Scientists working on Wnt proteins and those interested in stem cells now have good cause to celebrate. Active, pure Wnt has at last been isolated! A joint venture by the groups of Roel Nusse, Irving Weissman and Tannishtha Reya has shown that Wnts are palmitoylated and that Wnt signalling can induce self-renewal of haematopoietic stem cells (HSCs).

Wnt molecules regulate many aspects of development, such as stem-cell proliferation, but studying these processes ex vivo has so far been hampered by the inability to purify significant amounts of soluble, active Wnts. So Karl Willert and colleagues, from Nusse's laboratory, subjected medium from Wnt-expressing (and secreting) cells to chromatography in the presence of a detergent, size-exclusion chromatography, and then cation exchange to obtain fractions of Wnt (in this case Wnt3a) that was >95% pure. Importantly, this purified Wnt3a could still stabilize cytosolic β-catenin, a known target of Wnt.

That detergent was required throughout purification implied that Wnts were hydrophobic, and metabolic labelling indicated that Wnt3a was palmitoylated. Further analysis showed that the most amino-terminally conserved cysteine residue of Wnt-family members (C77 in Wnt3a) was the target. Mutation of this residue, although not affecting the level of secreted protein, conferred a loss of hydrophobicity and rendered the protein inactive in β-catenin stabilization assays. Consistent with the importance of this residue, natural loss-of-function alleles of Drosophila wg and of C. elegans egl-20 (which are homologues of Wnt) occur, both of which affect this conserved cysteine.

Weissman, Nusse and Reya then showed that purified Wnt3a induces proliferation of HSCs, cells that give rise to all lineages of the blood. Unpurified Wnt3a, by contrast, induced differentiation, emphasizing the importance of being able to purify Wnt. Wnt3a-treated HSCs were then transplanted into lethally irradiated mice and, 6 weeks later, 100% of the mice contained donor-derived cells. This frequency of reconsitution indicated that self-renewal had occurred in vitro.

In the second Nature paper, Weissman, Reya and colleagues showed the importance of the Wnt signalling pathway as a whole in HSC homeostasis in vitro and in vivo. In long-term experiments, expressing constitutively active β-catenin maintained HSCs in an immature state while allowing them to proliferate. Transplantation analysis again indicated that the expanded HSCs retained their function. Using a reporter assay, the authors verified that HSCs in their normal microenvironment respond to endogenous, canonical Wnt signalling. Wnt signalling is also required for normal HSC growth; expression of a soluble form of the Frizzled cysteine-rich domain, or ectopic expression of Axin (which increases β-catenin degradation) — both of which inhibit Wnt signalling — inhibited growth of HSCs. In vivo, transplantation of axin-expressing HSCs into irradiated mice inhibited reconstitution of the haematopoietic system of these mice. The final finding was that HoxB4 and Notch1 , genes that were previously implicated in HSC self-renewal, were upregulated in response to Wnt signalling.

Components of the Wnt signalling pathway have previously been implicated in regulating the proliferation of progenitor cells of the skin, gut and brain. The finding that soluble Wnt3a can induce proliferation of HSCs adds to the potential of using Wnt signalling as “...a general cue for self-renewal in stem and/or progenitor cells from diverse tissues”. Importantly, Wnt signalling might facilitate the expansion of a patient's or an allogeneic donor's HSCs as a potential future transplantation source. Finally, the authors propose that Wnt/β-catenin signalling might be an important regulator of cancer stem-cell self-renewal as well.