ANTIOXIDANTS PROTECT HEMATOPOIETIC STEM/PROGENITOR CELLS AGAINST OXIDATIVE STRESS AND LOSS OF STEMNESS PROPERTIES DURING EX VIVO MANIPULATION IN CELL AND GENE THERAPY PROTOCOLS
Author(s): ,
Elia Henry
Affiliations:
LSHL, IRCM,INSERM U1274/CEA,Fontenay-aux-Roses,France
,
Vilma Barroca
Affiliations:
IRCM,INSERM U1274/CEA,Fontenay-aux-Roses,France
,
Caroline Devanand
Affiliations:
IRCM,INSERM U1274/CEA,Fontenay-aux-Roses,France
,
Françoise Pflumio
Affiliations:
LSHL, IRCM,INSERM U1274/CEA,Fontenay-aux-Roses,France
Marie-Laure Arcangeli
Affiliations:
LSHL, IRCM,INSERM U1274/CEA,Fontenay-aux-Roses,France
(Abstract release date: 05/14/20) EHA Library. Henry E. 06/12/20; 293949; EP1463
Ms. Elia Henry
Ms. Elia Henry
Contributions
Abstract

Abstract: EP1463

Type: e-Poster

Background
Hematopoietic stem cells (HSC) are fundamental components of our hematopoietic system, able to produce every type of blood cell. They display self-renewal properties, which are essential to the maintenance of HSC pool during our life. These features make them ideal candidates for cell and gene therapy. Protocols involved in these therapies often comprise culture steps that allow expansion and/or genetic modification of HSC. However, these steps decrease HSC self-renewal potential, despite the addition of growth factors such as SCF, FLT3L or TPO.  This is partly due to reactive oxygen species (ROS) generated during culture, which can alter HSC and graft quality. 

Aims
In this work, we studied how ROS affect HSC functional properties during ex vivo culture, and how antioxidants, by decreasing oxidative stress, limit HSC loss of function.

Methods
This study was conducted with human hematopoietic stem and progenitor cells (HSPC) displaying a CD34+CD38lowCD45RA-CD90+ phenotype, isolated from umbilical cord blood. HSPC functionalities were evaluated after two days of culture in lentiviral transduction medium complemented with cytokines to mimic gene therapy protocols, after treatment with two different antioxidants (N-Acetylcysteine or NAC and Tempol). CFU-C and LTC-IC assays were used along with serial transplantation in immunodeficient NSG mice. Phenotype, cell cycle, mitochondrial activity, ROS generation and signalization pathway activation were studied by flow cytometry at different culture time points. A transcriptomic analysis (Affymetrix micro-array) was conducted to identify candidate genes to explain the functional effects observed.

Results

Adding antioxidants before the two days culture of HSPC significantly increased secondary and tertiary CFU-C numbers and LTC-IC frequency compared to untreated HSPC. Antioxidants-treated HSPC also displayed a better serial reconstitution capacity in NSG mice. This suggested a better preservation of HSPC long-term functional properties. In vitro, we confirmed using CellRox Orange that NAC and Tempol exerted antioxidant properties. Accordingly p38MAPK activation was decreased in antioxidant-treated HSPC after two days of culture. At longer time points (4 to 7 days), mitochondrial activity was found decreased while a larger proportion of cells displayed an immature phenotype in Tempol-treated samples. However, antioxidants did not modify the cell cycle status of HSPC. Finally, looking at gene expression, we witnessed that the transcriptomic signature of 2 days-cultured HSPC was drastically different to fresh HSPC, with pathways related to cell cycle, differentiation, DNA damage and oxidative stress being activated. Antioxidant-treated HSPC displayed expression differences of specific genes such as VEGFα or calcium response related genes, uncovering potential candidates to explain functional maintenance.

