scholarly journals Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors [see comments]

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5032-5039 ◽  
Author(s):  
T Watanabe ◽  
C Kuszynski ◽  
K Ino ◽  
DG Heimann ◽  
HM Shepard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Flow Cytometric Analysis ◽  
Human Bone ◽  
Uninfected Control ◽  
Human Bone Marrow ◽  
Flow Cytometric ◽  
Cd34 Cells ◽  
Adenovirus Vectors ◽  
Beta Galactosidase

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit- erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.

CD45 Gating for Routine Flow Cytometric Analysis of Human Bone Marrow Specimens

1993 ◽  
Vol 677 (1 Clinical Flow) ◽  
pp. 265-280 ◽  
Author(s):  
GREGORY T. STELZER ◽  
KEITH E. SHULTS ◽  
MICHAEL R. LOKEN
Keyword(s):  
Bone Marrow ◽  
Flow Cytometric Analysis ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Flow Cytometric

CD164, a Novel Sialomucin on CD34+ and Erythroid Subsets, Is Located on Human Chromosome 6q21

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 849-866 ◽  
Author(s):  
Suzanne M. Watt ◽  
Hans-Jörg Bühring ◽  
Irene Rappold ◽  
James Yi-Hsin Chan ◽  
Jane Lee-Prudhoe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Flow Cytometric ◽  
Cd34 Cells

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34+ hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34+ cells to bone marrow stroma and by negatively regulating CD34+hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34+ cells are CD164+, with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34+ human bone marrow and cord blood cells that were CD38lo/− or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34+CD71lo/− or CD34+CD33lo/− cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34+CD164(103B2/9E10)lo/−cells in bone marrow are mainly CD19+ B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34lo/−CD19+ and CD34lo/−CD20+ B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34lo/−CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21. © 1998 by The American Society of Hematology.

CD164, a Novel Sialomucin on CD34+ and Erythroid Subsets, Is Located on Human Chromosome 6q21

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 849-866 ◽  
Author(s):  
Suzanne M. Watt ◽  
Hans-Jörg Bühring ◽  
Irene Rappold ◽  
James Yi-Hsin Chan ◽  
Jane Lee-Prudhoe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Flow Cytometric ◽  
Cd34 Cells

Abstract CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34+ hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34+ cells to bone marrow stroma and by negatively regulating CD34+hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34+ cells are CD164+, with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (&gt;90%) of CD34+ human bone marrow and cord blood cells that were CD38lo/− or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34+CD71lo/− or CD34+CD33lo/− cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34+CD164(103B2/9E10)lo/−cells in bone marrow are mainly CD19+ B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34lo/−CD19+ and CD34lo/−CD20+ B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34lo/−CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21. © 1998 by The American Society of Hematology.

scholarly journals Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3693-3701 ◽  
Author(s):  
Ponnazhagan Veena ◽  
Christie M. Traycoff ◽  
David A. Williams ◽  
Jon McMahel ◽  
Susan Rice ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Retroviral Vector ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Transduction Efficiency ◽  
Cd34 Cells

Abstract Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

scholarly journals Flow cytometric analysis of the DNA content of bovine and human bone marrow cells

2000 ◽  
Vol 22 (2) ◽  
pp. 117-120
Author(s):  
D. Hoeben ◽  
C. Burvenich ◽  
M. Lenjou ◽  
G. Nijs ◽  
A.‐M. Massart‐Leëan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Content ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Flow Cytometric ◽  
Marrow Cells

scholarly journals Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3693-3701
Author(s):  
Ponnazhagan Veena ◽  
Christie M. Traycoff ◽  
David A. Williams ◽  
Jon McMahel ◽  
Susan Rice ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Retroviral Vector ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Transduction Efficiency ◽  
Cd34 Cells

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

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