scholarly journals Role of c-kit ligand in the expansion of human hematopoietic progenitor cells

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 634-641 ◽  
Author(s):  
J Brandt ◽  
RA Briddell ◽  
EF Srour ◽  
TB Leemhuis ◽  
R Hoffman
Keyword(s):  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Cell Numbers ◽  
Gm Csf ◽  
Kit Ligand ◽  
Marrow Cells

Abstract To test the hypothesis that the c-kit ligand plays an important role in the regulation of early events occurring during human hematopoiesis, we determined the effect of a recombinant form of c-kit ligand, termed mast cell growth factor (MGF), on the high-proliferative potential colony-forming cell (HPP-CFC) and the cell responsible for initiating long-term hematopoiesis in vitro (LTBMIC). MGF alone did not promote HPP-CFC colony formation by CD34+ DR- CD15- marrow cells, but synergistically augmented the ability of a combination of granulocyte- monocyte colony-stimulating factor (GM-CSF) interleukin (IL)-3 and a recombinant GM-CSF/IL-3 fusion protein (FP) to promote the formation of HPP-CFC-derived colonies. MGF had a similarly profound effect on in vitro long-term hematopoiesis. Repeated additions of IL-3, GM-CSF, or FP alone to CD34+ DR- CD15- marrow cells in a stromal cell-free culture system increased cell numbers 10(3)-fold by day 56 of long-term bone marrow culture (LTBMC), while combinations of MGF with IL-3 or FP yielded 10(4)- and 10(5)-fold expansion of cell numbers. Expansion of the number of assayable colony-forming unit-granulocyte-monocyte (CFU- GM) generated during LTBMC was also markedly enhanced when MGF was added in combination with IL-3 or FP. In addition, MGF, IL-3, and FP individually led to a twofold to threefold increase in HPP-CFC numbers after 14 to 21 days of LTBMC. Furthermore, the effects of these cytokines on HPP-CFC expansion during LTBMC were additive. Throughout the LTBMC, cells receiving MGF possessed a higher cloning efficiency than those receiving IL-3, GM-CSF, or FP alone. These data indicate that the c-kit ligand synergistically interacts with a number of cytokines to directly augment the proliferative capacity of primitive human hematopoietic progenitor cells.

scholarly journals Role of c-kit ligand in the expansion of human hematopoietic progenitor cells

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 634-641 ◽  
Author(s):  
J Brandt ◽  
RA Briddell ◽  
EF Srour ◽  
TB Leemhuis ◽  
R Hoffman
Keyword(s):  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Cell Numbers ◽  
Gm Csf ◽  
Kit Ligand ◽  
Marrow Cells

To test the hypothesis that the c-kit ligand plays an important role in the regulation of early events occurring during human hematopoiesis, we determined the effect of a recombinant form of c-kit ligand, termed mast cell growth factor (MGF), on the high-proliferative potential colony-forming cell (HPP-CFC) and the cell responsible for initiating long-term hematopoiesis in vitro (LTBMIC). MGF alone did not promote HPP-CFC colony formation by CD34+ DR- CD15- marrow cells, but synergistically augmented the ability of a combination of granulocyte- monocyte colony-stimulating factor (GM-CSF) interleukin (IL)-3 and a recombinant GM-CSF/IL-3 fusion protein (FP) to promote the formation of HPP-CFC-derived colonies. MGF had a similarly profound effect on in vitro long-term hematopoiesis. Repeated additions of IL-3, GM-CSF, or FP alone to CD34+ DR- CD15- marrow cells in a stromal cell-free culture system increased cell numbers 10(3)-fold by day 56 of long-term bone marrow culture (LTBMC), while combinations of MGF with IL-3 or FP yielded 10(4)- and 10(5)-fold expansion of cell numbers. Expansion of the number of assayable colony-forming unit-granulocyte-monocyte (CFU- GM) generated during LTBMC was also markedly enhanced when MGF was added in combination with IL-3 or FP. In addition, MGF, IL-3, and FP individually led to a twofold to threefold increase in HPP-CFC numbers after 14 to 21 days of LTBMC. Furthermore, the effects of these cytokines on HPP-CFC expansion during LTBMC were additive. Throughout the LTBMC, cells receiving MGF possessed a higher cloning efficiency than those receiving IL-3, GM-CSF, or FP alone. These data indicate that the c-kit ligand synergistically interacts with a number of cytokines to directly augment the proliferative capacity of primitive human hematopoietic progenitor cells.

