scholarly journals Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 914-920 ◽  
Author(s):  
DM Bodine ◽  
PS Crosier ◽  
SC Clark
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Cell Function ◽  
Hematopoietic Growth Factors ◽  
Gm Csf ◽  
Liquid Suspension ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein ◽  
Stimulating Factor

We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G- CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

scholarly journals Effects of hematopoietic growth factors on the survival of primitive stem cells in liquid suspension culture

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 914-920 ◽  
Author(s):  
DM Bodine ◽  
PS Crosier ◽  
SC Clark
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Cell Function ◽  
Hematopoietic Growth Factors ◽  
Gm Csf ◽  
Liquid Suspension ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein ◽  
Stimulating Factor

Abstract We have examined the effects of 10 different growth factors either alone or in combination on colony-forming unit-spleen (CFU-S) and repopulating stem cell survival in vitro. Either interleukin-3 (IL-3), granulocyte-colony-stimulating factor (G-CSF), or IL-4 alone support CFU-S in vitro. The effects of IL-3 or G-CSF could be neutralized by adding antibodies against IL-3 or G-CSF, respectively. However, the effects of IL-4 could be neutralized with antibodies to IL-4 as well as with antibodies to IL-3 and G-CSF. The combinations of IL-3 and IL-6, IL-3 and G-CSF, IL-3 and IL-1 alpha, IL-3 and granulocyte-macrophage CSF (GM-CSF), and IL-4 and IL-6 acted synergistically to increase CFU-S number. Addition of macrophage inflammatory protein-1 alpha (MIP-1 alpha) to IL-3 and IL-6 inhibited the increase in CFU-S number. Repopulating stem cell function was measured in a competitive repopulation assay. Either IL-3 or IL-4 alone could preserve stem cell function in vitro. The combinations of IL-3 and IL-6, and IL-3 and G- CSF increased stem cell function approximately twofold. The combinations of IL-3 + G-CSF + IL-6, and IL-4 and IL-6 (both of which increased CFU-S number fivefold to 10-fold) decreased stem cell function approximately fourfold. These results demonstrate that certain combinations of growth factors can increase CFU-S number at the expense of stem cell function.

scholarly journals Murine myeloid cells transformed by myb require fibroblast-derived or autocrine growth factors in addition to granulocyte-macrophage colony- stimulating factor for proliferation

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 209-216 ◽  
Author(s):  
EM Macmillan ◽  
TJ Gonda
Keyword(s):  
Growth Factors ◽  
Myeloid Cells ◽  
Colony Stimulating Factor ◽  
Hematopoietic Growth Factors ◽  
Autocrine Growth ◽  
Gm Csf ◽  
Absolute Dependence ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Murine myeloid cells can be transformed in vitro by infection with recombinant retroviruses carrying activated myb genes. While these myb- transformed hematopoietic cells (MTHCs) can proliferate continuously in culture, they exhibit several characteristics of progenitor cells of the granulocyte-macrophage (GM) lineage, including an absolute dependence on hematopoietic growth factors (HGFs) such as GM colony- stimulating factor (GM-CSF) for survival and growth. Whereas we have previously shown that MTHCs respond synergistically to certain combinations of HGFs, we report here that MTHCs apparently require two HGFs for proliferation, because GM-CSF alone appears insufficient to promote growth when MTHCs are cultured at very low densities. However, proliferation can be stimulated by either increasing the density at which MTHCs are cultured (implying the production of an autocrine growth factor) or by the presence of a feeder layer of irradiated fibroblasts. We find that the activity of such feeder layers is greatest when the MTHCs are allowed to contact them directly; and by using mutant fibroblast lines, that it depends on the production of CSF- 1, but not Steel factor (SLF). In contrast, the autocrine factor appears not to be either CSF-1 or SLF, and the possibility is raised that it may represent a novel HGF activity. Potential implications of these results for normal and leukemic hematopoiesis are discussed.

scholarly journals Differentiation state and responses to hematopoietic growth factors of murine myeloid cells transformed by myb

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2813-2822 ◽  
Author(s):  
TJ Gonda ◽  
EM Macmillan ◽  
PV Townsend ◽  
AJ Hapel
Keyword(s):  
Growth Factors ◽  
Hematopoietic Cells ◽  
Myeloid Cell ◽  
Surface Markers ◽  
Colony Stimulating Factor ◽  
Monocytic Differentiation ◽  
Hematopoietic Growth Factors ◽  
Gm Csf ◽  
Stimulating Factor

