Determining the role of matrix compliance in the differentiation of mammary stem cells

Multipotent stem cells maintain the structure and function of the mammary gland throughout its development and respond to the physiological demands associated with pregnancy and lactation. The ability of mammary stem cells to maintain themselves as well as to give rise to differentiated progeny is not only affected by soluble factors but has increasingly become linked to mechanical cues including the elastic modulus of the extracellular matrix (ECM). Here we describe a protocol for determining how the mechanical properties of the ECM regulate the fate of mammary stem or progenitor cells. This protocol includes detailed methods for the fabrication of substrata with varying stiffness, culture of mammary progenitor cells on synthetic substrata, pharmacological modulation of actomyosin contractility, and analysis of gene expression to define the resulting fate of human mammary stem cells.