3167 – A NOVEL MICROFLUIDIC DEVICE FOR INVESTIGATING NORMAL AND MALIGNANT HEMATOPOIETIC STEM CELL FATE UNDER COMPRESSIVE STRESSES

Abstract

Hematopoietic stem and progenitor cell (HSPC) transplantation and gene therapy protocols are limited by stem cell numbers. While decades of work into the chemical and molecular modifiers of hematopoietic differentiation have been fruitful, substantially less is known about the impact of physical factors that govern HSPC survival, differentiation and self-renewal, thus limiting our ability to scale-up production of human HSPC numbers ex vivo. HSPC are exposed to potential mediators of mechanical signals in the bone marrow microenvironment via neighbouring cells. To gain a better understanding of how mechanical forces might impact stem cell behaviour in development and regeneration, we developed a novel microfluidic device that is capable of immobilising and mechanically stimulating a range of isolated HSPC fractions. Heterogenous populations of hematopoietic stem cells (HSC) and granulocyte-monocyte progenitor cells (GMP) were subjected to time-varying shear stress independently from controlled static compressive forces by modulating applied gas pressure using PDMS-based pneumatic valves. Live cell monitoring of HSC and GMP from wild-type and TET2 deficient animals allowed measurement of cellular response to mechanical stimulation ranging from mild deformation through to complete cell lysis. Cells were extracted from the device for downstream cellular and molecular assays. Using single cell and bulk liquid culture assays we show, for the first time, that compressive stress can impact the viability, cloning efficiency and the differentiation capacity of HSPCs. Our approach suggests that mechanical signals can alter HSC fate in the absence of other changes and may be harnessed to improve HSC expansion protocols, while also prompting the need for a more detailed understanding of the physical role of niche cells in the context of leukaemia seeding and transformation.