Liposome-stabilized WNT3A Protein (L-WNT) Upregulates Adult Hepatic Stem Cells and Reduces Cell Death in Liver during Cold Preservation

Introduction: Transplantation of extended criteria livers are critical to address organ shortage, but is associated with inferior outcomes due to the susceptibility of such organs to ischemic injury during retrieval and storage. We studied the efficacy of L-WNT, potent activator of adult stem cells, in liver pre-conditioning to enhance the quality and viability of organs prior to transplantation.

Methods: Wild type (WT) and Axin2LacZ/+ Wnt reporter mice were used. To study apoptosis in vivo (Figure 1A), heart-beating WT animals received intraportal L-WNT (2.5ng/µL) or saline (control group) followed by liver retrieval 15 minutes post-injection. Livers were preserved for 2 hours in storage solution at 0-4oC followed by quantitative analysis using TUNEL staining. To explore L-WNT mechanism of action in vitro (Figure 2A), liver slices from Axin2LacZ/+ mice were incubated at 37oC with L-WNT (0.5ng/µL) or with saline for 48 hours, followed by staining with Xgal to detect Wnt-responsive stem cells. Apoptosis was evaluated using Caspase-3 immunohistochemistry analysis and TUNNEL staining.

Results:  Pre-conditioning livers with L-WNT in vivo significantly decreased the number of TUNEL+ apoptotic cells compared to controls (Figure 1B). Treatment of liver slices with L-WNT in vitro significantly increased the number of Xgal+ Wnt-responsive stem cells while simultaneously significantly reducing the number of apoptotic cells as evidenced by a significant decrease in Caspase-3 expression and a significant decrease in TUNEL staining compared to controls (Figure 2B).

Conclusion: Liver pre-conditioning with L-WNT induces upregulation of L-WNT-responsive adult stem cells and reduces cellular apoptosis, induced by ischemia during organ retrieval and cold storage. L-WNT represents a novel biologic for donor preconditioning and liver storage to optimize organ quality prior to transplantation of extended criteria livers with goal of improving post-transplant outcomes.