P5990In vivo tracking of long-term survival of xenogeneic porcine mesenchymal stem cells seeded on tissue-engineered heart valve implanted in sheep
Authors: Gyongyosi M , Lukovic D , Pavo N , Gugerell A , Winkler J , Spannbauer A , Pavo I J , Michel-Behnke I , Emmert M Y , Hoerstrup S P , Marian T , Balkay L , Trencsenyi G , Wu J C , Zlabinger K 
Published: October 2019

Abstract

Background
Long-term survival of xenogeneic transplanted cells in adults requires strong immunosuppression and/or encapsulation of the cells to achieve peripheral transplant tolerance.

Purpose
The aim of our project was to seed decellularized tissue engineered heart valves (TEHV) with xenogeneic (porcine) mesenchymal stem cells (pMSCs) transfected transiently (Lipofectamine) with a positron emission tomography (PET)-reporter gene (pMSC-PETr), followed by implantation as pulmonary valve replacement into sheep without immunosuppression. The fate of the seeded pMSC-PETr was tracked via serial in-vivo non-invasive PET-computed tomography (PET-CT).

Methods
Static cultivation of TEHV scaffold led to successful ingrowth of the pMSC-PETr. For enabling quantitative assessment of viable pMSC-PETr in the TEHV scaffold after in vivo implantation, vials containing 5x104, 2x105, and 4x105 pMSC-PETr were in vitro mixed with the [18F]-FHBG PET tracer for 1 hr, then the non-bound tracer was washed out and vials were in vitro PET-CT imaged, giving reference values. TEHV-pMSC-PETr were then implanted percutaneously into the pulmonary valve position of sheep (n=4) under general anesthesia, while an additional sheep with no valve implantation served as a control. Ten mCi of [18F]-FHBGPET radiotracer was produced for each procedure and serial PET-CT imaging of the sheep was performed at 3 hr, 24 hr, 2 or 3 weeks, and 5 and 6 months after valve implantation. The study followed the Principles of laboratory animal care.

Results
PET-CT of vials containing increasing number of pMSC-PETr showed dose-dependent tracer uptake in the transfected cells in vitro (Figure). PET-CT images of the sheep 3 hr after implantation of the TEHV-pMSC-PETr showed a clear signal of transfected cells, with a mean estimated number of viable pMSC-PETr of 5.18±1.19x106. No meaningful decrease of the amount of living cells occurred at 24 hr or 2 or 3 weeks. Interestingly, 5- and 6-month follow-up PET-CT images showed clear in vivo and in vitro (after explantation) PET signals of the pMSC-PETr on TEHV, indicating spontaneous stable transfection of the PET reporter plasmid (insertional mutagenesis). Histology confirmed the survival of the pMSC-PETr at 5 and 6-month after xenogeneic transplantation. Merged immunohistochemistry and fluorescence imaging of anti-pig SLA I and anti-sheep MHC I antibodies and PET-reporter gene (HSV1-tk) suggested in vivo inter-species lateral jump gene transfer between pig MSCs and host sheep cells.
Figure 1

Conclusions
This is the first report on serial non-invasive in vivo tracking of long-term survival of xenogeneic pMSCs-PETr seeded on TEHVs and percutaneously implanted into the pulmonary position of sheep. Long-term follow-up revealed spontaneous stable transfection of the plasmid PET-reporter gene, which suggests the risk of insertional mutagenesis induced by the plasmid (transposon), and PET-reporter gene shuttle from xenogeneic pig MSCs to sheep cells.

Acknowledgement/Funding
LifeValve EU project (grant number: 242008)