scholarly journals Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells

Blood ◽  
1996 ◽  
Vol 87 (8) ◽  
pp. 3229-3236 ◽  
Author(s):  
P Gupta ◽  
JB McCarthy ◽  
CM Verfaillie
Keyword(s):  
Stem Cell ◽  
Heparan Sulfate ◽  
Ex Vivo ◽  
Function Analysis ◽  
Soluble Form ◽  
Stromal Fibroblast ◽  
Colony Stimulating Factor ◽  
Long Term Culture ◽  
Stimulating Factor

We have recently demonstrated that 50% of primitive human long-term culture-initiating cells (LTC-IC) are maintained for up to 8 weeks in stroma-dependent cultures in which progenitor-stroma contact is prevented (stroma noncontact), or when progenitors are cultured in medium conditioned by stromal feeders. This indicates that factors responsible for LTC-IC maintenance are present in soluble form in stromal supernatant (SN). Although the picogram concentrations of cytokines present in stromal SN can induce the differentiation of CD34+/HLA-DR- (DR-) cells to clonogenic cells (colony forming cells; CFC), they maintain only 10% of LTC-IC for 5 weeks, suggesting that factors other than these cytokines are required for LTC-IC maintenance. To characterize the factor(s) in stromal SN responsible for LTC-IC maintenance, we purified glycoproteins and proteoglycans (PG) from the SN of the LTC-IC supportive murine marrow stromal fibroblast cell line M2–10B4 by ion exchange high performance liquid chromatography (HPLC). Culture of DR- cells in a combination of M2–10B4-derived PG, but not glycoproteins and picogram concentrations of recombinant human interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), granulocyte- macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1alpha (MIP-1alpha) resulted in the recovery of 96% +/- 8% of LTC-IC maintained in cultures supplemented with unfractionated stromal SN. LTC-IC maintenance was largely retained after digestion of the PG-rich fraction with proteinase K and after dissociative gel filtration chromatography, but was completely abolished following treatment with nitrous acid, which digests heparan sulfate glycosaminoglycans (HS GAG). As for M2–10B4-derived HS GAG, high concentrations of bovine kidney HS GAG, but not bovine tracheal chondroitin sulfate, significantly improved cytokine-mediated LTC-IC maintenance. Maintenance of LTC-IC by these nonmarrow-derived HS GAG was, however, significantly lower than that seen with M2–10B4-derived HS. These studies demonstrate a role for marrow stroma-derived HS GAG in the long-term in vitro maintenance of human LTC-IC. Further structure-function analysis of these HS GAG may have important implications for ex vivo stem cell expansion and gene transfer into hematopoietic progenitors.

scholarly journals Increased numbers of long-term culture-initiating cells in the apheresis product of patients randomized to receive increasing doses of stem cell factor administered in combination with chemotherapy and a standard dose of granulocyte colony-stimulating factor

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3323-3328 ◽  
Author(s):  
A Weaver ◽  
D Ryder ◽  
D Crowther ◽  
TM Dexter ◽  
NG Testa
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Standard Dose ◽  
Fold Increase ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Long Term Culture ◽  
To Receive ◽  
Stimulating Factor

Long-term culture-initiating cells (LTC-IC) are arguably the most primitive human hematopoietic cells detectable by in vitro functional assays. We have investigated the mobilization of these cells into the blood of patients with ovarian carcinoma randomized to receive granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg) plus different doses of stem cell factor (SCF; c-kit ligand) after chemotherapy or G-CSF alone after chemotherapy. We have shown a significant SCF dose response for the mobilization of LTC-IC, with a 5.8-fold increase in LTC-IC mobilization in those patients receiving chemotherapy, G-CSF, and 20 micrograms/kg of SCF, the highest dose used, compared with the patients receiving chemotherapy and G-CSF alone. We have shown a threefold increase in CD34+ cells and up to a 64-fold increase in CD34+/33- cells was seen in patients treated with chemotherapy, G-CSF, and 20 micrograms/kg of SCF compared with those patients treated with chemotherapy and G-CSF alone. However, significant numbers of CD34+/38- cells were only found in the patients receiving 20 micrograms/kg of SCF as part of their mobilization regimen. Patients receiving chemotherapy plus G-CSF and SCF have enhanced mobilization of primitive cells and of the more committed progenitor cells compared with those patients receiving chemotherapy followed by G-CSF alone.