scholarly journals Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 661-669 ◽  
Author(s):  
EF Srour ◽  
JE Brandt ◽  
RA Briddell ◽  
S Grigsby ◽  
T Leemhuis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Hla Dr

Abstract Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

scholarly journals Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 661-669 ◽  
Author(s):  
EF Srour ◽  
JE Brandt ◽  
RA Briddell ◽  
S Grigsby ◽  
T Leemhuis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Fold Increase ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Hla Dr

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.

scholarly journals Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2890-2897 ◽  
Author(s):  
C von Kalle ◽  
HP Kiem ◽  
S Goehle ◽  
B Darovsky ◽  
S Heimfeld ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Leukemia Virus ◽  
Hematopoietic Progenitor Cells ◽  
Transduction Efficiency ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Marrow Cells

Abstract Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2890-2897 ◽  
Author(s):  
C von Kalle ◽  
HP Kiem ◽  
S Goehle ◽  
B Darovsky ◽  
S Heimfeld ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Leukemia Virus ◽  
Hematopoietic Progenitor Cells ◽  
Transduction Efficiency ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Marrow Cells

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction efficiency has been low using amphotropic Moloney murine leukemia virus (MoMLV) vectors. In this study, we investigated modifications of gene transfer using amphotropic MoMLV vectors in cell-free supernatant for their ability to increase the currently low transduction of both committed hematopoietic progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and their precursors, long-term culture- initiating cells (LTC-IC). First, based on the observation that bone marrow cells express more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction efficiency was compared between CD34 enriched and nonenriched progenitor populations. Third, the duration of transduction in vitro was extended to increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed to identical titers of pseudotyped PG13/LN vector or PA317/LN vector in the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vector supernatant was refed daily. Hematopoietic progenitor cells as measured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antigen positive cells significantly improved gene transfer from 13.9% G418-resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor-supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Hematopoietic progenitor cells from patients with myelodysplastic syndromes: in vitro colony growth and long-term proliferation

1999 ◽  
Vol 23 (4) ◽  
pp. 385-394 ◽  
Author(s):  
Eugenia Flores-Figueroa ◽  
Guillermo Gutiérrez-Espindola ◽  
Susana Guerrero-Rivera ◽  
Javier Pizzuto-Chavez ◽  
Hector Mayani
Keyword(s):  
Progenitor Cells ◽  
Myelodysplastic Syndromes ◽  
Colony Growth ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

scholarly journals Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.

1992 ◽  
Vol 175 (4) ◽  
pp. 1151-1154 ◽  
Author(s):  
J H Jansen ◽  
J C Kluin-Nelemans ◽  
J Van Damme ◽  
G J Wientjens ◽  
R Willemze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Interleukin 6 ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Marrow Cells

Since monocytes and macrophages that arise during the culture of bone marrow progenitor cells are potential sources of interleukin 6 (IL-6), we investigated whether auto- or paracrine production of this factor is involved in colony formation by normal hematopoietic progenitor cells. We added a polyclonal anti-IL-6 antiserum and a monoclonal anti-IL-6 antibody to cultures of monocyte- and T cell-depleted bone marrow cells. Colony formation was stimulated with granulocyte/monocyte-colony-stimulating factor (GM-CSF), monocyte-CSF, or IL-3. Addition of anti-IL-6 antibody resulted in decreased numbers of monocytic colonies to 40-50% of control values, whereas the numbers of granulocytic colonies were not altered. The inhibitory effect was preserved in cultures of CD34(+)-enriched bone marrow cells. As a second approach, we added a monoclonal antibody directed against the IL-6 receptor to cultures of monocyte- and T cell-depleted bone marrow cells. This antibody almost completely inhibited the growth of monocytic colonies, again without decreasing the number of granulocytic colonies. Finally, the importance of IL-6 in monocytopoiesis was demonstrated in serum-deprived bone marrow cultures: addition of exogenous IL-6 to cultures stimulated with GM-CSF resulted in increased numbers of monocytic colonies. Our results indicate that the permissive presence of IL-6 is required for optimal monocytic colony formation by bone marrow progenitor cells.

scholarly journals Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor

Blood ◽  
1992 ◽  
Vol 79 (12) ◽  
pp. 3159-3167 ◽  
Author(s):  
RA Briddell ◽  
VC Broudy ◽  
E Bruno ◽  
JE Brandt ◽  
EF Srour ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Hematopoietic Progenitor Cells ◽  
Cloning Efficiency ◽  
Cell Cloning ◽  
Hematopoietic Progenitor ◽  
Kit Ligand ◽  
Hla Dr

