scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831 ◽  
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Abstract Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

scholarly journals Intracellular Immunization of Rhesus CD34+ Hematopoietic Progenitor Cells With a Hairpin Ribozyme Protects T Cells and Macrophages From Simian Immunodeficiency Virus Infection

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4822-4831
Author(s):  
Michael Rosenzweig ◽  
Douglas F. Marks ◽  
Donna Hempel ◽  
Marina Heusch ◽  
Günter Kraus ◽  
...  
Keyword(s):  
T Cells ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hairpin Ribozyme ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Cell Gene ◽  
Stem Cell Gene Therapy

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.

scholarly journals In vitro expansion of hematopoietic progenitor cells induces functional expression of Fas antigen (CD95)

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2871-2877 ◽  
Author(s):  
K Takenaka ◽  
K Nagafuji ◽  
M Harada ◽  
S Mizuno ◽  
T Miyamoto ◽  
...  
Keyword(s):  
Growth Factors ◽  
Progenitor Cells ◽  
Calf Serum ◽  
Functional Expression ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Progenitor ◽  
Fas Antigen ◽  
Cd34 Cells

Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis. To investigate the role of both molecules in hematopoiesis, we evaluated the expression of Fas Ag and Bcl-2 on CD34+ hematopoietic progenitor cells expanded in vitro. CD34+ cells isolated from bone marrow were cultured in iscove's modified Dulbecco's medium supplemented with 10% fetal calf serum, 1% bovine serum albumin, 50 ng/mL stem cell factor, 50 ng/mL interleukin-3 (IL-3), 50 ng/mL IL-6, 100 ng/mL granulocyte colony-stimulating factor, and 3 U/mL erythropoietin for 7 days. Colony-forming unit of granulocytes/macrophages (CFU-GM) and burst-forming unit of erythroids (BFU-E) were expanded 6.9-fold and 8.8-fold in number at day 5 of culture, respectively. Freshly isolated CD34+ cells did not express Fas Ag, whereas approximately half of them expressed Bcl-2. CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag and rapidly lost Bcl-2 expression. Furthermore, apoptosis was induced in the cultured CD34+ population when anti-Fan antibody (IgM; 1 microgram/mL) was added, as shown by significant decrease in the number of viable cells, morphologic changes, induction of DNA fragmentation, and significant decrease in the number of clonogenic progenitor cells including CFU. GM and BFU-E. These results indicate that functional expression of Fas Ag is induced on CD34+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and downregulation of Bcl-2 may be expressed as part of the differentiation program of hematopoietic cells and may be involved in the regulation of hematopoiesis.

scholarly journals Inhibition of purified CD34+ hematopoietic progenitor cells by human immunodeficiency virus 1 or gp120 mediated by endogenous transforming growth factor beta 1.

1996 ◽  
Vol 183 (1) ◽  
pp. 99-108 ◽  
Author(s):  
G Zauli ◽  
M Vitale ◽  
D Gibellini ◽  
S Capitani
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Transforming Growth Factor ◽  
Hematopoietic Progenitor Cells ◽  
Tgf Beta ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Immunodeficiency Virus ◽  
Hiv 1

Human CD34+ hematopoietic progenitor cells, stringently purified from the peripheral blood of 20 normal donors, showed an impaired survival and clonogenic capacity after exposure to either heat-inactivated human immunodeficiency virus (HIV) 1 (strain IIIB) or cross-linked envelope gp120. Cell cycle analysis, performed at different times in serum-free liquid culture, showed an accumulation in G0/G1 in HIV-1- or gp120-treated cells and a progressive increase of cells with subdiploid DNA content, characteristic of apoptosis. In blocking experiments with anti-transforming growth factor (TGF) beta 1 neutralizing serum or TGF-beta 1 oligonucleotides, we demonstrated that the HIV-1- or gp120-mediated suppression of CD34+ cell growth was almost entirely due to an upregulation of endogenous TGF-beta 1 produced by purified hematopoietic progenitors. Moreover, by using a sensitive assay on the CCL64 cell line, increased levels of bioactive TGF-beta 1 were recovered in the culture supernatant of HIV-1/gp120-treated CD34+ cells. Anti-TGF-beta 1 neutralizing serum or TGF-beta 1 oligonucleotides were also effective in inducing a significant increase of the plating efficiency of CD34+ cells, purified from the peripheral blood of three HIV-1-seropositive individuals, suggesting that a similar mechanism may be also operative in vivo. The relevance of these findings to a better understanding of the pathogenesis of HIV-1-related cytopenias is discussed.

scholarly journals Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4363-4368 ◽  
Author(s):  
Yasuhiro Ebihara ◽  
Kohichiro Tsuji ◽  
Stewart D. Lyman ◽  
Xingwei Sui ◽  
Makoto Yoshida ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Receptor Tyrosine Kinase ◽  
Synergistic Effects ◽  
Myeloid Cells ◽  
Synergistic Action ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cord Blood Cd34

We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3328-3337 ◽  
Author(s):  
Domenico Sansonno ◽  
Claudio Lotesoriere ◽  
Vito Cornacchiulo ◽  
Massimo Fanelli ◽  
Pietro Gatti ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Progenitor Cells ◽  
Hcv Infection ◽  
Hematopoietic Progenitor Cells ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

scholarly journals The Chemokine SDF-1 Is a Chemoattractant for Human CD34+ Hematopoietic Progenitor Cells and Provides a New Mechanism to Explain the Mobilization of CD34+ Progenitors to Peripheral Blood