Conclusion

These results demonstrate that antioxidants can protect HSC functional properties during ex vivo culture for gene and cell therapy protocols. Further experiments (qPCR, analysis of glucose uptake and calcium efflux, epigenetic analysis) are needed to better characterize the molecular mechanisms behind these functional effects. VEGFα involvement will be investigated with in vitro functional experiments. Since all these results were obtained with umbilical cord blood cells, we aim at testing the relevance of antioxidants to protect adult mobilized HSPC. Overall our results will allow designing new culture conditions to protect HSPC from ROS-driven exhaustion.

Session topic: 23. Hematopoiesis, stem cells and microenvironment

Keyword(s): Antioxidants, Ex vivo, Gene therapy, Hematopoietic stem and progenitor cells

Abstract: EP1463

Type: e-Poster

Background
Hematopoietic stem cells (HSC) are fundamental components of our hematopoietic system, able to produce every type of blood cell. They display self-renewal properties, which are essential to the maintenance of HSC pool during our life. These features make them ideal candidates for cell and gene therapy. Protocols involved in these therapies often comprise culture steps that allow expansion and/or genetic modification of HSC. However, these steps decrease HSC self-renewal potential, despite the addition of growth factors such as SCF, FLT3L or TPO.  This is partly due to reactive oxygen species (ROS) generated during culture, which can alter HSC and graft quality. 

Aims
In this work, we studied how ROS affect HSC functional properties during ex vivo culture, and how antioxidants, by decreasing oxidative stress, limit HSC loss of function.

Methods
This study was conducted with human hematopoietic stem and progenitor cells (HSPC) displaying a CD34+CD38lowCD45RA-CD90+ phenotype, isolated from umbilical cord blood. HSPC functionalities were evaluated after two days of culture in lentiviral transduction medium complemented with cytokines to mimic gene therapy protocols, after treatment with two different antioxidants (N-Acetylcysteine or NAC and Tempol). CFU-C and LTC-IC assays were used along with serial transplantation in immunodeficient NSG mice. Phenotype, cell cycle, mitochondrial activity, ROS generation and signalization pathway activation were studied by flow cytometry at different culture time points. A transcriptomic analysis (Affymetrix micro-array) was conducted to identify candidate genes to explain the functional effects observed.

Results

Adding antioxidants before the two days culture of HSPC significantly increased secondary and tertiary CFU-C numbers and LTC-IC frequency compared to untreated HSPC. Antioxidants-treated HSPC also displayed a better serial reconstitution capacity in NSG mice. This suggested a better preservation of HSPC long-term functional properties. In vitro, we confirmed using CellRox Orange that NAC and Tempol exerted antioxidant properties. Accordingly p38MAPK activation was decreased in antioxidant-treated HSPC after two days of culture. At longer time points (4 to 7 days), mitochondrial activity was found decreased while a larger proportion of cells displayed an immature phenotype in Tempol-treated samples. However, antioxidants did not modify the cell cycle status of HSPC. Finally, looking at gene expression, we witnessed that the transcriptomic signature of 2 days-cultured HSPC was drastically different to fresh HSPC, with pathways related to cell cycle, differentiation, DNA damage and oxidative stress being activated. Antioxidant-treated HSPC displayed expression differences of specific genes such as VEGFα or calcium response related genes, uncovering potential candidates to explain functional maintenance.

Conclusion

These results demonstrate that antioxidants can protect HSC functional properties during ex vivo culture for gene and cell therapy protocols. Further experiments (qPCR, analysis of glucose uptake and calcium efflux, epigenetic analysis) are needed to better characterize the molecular mechanisms behind these functional effects. VEGFα involvement will be investigated with in vitro functional experiments. Since all these results were obtained with umbilical cord blood cells, we aim at testing the relevance of antioxidants to protect adult mobilized HSPC. Overall our results will allow designing new culture conditions to protect HSPC from ROS-driven exhaustion.

Session topic: 23. Hematopoiesis, stem cells and microenvironment

Keyword(s): Antioxidants, Ex vivo, Gene therapy, Hematopoietic stem and progenitor cells

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