scholarly journals Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony- stimulating factor

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 430-435
Author(s):  
WE Fibbe ◽  
J van Damme ◽  
A Billiau ◽  
HM Goselink ◽  
PJ Voogt ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Culture Medium ◽  
Interleukin 1 ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Long Term Culture ◽  
Identical Manner ◽  
Immunologic Assays ◽  
Stimulating Factor

Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony- stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum. Stromal layers, once stimulated by IL 1, continued to release CSA into the culture medium in the absence of exogenous IL 1. A second IL 1 pulse induced CSA release in an identical manner, as did the primary stimulation, indicating that the CSA released was actively produced. Using specific immunologic assays, both granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) could be identified in the culture supernatants, and production of both factors was inducible by IL 1. Shortly after initiation of the long-term marrow cultures “spontaneous” G-CSF and M-CSF release occurred. The release of G-CSF diminished following addition of the anti-IL 1 antiserum, indicating that endogenous production of IL 1 by stromal cells had contributed to this effect. These results further support the role of IL 1 as an important modulator of CSF production by cells of the hematopoietic microenvironment.

scholarly journals Mobilization of hematopoietic stem and progenitor cell subpopulations from the marrow to the blood of mice following cyclophosphamide and/or granulocyte colony-stimulating factor

Blood ◽  
1993 ◽  
Vol 81 (7) ◽  
pp. 1960-1967 ◽  
Author(s):  
S Neben ◽  
K Marcus ◽  
P Mauch
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cell Content ◽  
Blood Stem Cells ◽  
Stimulating Factor

Committed progenitor cells and primitive stem cells mediate early and sustained engraftment, respectively, after lethal irradiation and stem cell transplantation. Peripheral blood stem cells (PBSC) from unstimulated mice are deficient in both cell types. To study techniques to mobilize both progenitor cells and primitive stem cells from the marrow to the blood, we collected peripheral blood from C57BL/6 mice 6 to 7 days after a single dose of cyclophosphamide (CY; 200 mg/kg intraperitoneally), after recombinant human granulocyte colony- stimulating factor (rhG-CSF) (250 micrograms/kg/d twice per day subcutaneously for 4 days), or after CY followed by G-CSF. Significant increases in white blood cell counts (1.6- to 2.7-fold) and circulating day 8 colony-forming unit spleen (CFU-S) (11- to 36-fold) were seen with all three mobilization methods compared with unstimulated control mice. Transplantation of mobilized blood stem cells into lethally irradiated hosts decreased the time to erythroid engraftment. Blood stem cells were analyzed for primitive stem cell content by Rs, an assay for CFU-S self-renewal, and competitive repopulation index (CRI), an assay of long-term repopulating ability. The primitive stem cell content of unstimulated blood was clearly deficient, but was significantly increased following mobilization, approaching normal bone marrow levels. These results were confirmed by an in vitro limiting dilution long-term culture assay that measures the frequency of progenitor cells and primitive stem cells. Mobilization following CY + G-CSF was accompanied by a marked loss of both progenitor cells and primitive stem cells in the marrow. In contrast, following G-CSF alone the progenitor cell and primitive stem cell content of the marrow was unchanged. Stem cell mobilization following CY + G-CSF was not affected by previous exposure of donors to cytosine arabinoside or cyclophosphamide, but was significantly reduced by previous exposure to busulfan. These data show that stem cell content in the blood may reach near-normal marrow levels after mobilization, the mobilization from the marrow to the blood is temporary and reversible, the specific technique used may mobilize different subpopulations of stem cells, and the type of prior chemotherapy may influence the ability to mobilize stem cells into the blood.

scholarly journals Enrichment of human hematopoietic stem cell activity in the CD34+Thy- 1+Lin- subpopulation from mobilized peripheral blood

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 368-378 ◽  
Author(s):  
L Murray ◽  
B Chen ◽  
A Galy ◽  
S Chen ◽  
R Tushinski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Colony Stimulating Factor ◽  
In Vivo Models ◽  
Hematopoietic Stem ◽  
Stimulating Factor ◽  
Mobilized Peripheral Blood

