Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation

1994 ◽  
Vol 14 (7) ◽  
pp. 4834-4842
Author(s):  
A Dubart ◽  
F Feger ◽  
C Lacout ◽  
F Goncalves ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Major Influence ◽  
Proliferative Potential ◽  
Steel Factor ◽  
Interleukin 3 ◽  
Marrow Cells

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

scholarly journals Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

1994 ◽  
Vol 14 (7) ◽  
pp. 4834-4842 ◽  
Author(s):  
A Dubart ◽  
F Feger ◽  
C Lacout ◽  
F Goncalves ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Major Influence ◽  
Proliferative Potential ◽  
Steel Factor ◽  
Interleukin 3 ◽  
Marrow Cells

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

scholarly journals Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1473-1481 ◽  
Author(s):  
LS Rusten ◽  
SE Jacobsen ◽  
O Kaalhus ◽  
OP Veiby ◽  
S Funderud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

scholarly journals Functional differences between CD38- and DR- subfractions of CD34+ bone marrow cells

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1473-1481 ◽  
Author(s):  
LS Rusten ◽  
SE Jacobsen ◽  
O Kaalhus ◽  
OP Veiby ◽  
S Funderud ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Cd34 Cells ◽  
Marrow Cells

Several studies have previously demonstrated enrichment in primitive progenitor cells in subfractions of CD34+ bone marrow (BM) cells not expressing CD38 or HLA-DR (DR) antigens. However, no studies have directly compared these two cell populations with regard to their content of primitive and more committed progenitor cells. Flow cytometric analysis of immunomagnetic isolated CD34+ cells demonstrated little overlap between CD34+CD38- and CD34+DR- progenitor subpopulations in that only 12% to 14% of total CD34+DR- and CD34+CD38- cells were double negative (CD34+CD38-DR-). Although the number of committed myeloid progenitor cells (colony-forming units granulocyte- macrophage) was reduced in both subpopulations, only CD34+CD38- cells were significantly depleted in committed erythroid progenitor cells (burst-forming units-erythroid). In single-cell assay, CD34+CD38- cells showed consistently poorer response to single as opposed to multiple hematopoietic growth factors as compared with unfractionated CD34+ cells, indicating that the CD34+CD38- subset is relatively enriched in primitive hematopoietic progenitor cells. Furthermore, CD34+CD38- and CD34+DR- cells, respectively, formed 3.2-fold and 1.6-fold more high proliferative potential colony-forming cell (HPP-CFC) colonies than did unfractionated CD34+ cells. Finally, CD34+CD38-DR- cells were depleted in HPP-CFCs as compared with CD34+CD38+DR+ cells. The results of the present study suggest that both the CD38- and DR- subfractions of CD34+ bone marrow cells are enriched in primitive hematopoietic progenitor cells, with the CD34+CD38- subpopulation being more highly enriched than CD34+DR- cells.

scholarly journals Interleukin-3 is significantly more effective than other colony- stimulating factors in long-term maintenance of human bone marrow- derived colony-forming cells in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1836-1841 ◽  
Author(s):  
M Kobayashi ◽  
BH Van Leeuwen ◽  
S Elsbury ◽  
ME Martinson ◽  
IG Young ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cultured Cells ◽  
Bone Marrow Cells ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Factor G ◽  
Marrow Cells

Abstract Human bone marrow cells cultured for 21 days in the presence of recombinant human interleukin-3 (IL-3) produced up to 28 times more colony-forming cells (CFC) than could be obtained from cultures stimulated with granulocyte colony stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF). IL-3-cultured cells retained a multipotent response to IL-3 in colony assays but were restricted to formation of granulocyte colonies in G-CSF and granulocyte or macrophage colonies in GM-CSF. Culture of bone marrow cells in IL-3 also led to accumulation of large numbers of eosinophils and basophils. These data contrast with the effects of G-CSF, GM-CSF, and IL-3 in seven-day cultures. Here both GM-CSF and IL-3 amplified total CFC that had similar multipotential colony-forming capability in either factor. G-CSF, on the other hand, depleted IL-3-responsive colony-forming cells dramatically, apparently by causing these cells to mature into granulocytes. The data suggest that a large proportion of IL-3- responsive cells in human bone marrow express receptors for G-CSF and can respond to this factor, the majority becoming neutrophils. Furthermore, the CFC maintained for 21 days in IL-3 may be a functionally distinct population from that produced after seven days culture of bone marrow cells in either IL-3 or GM-CSF.

scholarly journals Erythroid cell growth from normal and W/WV murine bone marrow on macrophage-coated membranes

