scholarly journals Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2821-2826 ◽  
Author(s):  
CM Verfaillie
Keyword(s):  
Bone Marrow ◽  
Direct Contact ◽  
Hematopoietic Progenitors ◽  
Proliferation And Differentiation ◽  
Bone Marrow Cultures ◽  
Marrow Stroma ◽  
Blood Elements

Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45- microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.

scholarly journals Direct contact between human primitive hematopoietic progenitors and bone marrow stroma is not required for long-term in vitro hematopoiesis

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2821-2826 ◽  
Author(s):  
CM Verfaillie
Keyword(s):  
Bone Marrow ◽  
Direct Contact ◽  
Hematopoietic Progenitors ◽  
Proliferation And Differentiation ◽  
Bone Marrow Cultures ◽  
Marrow Stroma ◽  
Blood Elements

Abstract Long-term bone marrow cultures support both differentiation and conservation of primitive human hematopoietic progenitors in the absence of exogenous cytokines. It is believed that hematopoiesis in such cultures requires direct contact between hematopoietic progenitors and stroma. In the present study, we demonstrate that primitive progenitors physically separated from the stromal layer by a 0.45- microns microporous membrane continue to generate differentiated progenitors for at least 8 weeks. Moreover, primitive progenitors are conserved to a greater extent under these conditions, as when cultured in direct contact with the stroma. However, excessive production of granulocyte-macrophage progenitors occurs when primitive progenitors are not allowed to interact directly with the stroma. Thus, direct contact between hematopoietic and stromal cells is not required for either differentiation or conservation of primitive hematopoietic progenitors but is essential for the regulated production of mature blood elements. These findings can now be used to define the role of diffusible factors and cell-cell or cell-extracellular matrix adhesion events in the regulation of conservation, proliferation, and differentiation of primitive human hematopoietic progenitors in vitro.

scholarly journals Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2045-2053 ◽  
Author(s):  
CM Verfaillie
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Terminal Differentiation ◽  
Hematopoietic Progenitors ◽  
Soluble Factors ◽  
Bone Marrow Cultures ◽  
Stromal Layer

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (“stroma-free”) or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane (“stroma-noncontact”). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a “stroma-free” culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in “stroma-noncontact” cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in “stroma-noncontact” cultures supplemented with or without IL-3 than in “stroma-free” cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.

scholarly journals Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 332-337 ◽  
Author(s):  
RA Briddell ◽  
JE Brandt ◽  
TB Leemhuis ◽  
R Hoffman
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Gm Csf ◽  
Liquid Suspension ◽  
Bone Marrow Cultures ◽  
Macrophage Colony

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

scholarly journals Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 332-337 ◽  
Author(s):  
RA Briddell ◽  
JE Brandt ◽  
TB Leemhuis ◽  
R Hoffman
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Interleukin 1 ◽  
Gm Csf ◽  
Liquid Suspension ◽  
Bone Marrow Cultures ◽  
Macrophage Colony

Abstract An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.

Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6

Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1438-1447
Author(s):  
RL Mei ◽  
SA Burstein
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Maturation In Vitro ◽  
Bone Marrow Cultures ◽  
Acetylcholinesterase Staining ◽  
Stromal Layer ◽  
Marrow Cultures

Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.

scholarly journals Megakaryocytic maturation in murine long-term bone marrow culture: role of interleukin-6

Blood ◽  
1991 ◽  
Vol 78 (6) ◽  
pp. 1438-1447 ◽  
Author(s):  
RL Mei ◽  
SA Burstein
Keyword(s):  
Bone Marrow ◽  
Interleukin 6 ◽  
Maturation In Vitro ◽  
Bone Marrow Cultures ◽  
Acetylcholinesterase Staining ◽  
Stromal Layer ◽  
Marrow Cultures

Abstract Megakaryocytic maturation was analyzed in long-term bone marrow cultures in the absence of added growth factors. Megakaryocytes could be observed for periods of up to 13 weeks in both the supernatant and stromal layer of these cultures. Using acetylcholinesterase staining for enumeration and sizing of megakaryocytes, and a novel rat antimurine platelet monoclonal antibody (MoAb) that detects only megakaryocytes in bone marrow, the number, volume, and ploidy of these cells were assessed microscopically and by flow cytometry. Correlation of these measurements with ambient interleukin-6 (IL-6) levels showed no relationship between IL-6 bioactivity and megakaryocyte number. Conversely, the relatively high IL-6 bioactivity present during the first 2 weeks of culture was correlated with increased megakaryocytic size and ploidy, while the relatively lower IL-6 bioactivity present after week 3 corresponded to decreased megakaryocytic size and ploidy. Addition of neutralizing anti-IL-6 MoAb decreased megakaryocytic size and ploidy at times when ambient IL-6 levels were relatively high, while the addition of exogenous IL-6 increased size and ploidy at times when endogenous IL-6 concentrations were low. The data show that long- term bone marrow cultures can be used as a means to evaluate megakaryocytic maturation in vitro, and suggest that, to some extent, IL-6 plays a role in the maturation process in this system.

scholarly journals Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2045-2053 ◽  
Author(s):  
CM Verfaillie
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Expansion ◽  
Terminal Differentiation ◽  
Hematopoietic Progenitors ◽  
Soluble Factors ◽  
Bone Marrow Cultures ◽  
Stromal Layer

Abstract We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (“stroma-free”) or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane (“stroma-noncontact”). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a “stroma-free” culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in “stroma-noncontact” cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in “stroma-noncontact” cultures supplemented with or without IL-3 than in “stroma-free” cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.

scholarly journals Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures.

1989 ◽  
Vol 9 (9) ◽  
pp. 3973-3981 ◽  
Author(s):  
G V Borzillo ◽  
C J Sherr
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chain Gene ◽  
Mouse Bone Marrow ◽  
Feeder Layers ◽  
Bone Marrow Cultures ◽  
B Lymphoid ◽  
Marrow Cultures

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.

Early pre-B-cell transformation induced by the v-fms oncogene in long-term mouse bone marrow cultures

1989 ◽  
Vol 9 (9) ◽  
pp. 3973-3981
Author(s):  
G V Borzillo ◽  
C J Sherr
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Chain Gene ◽  
Mouse Bone Marrow ◽  
Feeder Layers ◽  
Bone Marrow Cultures ◽  
B Lymphoid ◽  
Marrow Cultures

Murine long-term bone marrow cultures that support B-lymphoid-cell development were infected with a helper-free retrovirus containing the v-fms oncogene. Infection of B-lymphoid cultures resulted in the rapid clonal outgrowth of early pre-B cells, which grew to high cell densities on stromal cell feeder layers, expressed v-fms-coded glycoproteins, and underwent immunoglobulin heavy-chain gene rearrangements. Late-passage cultures gave rise to factor-independent variants that proliferated in the absence of feeder layers, developed resistance to hydrocortisone, and became tumorigenic in syngeneic mice. The v-fms oncogene therefore recapitulates known effects of the v-abl and bcr-abl oncogenes on B-lineage cells. The ability of v-fms to induce transformation of early pre-B cells in vitro underscores the capacity of oncogenic mutants of the colony-stimulating factor-1 receptor to function outside the mononuclear phagocyte lineage.

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

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