Contributions
Abstract: EP1298
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Hematopoietic Stem and Progenitor Cells (HSPCs) reside in a specialized microenvironment in the bone marrow (BM), named the hematopoietic niche. Mesenchymal Stromal Cells (MSCs) are key elements of the BM niche, regulating HSPC function by direct contact and through the release of paracrine factors. Due to these properties, MSCs have been employed in vitro to support HSPC expansion, and in-vivo to promote HSPC engraftment and hematopoietic reconstitution in preclinical models and clinical trials of HSPC transplantation (HSCT).
Aims
Based on these studies, in our work we aimed to improve the outcome of HSCT with CRISPR-Cas9 gene-edited (GE)-HSPCs, whose efficiency equires further advancement to fully exploit its therapeutic potential. Indeed, GE-HSPCs activate a DNA damage cascade in response to nuclease-induced double strand break and delivery of DNA repair template, which constrains HSPC proliferation and long-term repopulating capacity.
Methods
To this aim, we developed a novel MSC-based in-vitro approach to expand and recover GE-HSPCs. We analyzed the GE-HSPC phenotype by flow cytometry and the activation of a DNA stress response in GE-HSPCs by immunohistochemistry. We further evaluated the cell-cycle state, the level of inflammation and apoptosis by flow-cytometry and gene expression analysis. The fitness of HSPCs gene-editd according to standard procedures were compared to HSPCs gene-edited in the presence of MSCs (+MSCs). Preclinical mouse models of HSCT were used to prove the superior capability of GE-HSPCs + MSCs to long-term engraft and favor hematological reconstitution.
Results
We show that MSCs produced several hematopoietic supportive and anti-inflammatory factors, capable to reduce the proliferation arrest, and mitigate the apoptotic and inflammatory programs activated in GE-HSPCs, ultimately improving GE-HSPC expansion and clonogenic potential. This resulted into a superior GE-HSPC engraftment and hematological reconstitution in transplanted mice. Moreover, we demonstrated that MSCs favored the engraftment of limited doses of GE-HSPCs when co-transplanted in vivo.
Conclusion
In conclusion, our work poses the basis for the clinical use of MSCs as a novel, safe and efficient strategy to preserve the long-term repopulating capacity and promote engraftment of CRISPR-Cas9 gene corrected HSPCs.
Keyword(s): Gene therapy, HSCT, Mesenchymal stem cell
Abstract: EP1298
Type: E-Poster Presentation
Session title: Stem cell transplantation - Experimental
Background
Hematopoietic Stem and Progenitor Cells (HSPCs) reside in a specialized microenvironment in the bone marrow (BM), named the hematopoietic niche. Mesenchymal Stromal Cells (MSCs) are key elements of the BM niche, regulating HSPC function by direct contact and through the release of paracrine factors. Due to these properties, MSCs have been employed in vitro to support HSPC expansion, and in-vivo to promote HSPC engraftment and hematopoietic reconstitution in preclinical models and clinical trials of HSPC transplantation (HSCT).
Aims
Based on these studies, in our work we aimed to improve the outcome of HSCT with CRISPR-Cas9 gene-edited (GE)-HSPCs, whose efficiency equires further advancement to fully exploit its therapeutic potential. Indeed, GE-HSPCs activate a DNA damage cascade in response to nuclease-induced double strand break and delivery of DNA repair template, which constrains HSPC proliferation and long-term repopulating capacity.
Methods
To this aim, we developed a novel MSC-based in-vitro approach to expand and recover GE-HSPCs. We analyzed the GE-HSPC phenotype by flow cytometry and the activation of a DNA stress response in GE-HSPCs by immunohistochemistry. We further evaluated the cell-cycle state, the level of inflammation and apoptosis by flow-cytometry and gene expression analysis. The fitness of HSPCs gene-editd according to standard procedures were compared to HSPCs gene-edited in the presence of MSCs (+MSCs). Preclinical mouse models of HSCT were used to prove the superior capability of GE-HSPCs + MSCs to long-term engraft and favor hematological reconstitution.
Results
We show that MSCs produced several hematopoietic supportive and anti-inflammatory factors, capable to reduce the proliferation arrest, and mitigate the apoptotic and inflammatory programs activated in GE-HSPCs, ultimately improving GE-HSPC expansion and clonogenic potential. This resulted into a superior GE-HSPC engraftment and hematological reconstitution in transplanted mice. Moreover, we demonstrated that MSCs favored the engraftment of limited doses of GE-HSPCs when co-transplanted in vivo.
Conclusion
In conclusion, our work poses the basis for the clinical use of MSCs as a novel, safe and efficient strategy to preserve the long-term repopulating capacity and promote engraftment of CRISPR-Cas9 gene corrected HSPCs.
Keyword(s): Gene therapy, HSCT, Mesenchymal stem cell