Abstract The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy- 1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell- containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Granulocyte Colony-Stimulating Factor Enhances Bone Marrow Stem Cell Damage Caused by Repeated Administration of Cytotoxic Agents

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1950-1956 ◽  
Author(s):  
Ronald van Os ◽  
Simon Robinson ◽  
Tara Sheridan ◽  
John M.K. Mislow ◽  
Donald Dawes ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Experimental Model ◽  
Cytotoxic Agents ◽  
Cytotoxic Agent ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cell Content ◽  
Stimulating Factor

Despite the increasing use of cytokines to circumvent the acute dose-limiting myelotoxicity of cancer treatment, little is known about the combined effects of cytotoxic agents and cytokines on the primitive stem cells responsible for long-term hematopoiesis. In an experimental model, we administered cytotoxic agents that have variable effects on primitive stem cells in C57BL/6 (B6)-mice. Mice received six every-other-week doses of cyclophosphamide (CY, 84 mg/kg), VP-16 (24 mg/kg) + cisplatinum (2.4 mg/kg), carboplatinum (50 mg/kg), chlorambucil (12 mg/kg), BCNU (13.2 mg/kg), or TBI (80 cGy). Granulocyte colony-stimulating factor (G-CSF; 250 μg/kg/day) was administered subcutaneously twice daily on days 3 to 6 after each dose of the cytotoxic agent. Comparison with animals receiving the cytotoxic agent alone was made to investigate the effects of G-CSF on long-term hematopoiesis. Hematopoiesis was measured 20 weeks after the last dose of the cytotoxic agent by assessment of peripheral blood counts, marrow cellularity, progenitor cell content (colony-forming units-spleen; CFU-S), and primitive stem cell number (long-term repopulating ability and day 28 and day 35 cobblestone area-forming cell [CAFC] frequencies). Exposure to cytotoxic agents alone resulted in a significant decrease in primitive stem cells (as measured by repopulating units [RU] and day 28 and day 35 CAFC content) in animals given carboplatinum, chlorambucil, BCNU, and TBI, but not in animals treated with cyclophosphamide or VP-16 and cisplatinum. The addition of G-CSF resulted in a significant decrease in stem cell content when compared with no G-CSF administration in animals treated with chlorambucil, BCNU, or TBI. Thus, G-CSF administered after repeated exposure to cytotoxic agents, appeared to damage the primitive stem cell compartment when used in combination with agents known to damage primitive stem cells. These results, although obtained in an experimental model, should raise concerns for the indiscriminate use of G-CSF in the clinic. © 1998 by The American Society of Hematology.

scholarly journals Mobilization of long-term hematopoietic reconstituting cells in mice by the combination of stem cell factor plus granulocyte colony-stimulating factor

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 795-799 ◽  
Author(s):  
XQ Yan ◽  
R Briddell ◽  
C Hartley ◽  
G Stoney ◽  
B Samal ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Short Term ◽  
Term Survival ◽  
Group A ◽  
Group B ◽  
Stimulating Factor

Abstract In this study, we have compared the ability of recombinant human granulocyte colony-stimulating factor (rhG-CSF) alone and the combination of low doses of recombinant rat pegylated stem cell factor (rrSCF-PEG) plus rhG-CSF to mobilize peripheral blood progenitor cells (PBPCs) with long-term engrafting potential. Female recipient irradiated mice were transplanted with PBPCs from male mice that were mobilized with rhG-CSF alone (group A) or rrSCF-PEG plus rhG-CSF (group B). As previously shown, greater short-term survival resulted in group B compared with group A, with 80% and 40% survival at 30 days posttransplant, respectively. Both groups of animals showed long-term donor-derived engraftment in greater than 95% of animals, as determined by quantitative specific polymerase chain reaction amplification of a Y chromosome sequence from whole blood of the mice at 6 to 12 months posttransplantation. Analysis of individual granulocyte-macrophage colonies, picked up from semisolid methylcellulose culture of bone marrow cells from transplanted mice, resulted in detection of donor- derived DNA in 98% of colonies from group B mice compared with 81% from group A mice. These data show that cells with long-term potential are mobilized by rhG-CSF alone and the combination of rrSCF-PEG plus rhG- CSF. Furthermore, an increased number of cells with short-term and long- term engraftment potential was obtained with rrSCF-PEG plus rhG-CSF compared with rhG-CSF alone.

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