Blood ◽  
1977 ◽  
Vol 50 (5) ◽  
pp. 857-866
Author(s):  
BJ Torok-Starb ◽  
NS Wolf ◽  
DR Boggs
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Bone Marrow Cells ◽  
Erythroid Cell ◽  
Erythroid Progenitor ◽  
Murine Bone Marrow ◽  
Cellulose Acetate Membranes ◽  
Coated Membranes ◽  
Marrow Cells

Cellulose acetate membranes (CAM) placed in the peritoneal cavity of mice develop a macrophage layer capable of supporting in vivo hematopoietic colonies from intraperitoneally injected bone marrow cells. Modifications allowing for routine morphologic identification of colonies showed that both erythrocytic (E) and granulocytic (G) colonies occur with a consistent E:G ratio of 0.19 +/- 0.037. Stimulating recipients by bleeding or phenylhydrazine injection did not produce a significant change in the total number of colonies and a reduction in granulocytic colonies so that the E:G ratio significnatly increased. Hypertransfusion of donor animals had no effect on the number of erythroid colonies that grew on CAM of average recipients. The total colony-forming ability of bone marrow cells from genetically anemic W/WV mice was found not to differ from that of normal +/+ littermates; however, the E:G ratio of W/WV marrow in bled recipients was significantly lower (p less than 0.01) then that of +/+ marrow. These studies suggest that a CAM system supports an erythroid progenitor which is not affected by hypotransfusion of the donor animal, yet is dependent upon erythropoietin for colony formation, and that it is defective in the W/WV mouse.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34+ bone marrow cells

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 581-585 ◽  
Author(s):  
Vishwanath Bhattacharya ◽  
Peter A. McSweeney ◽  
Qun Shi ◽  
Benedetto Bruno ◽  
Atsushi Ishida ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Vascular Graft ◽  
Bone Marrow Cells ◽  
Venous Blood ◽  
Immunomagnetic Bead ◽  
Composite Grafts ◽  
Cd34 Cells ◽  
Marrow Cells

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34+ cells can differentiate into endothelial cells. The current study was designed to determine whether CD34+ progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm × 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34+ cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 ± 5.3 × 106. During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO3staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% ± 3.4% vs 26.6% ± 7.6%; P = .0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34+ cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications.

scholarly journals Calcium ionophore but not phorbol ester promotes eicosanoids release by proliferating interleukin-3-dependent bone marrow cells

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1586-1592 ◽  
Author(s):  
Y Shibata ◽  
PG McCaffrey ◽  
H Sato ◽  
Y Oghiso
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
Calcium Ionophore ◽  
Mouse Bone Marrow ◽  
Ionophore A23187 ◽  
Interleukin 3 ◽  
Marrow Cells

Abstract Eicosanoid release during multilineage hematopoiesis was assessed using freshly isolated mouse bone marrow cells cultured in the presence of interleukin-3 (IL-3) (10% WEHI-3 culture-conditioned medium). Cells that could release prostaglandin E2 (PGE2) when stimulated with calcium ionophore A23187, but not with phorbol ester (PMA), appeared within 4 days. The cells harvested on day 10 released 42 ng of PGE2/10(6) cells/mL after A23187 stimulation. Leukotriene B4 (LTB4) (4 ng/mL) was also detected after A23187 stimulation, but there was no detectable LTC4 (less than 0.5 ng/mL). Nonadherent bone marrow cells were isolated from 28-day cultures and cloned. All clones were strongly IL-3- dependent. Although other growth factors such as granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and CSF-1 failed to promote survival or support proliferation of the cells, three clones (11–1-A6, 3–2-D5, and 11–1-A1) showed significant increases in 3H-thymidine incorporation, respectively, after PMA treatment for 24 hours. Surviving cells displayed dominantly myeloid type morphology and phenotypic characteristics. The data suggest that IL-3 is important in the formation of PGE2-producing cells. In contrast to many macrophages (MO), neither the IL-3-dependent cell lines nor the IL-3-cultured bone marrow cells released significant amounts of PGE2 when stimulated with PMA or IL-3, although PMA and IL-3 both induced translocation of protein kinase C (PKC) to the membrane fraction. The lack of production of PGE2 and other eicosanoids by the PMA- and IL-3- stimulated cell lines was confirmed by measuring the release of 3H- arachidonic acid. The data suggest that in IL-3-dependent bone marrow cell lines the activation of eicosanoid metabolism requires elevated cellular Ca2+; PKC activation alone does not appear to be a sufficient stimulus.

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.

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