SAR-guided engineering of stem cell probes

Stem cells orchestrate tissue development and homeostasis and hold vast therapeutic potential for tissue regeneration and repair. However, the ability to study basic stem cell biology in vitro is hampered by the lack of robust probes allowing their identification and isolation. R-spondins (RSPOs) are ligands for LGRs, receptors which have emerged as faithful stem cell markers in a number of tissues.

We have developed an expression system for the generation of RSPO fusion proteins that bind LGRs with high affinity. We will combine pairwise in vitro binding data between RSPO and LGR isoforms with functional assays of RSPO activity to predict optimal amino acid substitutions along the RSPO binding interface conferring specificity to each of the three LGR isoforms. We will refine these predictions based on structural analysis of RSPO and LGR pairs. Structure-activity relationships based on amino acid substitutions in the RSPO binding interface will instruct the engineering of RSPOs (engRSPOs) that selectively bind each LGR isoform.

EngRSPOs will be used for the isolation of individual stem cell compartments which we will exploit for future studies uncovering basic mechanisms that maintain stem cell pluripotency.