Novel strategy for rapid functional in vivo validation of oncogenic drivers in haematological malignancies

In cancer research, it remains challenging to functionally validate putative novel oncogenic drivers and to establish relevant preclinical models for evaluation of novel therapeutic strategies. Here, we describe an optimized and efficient pipeline for the generation of novel conditional overexpression mouse models in which putative oncogenes, along with an eGFP/Luciferase dual reporter, are expressed from the endogenous ROSA26 (R26) promoter. The efficiency of this approach was demonstrated by the generation and validation of novel R26 knock-in (KI) mice that allow conditional overexpression of Jarid2, Runx2, MN1 and a dominant negative allele of ETV6. As proof of concept, we confirm that MN1 overexpression in the hematopoietic lineage is sufficient to drive myeloid leukemia. In addition, we show that T-cell specific activation of MN1 in combination with loss of Pten increases tumour penetrance and stimulates the formation of Lyl1(+) murine T-cell lymphoblastic leukemias or lymphomas (T-ALL/T-LBL). Finally, we demonstrate that these luciferase-positive murine AML and T-ALL/T-LBL cells are transplantable into immunocompromised mice allowing preclinical evaluation of novel antileukemic drugs in vivo

Human decellularized dermal matrix seeded with adipose-derived stem cells enhances wound healing in a murine model: Experimental study.

Objective: Full-thickness cutaneous wounds treated with split-thickness skin grafts often result in unaesthetic and hypertrophic scars. Dermal substitutes are currently used together with skin grafts in a single treatment to reconstruct the dermal layer of the skin, resulting in improved quality of scars. Adipose-derived stem cells (ASCs) have been described to enhance wound healing through structural and humoral mechanisms. In this study, we investigate the compatibility of xenogen-free isolated human ASCs seeded on human acellular dermal matrix (Glyaderm®) in a murine immunodeficient wound model. Methods: Adipose tissue was obtained from abdominal liposuction, and stromal cells were isolated mechanically and cultured xenogen-free in autologous plasma-supplemented medium. Glyaderm® discs were seeded with EGFP-transduced ASCs, and implanted on 8 mm full-thickness dorsal wounds in an immunodeficient murine model, in comparison to standard Glyaderm® discs. Re-epithelialization rate, granulation thickness and vascularity were assessed by histology on days 3, 7 and 12. Statistical analysis was conducted using the Wilcoxon signed-rank test. EGFP-staining allowed for tracking of the ASCs in vivo. Hypoxic culture of the ASCs was performed to evaluate cytokine production. Results: ASCs were characterized with flowcytometric analysis and differentiation assay. EGFP-tranduction resulted in 95% positive cells after sorting. Re-epithelialization in the ASC-seeded Glyaderm® side was significantly increased, resulting in complete wound healing in 12 days. Granulation thickness and vascularization were significantly increased during early wound healing. EGFP-ASCs could be retrieved by immunohistochemistry in the granulation tissue in early wound healing, and lining vascular structures in later stages. Conclusion: Glyaderm® is an effective carrier to deliver ASCs in full-thickness wounds. ASC-seeded Glyaderm® significantly enhances wound healing compared to standard Glyaderm®. The results of this study encourage clinical trials for treatment of full-thickness skin defects. Furthermore, xenogen-free isolation and autologous plasma-augmented culture expansion of ASCs, combined with the existing clinical experience with Glyaderm®, aid in simplifying the necessary procedures in a GMP-laboratory setting.status: Published onlin