1997 ◽  
Vol 185 (1) ◽  
pp. 111-120 ◽  
Author(s):  
A. Aiuti ◽  
I.J. Webb ◽  
C. Bleul ◽  
T. Springer ◽  
J.C. Gutierrez-Ramos
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stromal Cell Derived Factor ◽  
Transient Elevation

Hematopoietic progenitor cells migrate in vitro and in vivo towards a gradient of the chemotactic factor stromal cell-derived factor-1 (SDF-1) produced by stromal cells. This is the first chemoattractant reported for human CD34+ progenitor cells. Concentrations of SDF-1 that elicit chemotaxis also induce a transient elevation of cytoplasmic calcium in CD34+ cells. SDF-1-induced chemotaxis is inhibited by pertussis toxin, suggesting that its signaling in CD34+ cells is mediated by seven transmembrane receptors coupled to Gi proteins. CD34+ cells migrating to SDF-1 include cells with a more primitive (CD34+/CD38− or CD34+/DR−) phenotype as well as CD34+ cells phenotypically committed to the erythroid, lymphoid and myeloid lineages, including functional BFU-E, CFU-GM, and CFU-MIX progenitors. Chemotaxis of CD34+ cells in response to SDF-1 is increased by IL-3 in vitro and is lower in CD34+ progenitors from peripheral blood than in CD34+ progenitors from bone marrow, suggesting that an altered response to SDF-1 may be associated with CD34 progenitor mobilization.

Direct and Indirect Effects of Oxymetholone on Growth and Apoptosis of Hematopoietic Progenitor Cells In Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4127-4127
Author(s):  
Deog-Yeon Jo ◽  
Seong-Woo Kim ◽  
Jin-Hee Hwang ◽  
Hwan-Jung Yun ◽  
Samyong Kim
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Indirect Effects ◽  
Clonal Growth ◽  
Hematopoietic Progenitor Cells ◽  
Direct And Indirect Effects ◽  
Hematopoietic Progenitor ◽  
Colony Forming Unit ◽  
Cd34 Cells

Abstract Despite a tarnished reputation, androgens remain a common treatment for aplastic anemia, especially in the Orient. Increased apoptosis of hematopoietic cells is characteristic of aplastic anemia; however, it has not been established whether androgens affect apoptosis of hematopoietic progenitor cells. In most previous studies regarding the in vitro effects of androgens on hematopoietic cells, whole bone marrow cells were used, rather than purified hematopoietic progenitor cells such as CD34+ cells. With these questions in mind, we investigated the direct and indirect effects of oxymetholone and other androgens on apoptosis and growth of normal hematopoietic progenitor cells (HPCs) in vitro. Oxymetholone did not rescue normal BM CD34+ cells and colony-forming cells (CFCs), other than mature erythroid CFCs, from apoptosis induced by growth factor deprivation. Unexpectedly, both testosterone and 5-dihydrotestosterone (5-DHT) at a concentration of 10−5 M, but not oxymetholone, increased the percentage of annexin-positive apoptotic cells (62.2 ± 5.9%, P < 0.05; 61.7 ± 6.4%, P < 0.05, respectively) compared with the controls (52.6 ± 5.6%). The addition of either stromal cell-derived factor-1 (SDF-1) or stem cell factor (SCF) partially relieved the increase in apoptosis induced by 5-DHT, and the addition of both SDF-1 and SCF completely reversed it. Oxymetholone did not rescue CFCs from interferon-gamma (IFN-g)-induced inhibition of clonal growth of BM CD34+ cells in methylcellulose cultures. Furthermore, oxymetholone did not mitigate IFN-g-induced suppression of CD34+ cell survival in the presence of growth factors. In a methylcellulose clonogenic assay, oxymetholone stimulated the clonal growth of colony-forming unit-erythroid at low concentrations, while not affecting colony-forming unit-granulocyte/macrophage or burst-forming unit-erythroid. Oxymetholone did not reverse the IFN-g-induced inhibition of colony formation by CD34+ cells. Interestingly, oxymetholone stimulated the production of SCF and thrombopoietin in normal human bone marrow stromal cells (BMSCs) through transcriptional regulation while inhibiting the production of interleukin-6. In agreement with this, oxymetholone-treated BMSCs better supported the survival and growth of HPCs. These results suggest that oxymetholone exerts most of its myelostimulatory effects via the regulation of cytokine production in BMSCs, rather than by direct action on hematopoietic progenitor cells.

Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus

Blood ◽  
1990 ◽  
Vol 76 (12) ◽  
pp. 2476-2482 ◽  
Author(s):  
JM Molina ◽  
DT Scadden ◽  
M Sakaguchi ◽  
B Fuller ◽  
A Woon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Immunodeficiency Virus ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Marrow Cells

The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.

scholarly journals Synergistic Action of Flt3 and gp130 Signalings in Human Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4363-4368 ◽  
Author(s):  
Yasuhiro Ebihara ◽  
Kohichiro Tsuji ◽  
Stewart D. Lyman ◽  
Xingwei Sui ◽  
Makoto Yoshida ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Receptor Tyrosine Kinase ◽  
Synergistic Effects ◽  
Myeloid Cells ◽  
Synergistic Action ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

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