EVP, Head of US Operations & CSO
In vitro exposure of isolated porcine islets from Neu5Gc-knockout/GT-knockout/hCD46 transgenic donors to human blood/plasma
Rita Bottino1, Hidetaka Hara2, Hayato Iwase2, Whayoung Lee2, Mohamed Ezzelarab2, Martin Wijkstrom2, Suzanne Bertera1, Massimo Trucco1, Carol Phelps3, Jagdeece Ramsoondar3, David Ayares3, David KC Cooper2.
1Institute of Cellular Therapeutics, Allegheny Health Network, Pittsburgh, PA, United States; 2Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, United States; 3Revivicor, Blacksburg, VA, United States
Background: Early islet loss secondary to the instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to successful engraftment of pig islets following xenotransplantation. Events occurring as soon as the islets come into contact with the recipient blood in vivo include coagulation and complement activation, and an inflammatory reaction, resulting in islet damage. Pig islets exposed in vitro to human blood are subject to similar events, leading to islet loss. Use of genetically-engineered pig islet donors, such as α1,3-galactosyltransferase gene-knockout (GTKO) pigs transgenic for one or more human complement-regulatory proteins (e.g., hCD46), has improved islet performance in vivo. However, in vitro exposure of these islets to human blood has shown no evident protection against early islet loss.
Methods: Isolated islets from pancreases from juvenile Neu5GcKO/GTKO/hCD46 pigs (n=2) following short-term culture were exposed in vitro to blood or plasma from three blood-type matched human donors. Time to clotting was recorded. Thirty and 60 min after exposure of the islets to blood, release of C-peptide (by ELISA) was determined. Following 3 hours exposure to human plasma, the viability of islets was evaluated using a dual fluorescence dye (calcein-AM, propidium iodide) and quantification of viable/dead cells was measured using Image J software. As controls, islets from a GTKO/hCD46 pig were similarly tested.
Results: Regardless of their genetic background, all islets triggered coagulation of blood within 6min. Blood from all 3 human donors clotted between 3min 19sec and 4min 48sec when in combination with Neu5GcKO/GTKO/hCD46 islets, and between 5min 05sec and 5min 55sec when in combination with GTKO/hCD46 islets. A non-physiologic increase in C-peptide is typically an indication of islet cell leakage/damage. In 5 of 6 combinations of blood and GTKO/hCD46 islets (after 30 or 60 min incubation) C-peptide increased over control (autologous combination) by 5,177±2,639pmol/L (mean±STD). In contrast, C-peptide increased only in 3 of the 12 combinations when Neu5GcKO/GTKO/hCD46 islets were employed (mean±STD increase 6,263±6,230 pmol/L). During the first 3 hours following exposure of islets to plasma, the number of dead cells increased 23% (23±13%, n=6) over control (autologous combination) for GTKO/hCD46 islets, whereas the increase was lower (15±11%, n=12) for Neu5GcKO/GTKO/hCD46 islets.
Conclusions: Although these remain preliminary observations, knockout of Neu5Gc in addition to Gal may reduce early pig islet loss from IBMIR.
11:00 - 12:30
|Inflammation & Coagulation||In vitro exposure of isolated porcine islets from Neu5Gc-knockout/GT-knockout/hCD46 transgenic donors to human blood/plasma||Room 109|
15:30 - 17:00
|Genetic Engineering; Methodology||Six-gene multi-transgenic pigs serve as source animals to address innate and chronic rejection, inflammation, and coagulopathy in organ xenografts||Room 109|