Comparative effects of retroviral-mediated gene transfer into primary human stromal cells of flt3-ligand, interleukin 3 and gm-csf on production of cord blood progenitor cells in long-term culture

Abstract Abstract 3211 We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells. Disclosures: No relevant conflicts of interest to declare.

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Transduction of Primitive Human Marrow and Cord Blood-Derived Hematopoietic Progenitor Cells With Adeno-Associated Virus Vectors

Blood ◽

10.1182/blood.v93.6.1882.406k03_1882_1894 ◽

1999 ◽

Vol 93 (6) ◽

pp. 1882-1894 ◽

Cited By ~ 55

Author(s):

Saswati Chatterjee ◽

Wei Li ◽

Christie Ann Wong ◽

Grace Fisher-Adams ◽

Di Lu ◽

...

Keyword(s):

Gene Transfer ◽

Cord Blood ◽

Progenitor Cells ◽

Early Time ◽

Placental Alkaline Phosphatase ◽

Adeno Associated Virus ◽

Long Term Culture ◽

Vector Integration ◽

Wide Range

We evaluated the capacity of adeno-associated virus (AAV) vectors to transduce primitive human myeloid progenitor cells derived from marrow and cord blood in long-term cultures and long-term culture-initiating cell (LTC-IC) assays. Single-colony analyses showed that AAV vectors transduced CD34+ and CD34+38− clonogenic cells in long-term culture. Gene transfer was readily observed in LTC-ICs derived from 5-, 8-, and 10-week cultures. Recombinant AAV (rAAV) transduction was observed in every donor analyzed, although a wide range of gene transfer frequencies (5% to 100%) was noted. AAV transduction of LTC-ICs was stable, with week-8 and -10 LTC-ICs showing comparable or better transduction relative to week-5 LTC-ICs. Fluorescence in situ hybridization (FISH) analyses performed to determine the fate of AAV vectors in transduced cells showed that 9% to 28% of CD34+ and CD34+38− cells showed stable vector integration as evidenced by chromosome-associated signals in metaphase spreads. Comparisons of interphase and metaphase FISH suggested that a fraction of cells also contained episomal vector at early time points after transduction. Despite the apparent loss of the episomal forms with continued culture, the number of metaphases containing integrated vector genomes remained stable long term. Transgene transcription and placental alkaline phosphatase (PLAP) expression was observed in CD34+, CD34+38−LTC-ICs in the absence of selective pressure. These results suggest that primitive myeloid progenitors are amenable to genetic modification with AAV vectors.

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Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.529 ◽

1993 ◽

Vol 178 (2) ◽

pp. 529-536 ◽

Cited By ~ 89

Author(s):

T Moritz ◽

D C Keller ◽

D A Williams

Keyword(s):

Bone Marrow ◽

Gene Transfer ◽

Cord Blood ◽

Progenitor Cells ◽

Severe Combined Immunodeficiency ◽

Genetic Material ◽

Retroviral Vectors ◽

Transduction Efficiency ◽

Human Cord Blood

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.

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Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽

10.1182/blood.v80.12.3079.bloodjournal80123079 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3079-3089

Author(s):

J Mladenovic ◽

SM Anderson

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Progenitor Cells ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Conditioned Media ◽

Enzyme Linked Immunosorbent Assay ◽

Gm Csf

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

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A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Blood ◽

10.1182/blood.v114.22.696.696 ◽

2009 ◽

Vol 114 (22) ◽

pp. 696-696

Author(s):

Zejin Sun ◽

Yan Li ◽

Jingling Li ◽

Shanbao Cai ◽

Yi Zeng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Gene Transfer ◽

Cord Blood ◽

Progenitor Cells ◽

Peripheral Blood ◽

Ex Vivo ◽

Transduction Efficiency ◽

Gene Transfer Efficiency

Abstract Abstract 696 Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited. In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v96.1.100.013k41_100_108 ◽

2000 ◽

Vol 96 (1) ◽

pp. 100-108 ◽

Cited By ~ 1

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.

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Prenatal liver stromal cells: Favorable feeder cells for long‐term culture of hepatic progenitor cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.28921 ◽

2019 ◽

Vol 120 (10) ◽

pp. 16624-16633 ◽

Cited By ~ 1

Author(s):

Zahra Feizi ◽

Ensieh Zahmatkesh ◽

Zahra Farzaneh ◽

Abbas Piryaei ◽

Roberto Gramignoli ◽

...

Keyword(s):

Progenitor Cells ◽

Stromal Cells ◽

Hepatic Progenitor Cells ◽

Feeder Cells ◽

Long Term Culture

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Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture

Blood ◽

10.1182/blood.v80.12.3079.3079 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3079-3089 ◽

Cited By ~ 2

Author(s):

J Mladenovic ◽

SM Anderson

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Progenitor Cells ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Conditioned Media ◽

Enzyme Linked Immunosorbent Assay ◽

Gm Csf

Abstract The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.

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

Blood ◽

10.1182/blood.v96.1.100 ◽

2000 ◽

Vol 96 (1) ◽

pp. 100-108 ◽

Cited By ~ 27

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 < .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.

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