LTBMC-ICs, whereas cultured CD34+/CD33+bright cells contain only 0.1% +/- 0.03% LTBMC-ICs. Because LTBMC-ICs are maintained significantly better in stroma-noncontact cultures supplemented with macrophage inflammatory protein 1 alpha (MIP- 1 alpha) and interleukin-3 (IL-3) (Verfaillie et al, J Exp Med 179:643, 1994), we evaluated the frequency of LTBMC-ICs in CD34+/CD33- cells present in such cultures. (3) CD34+/CD33- cells present in MIP-1 alpha + IL-3-supplemented cultures contain up to 30% LTBMC-ICs. The increased frequency of LTBMC-ICs in cultured CD34+ subpopulations may be the result of terminal differentiation of less primitive progenitors, loss of cells that fail to respond to the culture conditions or recruitment of quiescent LTBMC-ICs. The capability to select progenitor populations containing up to 30% LTBMC-ICs should prove useful in studies examining the growth requirements, self-renewal, and multilineage differentiation capacity of human hematopoietic stem cells at the single-cell level.

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Induction of Adipocyte Differentiation and Reduction of Hematopoesis-Supporting Activity of Mouse Stromal Cells by Suppression of Transcription Factor GATA-2.

Blood ◽

10.1182/blood.v106.11.1392.1392 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1392-1392

Author(s):

Yoko Okitsu ◽

Hideo Harigae ◽

Masanori Seki ◽

Toru Fujiwara ◽

Shinichiro Takahashi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Colony Formation ◽

Adipocyte Differentiation ◽

Fetal Liver ◽

Expression Level ◽

Hematopoietic Stem

Abstract (Introduction) Aplastic anemia (AA) is characterized by peripheral pancytopenia and fatty bone marrow. An immunological attack to hematopoietic stem cells has been thought to be responsible for the development of the disease. Previously, we reported the expression of transcription factor GATA-2 is significantly decreased in CD34 positive cells in AA. Together with the phenotypes of hematopoietic stem cells in GATA-2 hetero-knockout mice, GATA-2 down-regulation may play a role in the reduction of a stem cell pool observed in AA. On the other hand, GATA-2 has been shown to be essential for the maintenance of immaturity of preadipocytes. If a pathological immune response in AA decreases the level of GATA-2 expression in not only hematopoietic stem cells but also stromal preadipocytes, it may accelerate the maturation of preadipocytes, leading to the formation of fatty bone marrow. To explore this possibility, the phenotypic change of stromal preadipocytes by suppression of GATA-2 was examined in this study. (Method) The GATA-2 expression level was suppressed by using siRNA for GATA-2 in mouse stromal preadipocyte cell lines, TBR9 and TBR343. After the treatment with siRNA, the adipocyte differentiation was induced by the incubation with insulin and dexamethasone for 7days. Then, the maturation level was examined by oil drops formation judged by oil red staining, and by the expression level of adipcin and PPAR-γ mRNA. Supporting activity of hematopoietic colony formation was also evaluated by using mouse fetal liver cells after siRNA treatment. (Results) By using designed siRNA, the GATA-2 expression was suppressed to 30% of control, whereas the expression level of GATA-3, which is co-expressed in preadipocytes, was unchanged. When GATA-2 was suppressed by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated in both of stromal cells. Furthermore, the number of fetal liver hematopoetic colonies was significantly decreased by suppression of GATA-2, suggesting that GATA-2 down-regulation in stromal preadipocytes results in not only the acceleration of the maturation but also the reduced supporting activity of hematopoietic colony formation (Conclusion) These results suggest that suppression of GATA-2 in hematopoietic tissues induces the characteristic features of AA, i.e., decreased the number of hematopoietic stem cells and increased number of mature adipocytes.

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Targeting Acute Myeloid Leukemia Stem Cells by MUC1-C Subunit Inhibition

Blood ◽

10.1182/blood.v116.21.848.848 ◽

2010 ◽

Vol 116 (21) ◽

pp. 848-848 ◽

Cited By ~ 1

Author(s):

Dina Stroopinsky ◽

Jacalyn Rosenblatt ◽

Keisuke Ito ◽

Li Yin ◽

Hasan Rajabi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Myeloid Leukemia ◽

