Exploring the role of Wnt proteins in the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation

Based on their unique capacity to self-renew and to differentiate into blood cells of all lineages, hematopoietic stem cells (HSCs) hold many promises for treatment of many diseases. However, unsuccessful in vitro expansion and improper differentiation of HSCs limit the therapeutic potential of these cells. Over the past decades, a variety of studies have implicated the role of Wnt signalling in control of HSC function in vivo. Wnt signalling in the regulation of HSCs expansion was first demonstrated by in vitro studies where activation of Wnt signalling was achieved by retroviral transduction of a constitutively active form of β-catenin and recombinant Wnt3a protein in an anti-apoptotic Bcl2-transgenic background (Reya et al., 2003; Willert et al., 2003). However, with enforced, continuous Wnt activation, mice models displayed impaired HSC self-renewal and failure in multilineage differentiation (Kirstetter et al., 2006; Scheller et al., 2006). These studies led to a controversy in the field, and furthermore raised the question whether there is necessity for cross talk between Wnt signals and anti-apoptotic signals. Adding up to the controversy, several loss-of-function studies result in conflicting results either showing no effect in hematopoiesis (Cobas et al., 2004, Jeannet et al., 2008, Koch et al., 2008) or defect in HSCs repopulating capacity (Zhao et al., 2007). Thus, the precise role of Wnt pathway in HSC regulation is still an open debate, and requires a deeper investigation. More insights into the role of Wnt proteins in the regulation of HSCs could possibly be obtained from treating HSCs with purified Wnt3a protein in vitro. However, technical obstacles, in particular, short half-life and detergent associated toxicity of purified Wnt3a protein limits its use for in vitro assays. To increase the half-life and reduce toxicity of purified Wnt3a, we explored the ability of Wnt3a to associate with lipid vesicles. Firstly, we have found out that Wnt3a incorporated into several types of liposomes at a high level, and the association with these vesicles stabilized the activity. Additionally, we developed a dialysis-based method to remove the detergent from the Wnt3a protein. Importantly, we found that liposomal Wnt3a proteins retain their activity even in the absence of detergent. This greatly increases the potential use of Wnt3a protein for in vitro assays to study the role of Wnt signalling in hematopoiesis.