Bone morphogenetic proteins (BMPs) are growth factors belonging to the transforming growth factor β (TGF-β) superfamily, involved in a wide range of physiological and pathological processes. In the bone marrow, BMPs regulate the differentiation of mesenchymal stem cell (MSCs) into osteoblasts, adipocytes and chondrocytes, as well as the differentiation of hematopoietic stem cell (HSCs) into different blood lineages. In pathological conditions such as acute myeloid leukemia (AML), characterized by the abnormal accumulation of immature blood cells in the bone marrow, BMPs are upregulated in the extracellular matrix. Although HSC dysregulation has been studied in this BMP-enriched context, the impact of elevated BMP levels in the surrounding microenvironment remains unclear. This thesis investigates the dysregulation of MSCs in the leukemic bone marrow niche, focusing on the effects of the high BMP levels. To mimic their matrix-bound presentation in the extracellular matrix, BMPs were immobilized on a biomimetic film made of poly-L-lysine and hyaluronic acid. In the first part of the project, the BMP pathway was investigated in both human healthy and AML-derived MSCs, after stimulation with various concentrations and time points of matrix-bound BMP-2, BMP-4, BMP-7, BMP-9 and TGF-β1. The Smad-dependent pathway and BMP receptor expression and functional role were assessed using immunofluorescence staining at high-content screening (HCS). Given the established role of BMPs in cell adhesion, the expression and functional roles of β integrins and BMP-mediated adhesion were further analyzed using HCS. In a second part, a comparative analysis of BMP-responsive cells was assessed upon matrix-bound BMP stimulation (BMP-2, BMP-4, BMP-7 and BMP-9). C2C12 cells and human periosteum derived stem cells (hPDSCs), previously reported results (Sales et al. 2022), and healthy MSCs, results from the first part of this work, were compared thanks to the similar HSC methodologies used. Comparisons included BMP receptor and β integrin expression levels, pSmad1/5/9 signaling, and adhesive responses, along with the contribution of BMP receptors and integrins to these processes. Finally, the differentiation potential of healthy and AML-derived MSCs following BMP treatment was evaluated. Osteogenic, adipogenic, and chondrogenic differentiation capacities were assessed in both healthy and AML-derived MSCs using colorimetric assays, quantitative PCR and HCS immunofluorescence techniques. This work contributes to a deeper understanding of MSC behavior in the leukemic microenvironment and opens perspectives for the development of targeted therapeutic strategies in AML.

Supervision of the thesis :

Elisa MIGLIORINI

Catherine PICART