scholarly journals Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3290-3297 ◽  
Author(s):  
GM Crooks ◽  
DB Kohn
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Virus Binding ◽  
Steel Factor ◽  
Human Cd34 ◽  
Cd34 Cells

Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.

scholarly journals Growth factors increase amphotropic retrovirus binding to human CD34+ bone marrow progenitor cells

Blood ◽  
1993 ◽  
Vol 82 (11) ◽  
pp. 3290-3297 ◽  
Author(s):  
GM Crooks ◽  
DB Kohn
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Gene Transfer ◽  
Hematopoietic Cells ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Virus Binding ◽  
Steel Factor ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Gene transfer into human cells using murine amphotropic retroviral vectors is the basic technique used in most current gene therapy studies. The identity of the cell surface receptor for the amphotropic envelope remains unknown and thus its importance in gene transfer is poorly understood. We have measured specific retrovirus binding to cells to study amphotropic virus receptor regulation in human CD34+ bone marrow (BM) progenitors and primitive CD34+CD38- human hematopoietic cells. The rat monoclonal antibody 83A25 recognizes an epitope common to the envelope glycoprotein of all classes of Moloney murine leukemia virus. Indirect fluorescent labeling of 83A25 allows flow cytometric analysis of specific virus-cell interactions and is an indirect measure of specific receptors. Using this assay, amphotropic virus binding to fresh CD34+ cells was minimal. However, when CD34+ cells were cultured with or without growth factors for 4 days, specific binding of amphotropic retrovirus was readily shown. Inclusion of interleukin-3 (IL-3), IL-6, and Steel factor in cultures increased the fluorescence associated with amphotropic virus binding by twofold to four-fold (mean fold increase 2.7 +/- 0.84). Virus binding to CD34+CD38- cells was shown only in those cells culture in IL-3, IL-6, and Steel factor. These results suggest that certain cytokines may cause an increase in the number and/or affinity of amphotropic receptors on primitive human hematopoietic cells. Upregulation of viral receptor expression may be one of the mechanisms by which cytokines enhance gene transfer into primitive BM cells.

Expression of Macrophage Inflammatory Protein-1 Receptors in Human CD34+ Hematopoietic Cells and Their Modulation by Tumor Necrosis Factor- and Interferon-γ

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3073-3081 ◽  
Author(s):  
Jan Dürig ◽  
Erika A. de Wynter ◽  
Christoph Kasper ◽  
Michael A. Cross ◽  
James Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Necrosis ◽  
Hematopoietic Cells ◽  
Interferon Γ ◽  
Receptor Expression ◽  
Receptor Subtypes ◽  
Inflammatory Protein ◽  
Cd34 Cells ◽  
Macrophage Inflammatory Protein ◽  
Necrosis Factor

Abstract Macrophage inflammatory protein-1 (MIP-1) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1 may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1 receptors on CD34+ cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1 receptors on CD34+cells was analyzed by two-color flow cytometry using a biotinylated MIP-1 molecule. The mean percentage of LP CD34+ cells expressing the MIP-1 receptors was 67.7 ± 7.2% (mean ± SEM; n = 22) as compared with 89.9 ± 2.6% (n = 10) and 74.69 ± 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1 receptor subtypes on LP CD34+ cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34+ cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34+ cells for 24 to 36 hours in the presence of tumor necrosis factor- (TNF-) and interferon-γ (IFN-γ) resulted in a significant increase in MIP-1 receptor expression. TNF- induced MIP-1 receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1 receptor expression on hematopoietic cells. © 1998 by The American Society of Hematology.

scholarly journals Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3736-3749 ◽  
Author(s):  
Wanda Piacibello ◽  
Fiorella Sanavio ◽  
Antonella Severino ◽  
Alessandra Danè ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Hematopoietic Cells ◽  
Scid Mice ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Expression of Macrophage Inflammatory Protein-1 Receptors in Human CD34+ Hematopoietic Cells and Their Modulation by Tumor Necrosis Factor- and Interferon-γ