Murine hematopoietic cells can be transformed in vitro by recombinant retroviruses that express the myb oncogene, and hematopoietic growth factor (HGF)-dependent myeloid cell lines can be derived from these transformed primary cells. In this study, the differentiation state and responses of myb-transformed hematopoietic cells (MTHCs) have been investigated. We find that MTHCs exhibit properties of early myeloid progenitors including synergistic responses to combinations of HGFs and expression of certain surface markers. As reported previously, MTHCs respond well to granulocyte-macrophage colony-stimulating factor (GM- CSF) but can also respond to interleukin-3 (IL-3); the response to the latter factor depends on the mouse strain from which the cells are derived. Although these single factors stimulate MTHCs, combinations of these factors with colony-stimulating factor-1 (CSF-1 or M-CSF) or Steel factor (SLF or SCF) act synergistically to promote colony formation. The surface markers expressed by MTHCs include both granulocyte-macrophage lineage specific antigens Gr-1, 7/4, F4/80, and Mac-1, as well as two antigens found on early progenitors and stem cells--Thy-1 and Sca-1 (Ly6E). Expression of the latter markers is often heterogeneous and can be modulated by the growth factors to which the cells are exposed. Finally, we show that monocytic differentiation of MTHCs can be induced by exposure to tumor necrosis factor (TNF alpha). Taken together, these results suggest that MTHCs will be a useful model for studying HGF/cytokine responses in both proliferation and differentiation.

Differentiation state and responses to hematopoietic growth factors of murine myeloid cells transformed by myb

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2813-2822 ◽  
Author(s):  
TJ Gonda ◽  
EM Macmillan ◽  
PV Townsend ◽  
AJ Hapel
Keyword(s):  
Growth Factors ◽  
Hematopoietic Cells ◽  
Myeloid Cell ◽  
Surface Markers ◽  
Colony Stimulating Factor ◽  
Monocytic Differentiation ◽  
Hematopoietic Growth Factors ◽  
Gm Csf ◽  
Stimulating Factor

Abstract Murine hematopoietic cells can be transformed in vitro by recombinant retroviruses that express the myb oncogene, and hematopoietic growth factor (HGF)-dependent myeloid cell lines can be derived from these transformed primary cells. In this study, the differentiation state and responses of myb-transformed hematopoietic cells (MTHCs) have been investigated. We find that MTHCs exhibit properties of early myeloid progenitors including synergistic responses to combinations of HGFs and expression of certain surface markers. As reported previously, MTHCs respond well to granulocyte-macrophage colony-stimulating factor (GM- CSF) but can also respond to interleukin-3 (IL-3); the response to the latter factor depends on the mouse strain from which the cells are derived. Although these single factors stimulate MTHCs, combinations of these factors with colony-stimulating factor-1 (CSF-1 or M-CSF) or Steel factor (SLF or SCF) act synergistically to promote colony formation. The surface markers expressed by MTHCs include both granulocyte-macrophage lineage specific antigens Gr-1, 7/4, F4/80, and Mac-1, as well as two antigens found on early progenitors and stem cells--Thy-1 and Sca-1 (Ly6E). Expression of the latter markers is often heterogeneous and can be modulated by the growth factors to which the cells are exposed. Finally, we show that monocytic differentiation of MTHCs can be induced by exposure to tumor necrosis factor (TNF alpha). Taken together, these results suggest that MTHCs will be a useful model for studying HGF/cytokine responses in both proliferation and differentiation.

scholarly journals Murine myeloid cells transformed by myb require fibroblast-derived or autocrine growth factors in addition to granulocyte-macrophage colony- stimulating factor for proliferation

Blood ◽  
1994 ◽  
Vol 83 (1) ◽  
pp. 209-216 ◽  
Author(s):  
EM Macmillan ◽  
TJ Gonda
Keyword(s):  
Growth Factors ◽  
Myeloid Cells ◽  
Colony Stimulating Factor ◽  
Hematopoietic Growth Factors ◽  
Autocrine Growth ◽  
Gm Csf ◽  
Absolute Dependence ◽  
Macrophage Colony ◽  
Stimulating Factor

