Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling 
are necessary to stimulate proliferative processes in adult human β-cells. 
Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt 
receptor, increases β-catenin nuclear translocation and β-cell proliferation but 
results in lower insulin content. Our current goal was to engage canonical and 
non-canonical Wnt signaling at the receptor level to significantly increase 
human β-cell proliferation while maintaining a β-cell phenotype in intact 
islets. We adopted a system that utilized conditioned medium from L cells that 
expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition 
we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA 
inhibition) in these cultures. Treatment of intact human islets with L-WRN 
conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold 
in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased 
human β-cell proliferation ∼20 fold above glucose alone. Only the combination of 
L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial 
proliferation. Transcriptome-wide gene expression profiling demonstrated that 
L-WRN medium provoked robust changes in several signaling families, including 
enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment 
also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of 
Akt. Importantly, glucose-stimulated insulin secretion and content were not 
downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt 
signaling at the receptor level by this method leads to necessary crosstalk 
between multiple signaling pathways including activation of Akt, mTOR, 
Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase 
human β-cell proliferation while maintaining the β-cell phenotype.