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3073-3081 ◽  
Author(s):  
Jan Dürig ◽  
Erika A. de Wynter ◽  
Christoph Kasper ◽  
Michael A. Cross ◽  
James Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Necrosis ◽  
Hematopoietic Cells ◽  
Interferon Γ ◽  
Receptor Expression ◽  
Receptor Subtypes ◽  
Inflammatory Protein ◽  
Cd34 Cells ◽  
Macrophage Inflammatory Protein ◽  
Necrosis Factor

Macrophage inflammatory protein-1 (MIP-1) can stimulate growth inhibitory and potent chemotactic functions in hematopoietic cells. To investigate whether the action of MIP-1 may be regulated at the cellular receptor level, we studied the expression and modulation of MIP-1 receptors on CD34+ cells isolated from normal bone marrow (NBM), umbilical cord blood (CB), and leukapheresis products (LP). Expression of MIP-1 receptors on CD34+cells was analyzed by two-color flow cytometry using a biotinylated MIP-1 molecule. The mean percentage of LP CD34+ cells expressing the MIP-1 receptors was 67.7 ± 7.2% (mean ± SEM; n = 22) as compared with 89.9 ± 2.6% (n = 10) and 74.69 ± 7.04% (n = 10) in CB and NBM, respectively (P = .4). The expression of the MIP-1 receptor subtypes on LP CD34+ cells was studied by indirect immunofluorescence using specific antibodies for the detection of CCR-1, CCR-4, and CCR-5. Microscopical examination revealed a characteristic staining of the cytoplasmic cell membrane for all three receptor subtypes. Detailed analysis of two LP samples showed that 65.8%, 4.4%, and 30.5% of CD34+ cells express CCR-1, CCR-4, and CCR-5, respectively. Culture of LP CD34+ cells for 24 to 36 hours in the presence of tumor necrosis factor- (TNF-) and interferon-γ (IFN-γ) resulted in a significant increase in MIP-1 receptor expression. TNF- induced MIP-1 receptor upregulation in a time- and concentration-dependent manner. Our results suggest that inhibitory cytokines produced by the bone marrow microenvironment are likely to be involved in the regulation of MIP-1 receptor expression on hematopoietic cells. © 1998 by The American Society of Hematology.