Murine myeloid cells can be transformed in vitro by infection with recombinant retroviruses carrying activated myb genes. While these myb- transformed hematopoietic cells (MTHCs) can proliferate continuously in culture, they exhibit several characteristics of progenitor cells of the granulocyte-macrophage (GM) lineage, including an absolute dependence on hematopoietic growth factors (HGFs) such as GM colony- stimulating factor (GM-CSF) for survival and growth. Whereas we have previously shown that MTHCs respond synergistically to certain combinations of HGFs, we report here that MTHCs apparently require two HGFs for proliferation, because GM-CSF alone appears insufficient to promote growth when MTHCs are cultured at very low densities. However, proliferation can be stimulated by either increasing the density at which MTHCs are cultured (implying the production of an autocrine growth factor) or by the presence of a feeder layer of irradiated fibroblasts. We find that the activity of such feeder layers is greatest when the MTHCs are allowed to contact them directly; and by using mutant fibroblast lines, that it depends on the production of CSF- 1, but not Steel factor (SLF). In contrast, the autocrine factor appears not to be either CSF-1 or SLF, and the possibility is raised that it may represent a novel HGF activity. Potential implications of these results for normal and leukemic hematopoiesis are discussed.

scholarly journals Characterization of the human burst-forming unit-megakaryocyte

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 145-151 ◽  
Author(s):  
RA Briddell ◽  
JE Brandt ◽  
JE Straneva ◽  
EF Srour ◽  
R Hoffman
Keyword(s):  
Colony Formation ◽  
Progenitor Cell ◽  
Recombinant Erythropoietin ◽  
Hematopoietic Growth Factors ◽  
Gm Csf ◽  
Hla Dr ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Two classes of human marrow megakaryocyte progenitor cells are described. Colony-forming unit-megakaryocyte (CFU-MK)-derived colonies appeared in vitro after 12-day incubation; burst-forming unit- megakaryocyte (BFU-MK)-derived colonies appeared after 21 days. CFU-MK- derived colonies were primarily unifocal and composed of 11.6 +/- 1.2 cells/colony; BFU-MK-derived colonies were composed of 2.3 +/- 0.4 foci and 108.6 +/- 4.4 cells/colony. CFU-MK and BFU-MK were separable by counterflow centrifugal elutriation. CFU-MK colony formation was diminished by exposure to 5-fluorouracil (5-FU); BFU-MK colony formation was unaffected. CFU-MK and BFU-MK were immunologically phenotyped. CFU-MK expressed the human progenitor cell antigen-1 (HPCA- 1, CD34, clone My10) and a major histocompatibility class II locus, HLA- DR, and BFU-MK expressed only detectable amounts of CD34. BFU-MK colony formation was entirely dependent on addition of exogenous hematopoietic growth factors. Recombinant granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) possessed such colony- stimulating activity, whereas recombinant erythropoietin (Epo), G-CSF, IL-1 alpha, IL-4, and purified thrombocytopoiesis-stimulating factor did not. These studies indicate the existence of a human megakaryocyte progenitor cell, the BFU-MK, which has unique properties allowing it to be distinguished from the CFU-MK.

scholarly journals In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3155-3161 ◽  
Author(s):  
RM Schwartz ◽  
SG Emerson ◽  
MF Clarke ◽  
BO Palsson
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Culture Medium ◽  
Culture Conditions ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Medium Exchange ◽  
Proliferation And Differentiation

Abstract We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

scholarly journals Interleukin-2-activated natural killer cells can support hematopoiesis in vitro and promote marrow engraftment in vivo

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 670-677 ◽  
Author(s):  
WJ Murphy ◽  
JR Keller ◽  
CL Harrison ◽  
HA Young ◽  
DL Longo
Keyword(s):  
Growth Factors ◽  
Nk Cells ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Hematopoietic Growth Factors ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Growth Promoting

