scholarly journals Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 492-504 ◽  
Author(s):  
G Fisher-Adams ◽  
KK Jr Wong ◽  
G Podsakoff ◽  
SJ Forman ◽  
S Chatterjee
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Chromosomal Dna ◽  
Hematopoietic Progenitor ◽  
Adeno Associated Virus ◽  
Cd34 Cells ◽  
Vector Sequences

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

scholarly journals Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2059-2068 ◽  
Author(s):  
CM Traycoff ◽  
ST Kosak ◽  
S Grigsby ◽  
EF Srour
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Cell Tracking ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cells In Culture ◽  
Cd34 Cells ◽  
Limiting Dilution

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood.

1994 ◽  
Vol 179 (6) ◽  
pp. 1867-1875 ◽  
Author(s):  
S Z Zhou ◽  
S Cooper ◽  
L Y Kang ◽  
L Ruggieri ◽  
S Heimfeld ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Recombinant Virus ◽  
High Efficiency ◽  
Hematopoietic Progenitor Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Progenitor ◽  
Adeno Associated Virus

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Karen L. MacKenzie ◽  
Neil R. Hackett ◽  
Ronald G. Crystal ◽  
Malcolm A. S. Moore
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Adenoviral Vector ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Infected Cells

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P < .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Adenoviral vector–mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Karen L. MacKenzie ◽  
Neil R. Hackett ◽  
Ronald G. Crystal ◽  
Malcolm A. S. Moore
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Adenoviral Vector ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Infected Cells

Abstract Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level transgene expression is required. In the present investigations, we have used an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated toxicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34+ cells and primary colony forming unit–cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34+ cells. Transduction of CFU-Cs and more primitive HPCs was quantified following fluorescence activated cell sorting for green flourescence protein expression. Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of week-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more primitive cells. Although there was no apparent quantitative change in week-5 CAFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P &lt; .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU-Cs and more accurately indicated by week-5 CAFCs. These studies provide the first functional and quantitative evidence of adenoviral transduction of primitive HPCs. However, further investigations will be necessary to overcome adenoviral toxicity toward primitive HPCs before adenoviral vectors can be considered a safe option for gene therapy.

Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3367-3367
Author(s):  
Hal E. Broxmeyer ◽  
Edward F. Srour ◽  
Scott Cooper ◽  
Carrie T. Wallace ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Gm Csf ◽  
Cd34 Cells

Abstract Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.

scholarly journals Orderly Process of Sequential Cytokine Stimulation Is Required for Activation and Maximal Proliferation of Primitive Human Bone Marrow CD34+ Hematopoietic Progenitor Cells Residing in G0

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 658-668 ◽  
Author(s):  
Amy C. Ladd ◽  
Robert Pyatt ◽  
Andre Gothot ◽  
Susan Rice ◽  
Jon McMahel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase Ii ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
Cytokine Stimulation ◽  
Phase Iv

Abstract Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+ ). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with &lt;20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0 /G1 , unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

scholarly journals Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy- 1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2842-2848 ◽  
Author(s):  
M Korbling ◽  
YO Huh ◽  
A Durett ◽  
N Mirza ◽  
P Miller ◽  
...  
Keyword(s):  
Body Weight ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Allogeneic Transplantation ◽  
Hematopoietic Progenitor Cells ◽  
Donor Blood ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells ◽  
The Mean ◽  
Single Bone

Abstract Apheresis-derived hematopoietic progenitor cells have recently been used for allogeneic transplantation. Forty-one normal donors were studied to assess the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (12 micrograms/kg/d) on the peripheralization of hematopoietic progenitor cells and lymphoid subsets. The white blood cell, polymorphonuclear cell (PMNC), and lymphocyte concentrations at the peak of rhG-CSF effect in the donor's peripheral blood (PB) exceeded baseline by 6.4-, 8.0-, and 2.2-fold, respectively. Corresponding concentrations of PB CD34+ cells and primitive subsets such as CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells increased by 16.3-fold, 24.2-fold, and 23.2-fold, respectively in eight normal donors. The percentage of CD34+ Thy-1dim and CD34+ Thy- 1dim CD38- cells among CD34+ cells increased as well, suggesting an additional peripheralization effect of rhG-CSF on primitive CD34+ subsets. The preapheresis PB CD34+ and CD34+ Thy-1dim cell concentrations were predictive of their corresponding apheresis yield per liter of donor blood processed PB lymphoid subsets were not significantly affected by rhG-CSF treatment. The mean apheresis-derived yield of CD34+, CD34+ Thy-1dim, and CD34+ Thy-1dim CD38- cells per kilogram of recipient body weight and per liter of donor blood processed was 48.9 x 10(4) (n = 41), 27.2 x 10(4) (n = 10), and 1.9 x 10(4) (n = 10), respectively. As compared with 43 single bone marrow (BM) harvest, the CD34+ cell yield of peripheral blood progenitor cell allografts of 41 normal donors exceeded that of BM allografts by 3.7- fold and that of lymphoid subsets by 16.1-fold (CD3+), 13.3-fold (CD4+), 27.4-fold (CD8+), 11.0-fold (CD19+), and 19.4-fold (CD56+CD3-). All PBPC allografts were cryopreserved before transplantation. The mean recovery of CD34+ cells after freezing, thawing, and washing out dimethylsulfoxide was 86.6% (n = 31) and the recovery of lymphoid subsets was 115.5% (CD3+), 121.4% (CD4+), 105.6% (CD8+), 118.1% (CD19+), and 102.4% (CD56+CD3-). All donors were related to patients: 39 sibling-to-sibling, 1 parent-to-child, and 1 child-to-parent transplant. Thirty-eight transplants were HLA fully identical, two transplants differed in one and two antigens. Engraftment occurred in 38 recipients; two patients died too early to be evaluated, and one patient did not engraft. The lowest CD34+ cell dose transplanted and resulting in complete and sustained engraftment was 2.5 x 10(6)/kg of recipient body weight.(ABSTRACT TRUNCATED AT 400 WORDS)

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