Muc1 Expression ◽

Hematopoietic Stem ◽

Flow Cytometric ◽

Control Peptide ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 848 Introduction: Acute myeloid leukemia (AML) arises from a malignant stem cell population that is resistant to cytotoxic therapy and represents a critical reservoir of conferring disease recurrence. A major focus of investigation is the identification of unique markers on leukemia stem cells (LSCs) that differentiate them from normal hematopoietic stem cells and thereby serve as potential therapeutic targets. MUC1 is a high molecular weight transmembrane glycoprotein that is aberrantly expressed in many epithelial tumors and confers cell growth and survival. We have developed an inhibitor of the MUC1-C receptor subunit that blocks oligomer formation and nuclear localization. In the present study, we have examined expression of MUC1 on LSCs as compared to normal hematopoietic stem cells and studied the effect of MUC1-C inhibition on the functional properties of LSCs. Methods and Results: Using multichannel flow cytometric analysis, we isolated the LSC compartment as defined by CD34+/CD38-/lineage- cells from bone marrow specimens obtained from patients with active AML. The majority of LSCs strongly expressed MUC1 with a mean percentage of 77% (n=6). These findings were confirmed by immunocytochemical staining of LSCs isolated by flow cytometric sorting. MUC1 expression was not detectable on the CD34- fraction of AML cells, but was present on the granulocyte-macrophage progenitor (GMP) fraction (CD34+/CD38+ cells) (mean=83%; n=6). In contrast, MUC1 expression was not observed on CD34+ progenitors isolated from normal donors (18%, n=6). In concert with these findings, RT-PCR analysis for MUC1 RNA demonstrated expression in CD34+ cells isolated from AML patients, but not normal volunteers. Notably, we also found that MUC1 expression selectively identifies malignant hematopoietic progenitors in a patient with chimerism between normal and leukemia derived stem cells. The presence of MUC1+CD34+ cells was detected in a patient with AML who achieved a morphologic complete remission following sex mismatched allogeneic transplantation. Using Bioview technology, we found that MUC1 is expressed only in the recipient (XX) CD34+ cells, representing residual malignant cells, whereas the donor (XY) derived CD34+ cells, representing the majority of the progenitors, lacked MUC1 expression. We subsequently examined the effects of MUC1-C inhibition on the capacity of leukemic progenitors to proliferate and support colony formation. MUC1-C inhibition with the GO-203 cell-penetrating peptide resulted in downregulation of the β-catenin pathway, an important modulator of cell division and survival, which is known to support the LSC phenotype. No significant change was detected with a control peptide, or with MUC1-C inhibition of progenitors isolated from a normal control. Furthermore, MUC1-C inhibition resulted in apoptosis, as demonstrated by flow cytometric staining for AnnexinV in AML CD34+ cells, but not in CD34+ progenitors isolated from normal volunteers (mean Annexin positive cells 53% and 5%, respectively, n=4). Consistent with these findings, the MUC1-C inhibitor, but not the control, peptide resulted in cell death of CD34+ cells isolated from AML patients, but not normal controls. Most significantly, exposure of CD34+ AML cells to the MUC1-C inhibitor resulted in loss of their capacity for colony formation in vitro with mean colonies of 4 and 40 for those cells exposed to the MUC1 inhibitor and a control peptide (n=2). In contrast, colony formation by normal hematopoietic stem cells was unaffected. Conclusions: MUC1 is selectively expressed by leukemic progenitors and may be used to differentiate malignant from normal hematopoietic stem cell populations. MUC1-C receptor subunit inhibition results in (i) downregulation of b-catenin signaling, (ii) induction of apoptosis and cell death, and (iii) disruption of the capacity to induce leukemia colony formation. Disclosures: Stone: genzyme: Consultancy; celgene: Consultancy; novartis: Research Funding. Kufe:Genus Oncology: Consultancy, Equity Ownership.

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The Tao of Hematopoietic Stem Cells: Toward a Unified Theory of Tissue Regeneration

The Scientific World JOURNAL ◽

10.1100/tsw.2002.162 ◽

2002 ◽

Vol 2 ◽

pp. 983-995 ◽

Cited By ~ 4

Author(s):

Kevin D. Bunting ◽

Robert G. Hawley

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Adult Stem Cells ◽

Developmental Plasticity ◽

Marrow Cell ◽

Cell Types ◽

Therapeutic Benefit ◽

Hematopoietic Stem ◽

The Individual

Hematopoietic stem cells (HSCs) are the best studied of the tissue-specific stem cells. By definition, HSCs have long been regarded as restricted to formation of blood cells of both the lymphoid and myeloid lineages. HSCs residing in the bone marrow microenvironment have self-renewal capacity and can repopulate the hematopoietic system of irradiated transplant recipients for the lifetime of the individual. Therefore, HSCs are extremely important targets for gene therapy applications aimed toward the treatment of inherited and acquired blood disorders. However, recent studies have suggested that a subpopulation of HSCs may have the ability to contribute to diverse cell types such as hepatocytes, myocytes, and neuronal cells, especially following induced tissue damage. Preclinical amelioration of liver disease and myocardial infarcts by HSC-enriched bone marrow cell populations raises the possibility that HSC transplants have the potential to provide therapeutic benefit for a wide variety of diseases. These surprising findings contradict the dogma that adult stem cells are developmentally restricted. Extrapolation of these findings to the clinic will be facilitated by prospective identification of the stem cells that possess this developmental plasticity. Furthermore, characterization of the signaling pathways and molecular determinants regulating the remarkable transdifferentiation capacity of these stem cells may provide insight into novel approaches for modulating frequency of differentiative potential.

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