Abstract Purified natural killer (NK) cells were obtained from mice with severe combined immune deficiency (SCID) to ascertain their effect on hematopoiesis. When activated and propagated with recombinant human interleukin-2 (rhIL-2) in vitro, SCID spleen cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. When added with syngeneic bone marrow cells (BMC) in soft agar, the activated NK cells could support hematopoietic growth in vitro without the addition of exogenous hematopoietic growth factors. However, when syngeneic BMC were added along with cytokines to produce optimal growth conditions, the addition of NK cells was then inhibitory for hematopoietic colony formation. Antibodies to interferon- gamma (IFN-gamma) partially reversed the inhibitory effects. Supernatants from the NK-cell cultures could also exert these effects on hematopoiesis, although to a lesser extent. Analysis of the NK cell RNA demonstrated that activated NK cells express genes for hematopoietic growth factors such as granulocyte-macrophage colony- stimulating factor (GM-CSF), granulocyte CSF (G-CSF), and IL-1 beta. The NK cells were also found to express IFN-gamma, transforming growth factor-beta 1 (TGF-beta 1), and tumor necrosis factor-alpha (TNF-alpha) mRNA. Analysis of the NK-cell supernatants using factor-dependent myeloid progenitor cell lines showed that the NK cells were producing G- CSF and growth-promoting activity that could not be attributed to IL-1, IL-3, IL-4, IL-5, IL-6, GM-CSF, G-CSF, macrophage CSF (M-CSF), or stem cell factor. The transfer of activated NK cells with BMC into lethally irradiated syngeneic mice resulted in greater BMC engraftment in the recipients. Thus, these results using a pure population of activated NK cells indicate that when activated, these cells can produce a variety of growth factors for hematopoiesis and exert significant hematopoietic growth-promoting effects in vivo.

Pre-Stimulation of CML CD34+ Cells with High Concentrations of Growth Factors Counters Imatinib-Mediated Proliferation Suppression and Enhances Apoptosis of Non-Dividing Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2479-2479
Author(s):  
Melissa S. Holtz ◽  
Stephen J. Forman ◽  
Ravi Bhatia
Keyword(s):  
Growth Factors ◽  
Imatinib Treatment ◽  
Hematopoietic Growth Factors ◽  
Inhibition Of Proliferation ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Dividing Cells ◽  
High Concentrations

Abstract While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p<0.0001) and 7±3% in high GF (p=0.064)]. However, although overall apoptosis decreased, pre-stimulation resulted in increased apoptosis of undivided cells exposed to imatinib in either low GF (14±5%; p=0.022) or high GF (13±6%, p=0.065). These results are notable since increased apoptosis of undivided cells was not previously observed in any other condition. Importantly, the percent of input cells remaining viable and undivided decreased significantly for pre-stimulated cells exposed to imatinib in low GF (19±3%; p<0.0001) or high GF (7±2%; p=0.016). These results highlight the potential use of GF stimulation to enhance targeting of CML HSC. Additional studies examined whether GF readily available for clinical use (G-CSF and/or GM-CSF) could also enhance imatinib targeting of quiescent CML progenitors. CML CD34+ cells were exposed to 1mM imatinib for 96 hours in a basal low GF cocktail (250pg/ml G-CSF, 10pg/ml GM-CSF, 200pg/ml SCF, 1ng/ml IL6, 200pg/ml MIP1α, 50pg/ml LIF) alone or with the addition of G-CSF (50ng/ml) and/or GM-CSF (10ng/ml). While overall apoptosis decreased, apoptosis of undivided cells significantly increased for cells exposed to imatinib in high concentrations of G-CSF + GM-CSF compared to those in basal GF alone (8.7±1.3%; p=0.007). The percent of input cells remaining viable and undivided in the presence of imatinib significantly decreased with high G-CSF + GM-CSF compared to basal GF alone (7.2%±1.1; p=0.007). In conclusion, pre-stimulation with high concentrations of GF can lead to increased proliferation and enhance reduction of non-dividing CML CD34+ cells by imatinib. These results are of significance because non-dividing primitive cells have previously proven highly resistant to elimination by imatinib and support translational clinical studies to investigate whether intermittent GF administration can enhance elimination of residual CML stem and progenitor cells in patients in remission on imatinib treatment.

scholarly journals Growth factor requirements of childhood acute T-lymphoblastic leukemia: correlation between presence of chromosomal abnormalities and ability to grow permanently in vitro

Blood ◽  
1991 ◽  
Vol 77 (7) ◽  
pp. 1534-1545 ◽  
Author(s):  
R O'Connor ◽  
A Cesano ◽  
B Lange ◽  
J Finan ◽  
PC Nowell ◽  
...  
Keyword(s):  
Growth Factors ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Chromosomal Abnormalities ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Hematopoietic Growth Factors ◽  
Gm Csf

Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL- 103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, - 105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.

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