1068. A Murine Model for Lentiviral Gene Transfer to Hematopoietic Stem Cells - Minimizing Multi Copy Integration under Clinically Relevant Ex Vivo Conditions

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs ofFancC−/−mice. Using this approach, we show thatFancC−/−HSCs were efficiently corrected. Intrafemoral gene transfer of theFancCgene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

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Retroviral-mediated expression of recombinant Fancc enhances the repopulating ability of Fancc−/−hematopoietic stem cells and decreases the risk of clonal evolution

Blood ◽

10.1182/blood-2002-08-2404 ◽

2003 ◽

Vol 101 (4) ◽

pp. 1299-1307 ◽

Cited By ~ 58

Author(s):

Laura S. Haneline ◽

Xiaxin Li ◽

Samantha L. M. Ciccone ◽

Ping Hong ◽

Yanzhu Yang ◽

...

Keyword(s):

Stem Cells ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Clonal Evolution ◽

Wild Type ◽

Hematopoietic Stem ◽

Tnf Α ◽

Ifn Γ ◽

Marked Resistance

Fanconi anemia (FA) is a chromosomal instability disorder characterized by a progressive bone marrow (BM) failure and an increased incidence of myeloid leukemias. Children with FA are currently being enrolled in clinical trials to evaluate the safety of retroviral-mediated gene transfer. Previously, we usedFancc−/− mice to show thatFancc−/− hematopoietic stem cells (HSCs) have a profound defect in repopulating ability. Here, we examined whether retroviral-mediated gene transfer of recombinantFancc (rFancc) would restore the repopulating ability of Fancc−/− HSC to wild-type levels. Fancc−/− HSCs transduced with a retrovirus encoding rFancc exhibited a repopulating ability that approached wild-type levels. Interestingly, ∼30% of primary recipients (7 of 22) transplanted with uncorrectedFancc−/− cells developed a range of hematopoietic abnormalities including pancytopenia and BM hypoplasia similar to individuals with FA. Hematopoietic abnormalities were detected in only 1 of 22 mice transplanted withFancc−/− cells transduced with a retrovirus encoding rFancc. Moreover, several mice with hematopoietic defects had progenitors that displayed a marked resistance to IFN-γ, TNF-α, and MIP-1α compared to both Fancc−/−progenitors, which are uniquely hypersensitive to these cytokines, and wild-type progenitors. These data are analogous to studies using progenitors from patients with myelodysplasia and provide functional support for clonal evolution in these mice. Collectively, these data show that gene transfer can enhance HSC repopulating ability and suppresses the tendency for clonal evolution. These studies also reveal potential detrimental effects of ex vivo manipulation for untransducedFancc−/− HSCs.

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Polyclonal fluctuation of lentiviral vector–transduced and expanded murine hematopoietic stem cells

Blood ◽

10.1182/blood-2010-08-303222 ◽

2011 ◽

Vol 117 (11) ◽

pp. 3053-3064 ◽

Cited By ~ 38

Author(s):

Tobias Maetzig ◽

Martijn H. Brugman ◽

Stefan Bartels ◽

Niels Heinz ◽

Olga S. Kustikova ◽

...

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

De Novo ◽

Lentiviral Vector ◽

Ex Vivo ◽

Late Time ◽

Hematopoietic Stem ◽

The Impact

Abstract Gene therapy has proven its potential to cure diseases of the hematopoietic system. However, severe adverse events observed in clinical trials have demanded improved gene-transfer conditions. Whereas progress has been made to reduce the genotoxicity of integrating gene vectors, the role of pretransplantation cultivation is less well investigated. We observed that the STIF (stem cell factor [SCF], thrombopoietin [TPO], insulin-like growth factor-2 [IGF-2], and fibroblast growth factor-1 [FGF-1]) cytokine cocktail developed to effectively expand murine hematopoietic stem cells (HSCs) also supports the expansion of leukemia-initiating insertional mutants caused by gammaretroviral gene transfer. We compared 4 protocols to examine the impact of prestimulation and posttransduction culture in STIF in the context of lentiviral gene transfer. Observing 56 transplanted mice for up to 9.5 months, we found consistent engraftment and gene-marking rates after prolonged ex vivo expansion. Although a lentiviral vector with a validated insertional-mutagenic potential was used, longitudinal analysis identifying > 7000 integration sites revealed polyclonal fluctuations, especially in “expanded” groups, with de novo detection of clones even at late time points. Posttransduction expansion in STIF did not enrich clones with insertions in proto-oncogenes but rather increased clonal diversity. Our data indicate that lentiviral transduction in optimized media mediates intact polyclonal hematopoiesis without selection for growth-promoting hits by posttransduction expansion.

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Faculty Opinions recommendation of Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1016469.201396 ◽

2003 ◽

Author(s):

Miles Wilkinson

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Direct Delivery

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Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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A Review of Evaluating Hematopoietic Stem Cells Derived from Umbilical Cord Blood's Expansion and Homing

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200124115444 ◽

2020 ◽

Vol 15 (3) ◽

pp. 250-262

Author(s):

Maryam Islami ◽

Fatemeh Soleimanifar

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Umbilical Cord ◽

Stromal Cells ◽

Ex Vivo ◽

Hematologic Malignancies ◽

Future Directions ◽

Hematopoietic Stem ◽

Efficient Procedure ◽

Hematopoietic Recovery

Transplantation of hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB) has been taken into account as a therapeutic approach in patients with hematologic malignancies. Unfortunately, there are limitations concerning HSC transplantation (HSCT), including (a) low contents of UCB-HSCs in a single unit of UCB and (b) defects in UCB-HSC homing to their niche. Therefore, delays are observed in hematopoietic and immunologic recovery and homing. Among numerous strategies proposed, ex vivo expansion of UCB-HSCs to enhance UCB-HSC dose without any differentiation into mature cells is known as an efficient procedure that is able to alter clinical treatments through adjusting transplantation-related results and making them available. Accordingly, culture type, cytokine combinations, O2 level, co-culture with mesenchymal stromal cells (MSCs), as well as gene manipulation of UCB-HSCs can have effects on their expansion and growth. Besides, defects in homing can be resolved by exposing UCB-HSCs to compounds aimed at improving homing. Fucosylation of HSCs before expansion, CXCR4-SDF-1 axis partnership and homing gene involvement are among strategies that all depend on efficiency, reasonable costs, and confirmation of clinical trials. In general, the present study reviewed factors improving the expansion and homing of UCB-HSCs aimed at advancing hematopoietic recovery and expansion in clinical applications and future directions.

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