scholarly journals Knockdown of HSPA9 Induces Apoptosis and Increases TP53 Levels in Human CD34+ Hematopoietic Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 526-526
Author(s):  
Tuoen Liu ◽  
Kilannin Krysiak ◽  
Cara Lunn Shirai ◽  
Matthew Ndonwi ◽  
Matthew J. Walter
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Fold Change ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract HSPA9, a gene located on chromosome 5q31.2, is commonly deleted in patients with myelodysplastic syndromes (MDS). MDS patients with a deletion of the long arm of chromosome 5 [del(5q)] typically present with cytopenias, including anemia, and have increased levels of apoptosis in their bone marrow contributing to ineffective hematopoiesis. Recent evidence suggests that upregulation of TP53 in MDS bone marrow cells may contribute to the cytopenias and accererated apoptosis observed in patients. While the mechanisms of TP53 activation in MDS are likely to be multifactorial, gene haploinsufficiency has been shown to contribute. Previous reports have shown that knockdown of RPS14, a chromosome 5q33.1 gene, in human CD34+ cells (or heterozygous knockout in mouse bone marrow cells) results in upregulation of TP53 and an increase in apoptosis. It is not known whether additional del(5q) candidate genes contribute to TP53 activation in del(5q)-associated MDS. In order to determine whether HSPA9 gene deletion also results in TP53 activation, we used lentiviral shRNA vectors to knockdown the expression of HSPA9 in primary human CD34+ hematopoietic progenitor cells. The HSPA9 protein level was reduced to ~20% (sh960) and ~50% (sh433) compared to the control lentiviral shRNA (shGFP). Knockdown of HSPA9 significantly inhibited the growth (fold change sh960 compared to shGFP = 0.16, p<0.01; sh433 compared to shGFP = 0.39, p=0.06, N=3) and erythroid differentiation (CD71+ expression fold change sh960 compared to shGFP =0.26, p<0.001; sh433 compared to shGFP = 0.52, p<0.01, N=3) of human CD34+ cells grown in media promoting erythroid differentiation after 7 days in culture. In addition, knockdown of HSPA9 by sh960 significantly increased apoptosis (AnnexinV+ cells) in CD34+ cells compared to shGFP (fold change = 2, p<0.01, N=3). The increased apoptosis observed following HSPA9 knockdown was associated with increased TP53 expression (fold change sh960 compared to shGFP = 2 by intracellular flow cytometry, p<0.05, N=3), TP53 activity (sh960 compared to shGFP, p<0.05; sh433 compared to shGFP, p=0.06 by TP53 luciferase reporter assay), and increased mRNA expression of the pro-apoptotic TP53 target gene BAX (fold change sh960 compared to shGFP = 1.8 by Q-RT-PCR, p<0.05). Gene expression profiling of CD34+ cells transduced with sh433 vs. control knockdown revealed that 26 well-annotated TP53-induced genes are also expressed higher in HSPA9 knockdown cells compared to controls by gene set enrichment (FWER p=0.01), further implicating TP53 activation in HSPA9 knockdown cells. We show that treatment of primary human CD34+ cells with MKT-077, a rhodacyanine dye with inhibitory effects on HSPA9 protein by binding to its nucleotide binding domain, also results in dose-dependent growth inhibition, enhanced apoptosis, and reduced erythroid differentiation, similar to that observed following HSPA9 knockdown. HSPA9 has been shown to physically interact with TP53 in the cytoplasm, and knockdown of HSPA9 changes localization of TP53 from the cytoplasm to the nucleus in non-hematopoietic cells. The loss of cytoplasmic sequestration of TP53 by HSPA9 following HSPA9 knockdown is thought to contribute to many of the cellular phenotypes induced by HSPA9 knockdown. We show by immunoprecipitation that HSPA9 also interacts with TP53 in primary human CD34+ cells, providing a possible mechanism for regulation of TP53 by HSPA9 in hematopoietic cells. These findings indicate that HSPA9 knockdown may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS. The data also suggest that genetic (i.e., mutation or deletion) or functional inactivation of TP53 may be necessary for disease progression to occur in MDS patients with del(5q). Disclosures No relevant conflicts of interest to declare.

scholarly journals Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3736-3749 ◽  
Author(s):  
Wanda Piacibello ◽  
Fiorella Sanavio ◽  
Antonella Severino ◽  
Alessandra Danè ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Hematopoietic Cells ◽  
Scid Mice ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Anna Jetmore ◽  
P. Artur Plett ◽  
Xia Tong ◽  
Frances M. Wolber ◽  
Robert Breese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
G1 Cells ◽  
Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P &lt; .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

Adhesion receptor expression by CD34+ cells from peripheral blood or bone marrow grafts: Correlation with time to engraftment

2006 ◽  
Vol 34 (5) ◽  
pp. 680-687 ◽  
Author(s):  
Jack Gold ◽  
Helen M. Valinski ◽  
Adrianne N. Hanks ◽  
Karen K. Ballen ◽  
Chung-Cheng Hsieh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Receptor Expression ◽  
Adhesion Receptor ◽  
Cd34 Cells

scholarly journals Characterization of Retroviral Gene Transfer into Highly Purified Human CD34− Cells with Primitive Hematopoietic Capacity

2002 ◽  
Vol 5 (5) ◽  
pp. 635-643 ◽  
Author(s):  
Barbara Murdoch ◽  
Lisa Gallacher ◽  
Kristin Chadwick ◽  
Mickie Bhatia
Keyword(s):  
Gene Transfer ◽  
Retroviral Gene Transfer ◽  
Human Cd34 ◽  
Cd34 Cells

scholarly journals Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.

1987 ◽  
Vol 166 (1) ◽  
pp. 219-234 ◽  
Author(s):  
P W Kantoff ◽  
A P Gillio ◽  
J R McLachlin ◽  
C Bordignon ◽  
M A Eglitis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Adenosine Deaminase ◽  
Nonhuman Primates ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Retroviral Vector ◽  
Infected Cells ◽  
Low Levels ◽  
Neomycin Resistance

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.

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