Abstract
Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson’s disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced—cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.
Highlights
Despite advancements in human pluripotent stem cells differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson’s disease, resultant grafts contain low proportions of dopamine neurons
FailSafeTM human pluripotent stem cells (hPSCs) competently differentiated to ventral midbrain (VM) progenitors, as confirmed by the high proportion of cells co-expressing OTX2+ and FOXA2+ (81.5 ± 5.0%) at D11 of differentiation (Supplementary Fig. 1A, D), with minimal contamination of off-target BARHL1+, PITX2+ diencephalic progenitors (Supplementary Fig. 1B)
To determine the capability of the FailSafeTM system to ablate proliferative VM progenitors, we initially tracked the proportion of KI67+ proliferative cells during the VM differentiation
Summary
Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson’s disease, resultant grafts contain low proportions of dopamine neurons. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced—cell types that may pose adverse or unknown influences on graft and host function These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism. A plausible explanation, and recognised shortcoming of suicide gene therapy, is the risk of silencing or downregulation of the transgene and/or homologous recombination events that have been identified in both PSC cultures and teratomas[17,18,21] To circumvent this risk, we recently engineered a hPSC line incorporating a transcriptional link between the suicide gene and a gene essential for cell cycle progression (cyclinD1), subsequently referred to as the FailSafeTM hPSC line[18]. With hPSC therapies rapidly progressing towards the clinic, these findings have important implications for ensuring the safety and predictability of hPSC-derived DA grafts, targeted at restoring DA transmission in PD patients
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