A mouse antihuman monoclonal IgG2a antibody, termed stem cell receptor- 1 (SR-1), specific for a determinant of the c-kit ligand receptor (KR), was used as an immunologic probe to analyze KR expression by human bone marrow hematopoietic progenitor cells. Monoclonal antibodies to CD34 and HLA-DR were used in a multicolor staining protocol in conjunction with SR-1 to further define the phenotypes of various classes of hematopoietic progenitor cells. Expression of KR (SR-1+) on hematopoietic progenitor cells identified subpopulations of cells expressing CD34 (CD34+). While one-half of the CD34- and HLA-DR- expressing cells (CD34+ HLA-DR+) expressed the KR (SR-1+), one-third of the CD34+ cells that lacked HLA-DR expression (CD34+ HLA-DR-) were SR- 1+. The CD34+ HLA-DR+ SR-1+ cell population contained the vast majority of the more differentiated progenitor cells, including the colony- forming unit (CFU) granulocyte-macrophage; burst-forming unit- erythrocyte; CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; and the CFU-megakaryocyte. The overall progenitor cell cloning efficiency of this subpopulation was greater than 31%. By contrast, the CD34+ HLA-DR- SR-1+ cell population contained fewer of these more differentiated progenitor cells but exclusively contained the more primitive progenitor cells, the BFU-megakaryocyte, high proliferative potential-colony-forming cell, and long-term bone marrow culture- initiating cell. The overall progenitor cell cloning efficiency of this subpopulation was greater than 7%. Both the CD34+ HLA-DR- and CD34+ HLA- DR+ cell subpopulations lacking KR expression contained few assayable hematopoietic progenitor cells. Long-term bone marrow cultures initiated with CD34+ HLA-DR- SR-1+ but not CD34+ HLA-DR- SR-1- cells, which were repeatedly supplemented with c-kit ligand (KL) and interleukin-3, generated assayable progenitor cells of at least 2 lineages for 10 weeks. These experiments demonstrate the expression of the KR throughout the hierarchy of human hematopoietic progenitor cell development. We conclude from our data that the KL and KR play a pivotal role in cytokine regulation of both the primitive and more differentiated human hematopoietic progenitor cells.

Transcriptional Profiling Reveals a Critical Role for Gfi1 in STAT3-Dependent Dendritic Cell Development and -Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3555-3555
Author(s):  
Chozhavendan Rathinam ◽  
Robert Geffers ◽  
Raif Yuecel ◽  
Jan Buer ◽  
Karl Welte ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Progenitor Cells ◽  
Mhc Class Ii ◽  
Transcriptional Profiling ◽  
Critical Role ◽  
Hematopoietic Progenitor Cells ◽  
Developmental Defect ◽  
Hematopoietic Progenitor ◽  
Gm Csf

Abstract Dendritic cells (DCs) comprise heterogenous and functionally diverse populations of antigen presenting cells. Their developmental pathways remain largely unknown. Using a transcriptional profiling approach, we identified Gfi1 as a novel critical transcription factor in GM-CSF-dependent DC differentiation. Gfi1 is expressed in precursor and mature DCs, as seen in Gfi+/GFP mice, in which one Gfi1 allele is replaced by the GFP cDNA. Gfi1−/ − mice showed a global reduction of myeloid and lymphoid DCs in all lymphoid organs whereas epidermal Langerhans cells were enhanced in number. Gfi1−/ − DCs showed marked phenotypic and functional alterations, as exemplified by decreased MHC class II expression, absent upregulation of costimulatory molecules upon stimulation and reduced ability to stimulate specific T-cell responses. In contrast, Gfi1−/ − DCs exhibited an increased activation profile as assessed by enhanced secretion of IL12. In vitro, Gfi1−/ − hematopoietic progenitor cells were unable to develop into DCs in the presence of GM-CSF or Flt3L. Instead, they differentiated into macrophages, as evidenced by morphology, expression of cell surface markers, and functional properties. These findings suggest that Gfi1 is a critical modulator of DC versus macrophage development. Analysis of hematopoietic chimeras upon transplantation into congenic recipient mice established a cell-autonomous and non-redundant role for Gfi1 in DC development. Furthermore, upon retroviral gene transfer into Gfi1−/ − progenitor cells, the developmental defect could be reconstituted in vitro and in vivo. The inability of Gfi1−/ − hematopoietic progenitor cells to develop into DCs was associated with decreased STAT3 activation, as shown by Western blot and EMSA assays. In conclusion, we have identified Gfi1 as a critical transcription factor that controls DC versus macrophage development and dissociates DC maturation and -activation.

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