Biopreservation of a tissue engineered nigrostriatal pathway for tract reconstruction in Parkinson's disease.

Abstract

The typical motor symptoms of Parkinson's disease (PD) are caused by selective loss of dopaminergic neurons in the substantia nigra (SN). Although conventional pharmacotherapies can temporarily alleviate symptoms, no approved therapies exist to slow or reverse the underlying pathologic processes. To address this gap, cell transplantation therapies are being pursued; however, restoration of the original neuroanatomical circuit is not a goal of traditional ectopic intra-striatal neuronal transplantations. To address this, we developed an implantable tissue engineered nigrostriatal pathway (TE-NSP) containing human stem cell derived dopaminergic neurons with pre-formed long-projecting axonal bundles to replace the circuitry connecting the SN and the striatum. However, a challenge in the translation of tissue engineered medical products is the need for storage and transportation following biofabrication to enable point-of-care surgical implantation. Herein, we describe successful creation of TE-NSPs using a commercially-available human iPSC-derived dopaminergic neuronal source and describe growth characteristics for a range of neuronal and axonal densities. We also established protocols for the biopreservation of these fully-grown, human neuron-based TE-NSPs under hypothermic (4 °C) conditions for up to 2 days. Subsequent assessment of neuronal viability and maintenance of axonal-tract architecture out to 12-weeks in vitro demonstrate that short-term hibernation of TE-NSPs consisting of 55,000 neurons did not reduce neuronal viability, axonal health, structural integrity, or survival in physioxia conditions (5 % O₂) when compared to non-biopreserved controls. The long-term survival of biopreserved TE-NSPs in vitro provides proof-of-concept supporting hypothermic storage during transportation for future safety and efficacy studies. STATEMENT OF SIGNIFICANCE: Degeneration of dopaminergic neurons and their axonal projections comprising the nigrostriatal pathway leads to Parkinson's disease, the second most common neurodegenerative disease globally. To address shortcomings of current cell transplantation therapies that primarily focus on ectopic cell transplantation, we have generated an engineered microtissue with pre-formed axon tracts using human neurons. Our tissue engineered nigrostriatal pathways (TE-NSPs) are implantable engineered microtissue that structurally and functionally resemble the native nigrostriatal pathway. For future translational applications, we established their preservation under hypothermic conditions, facilitating storage and transportation to the clinical setting. The study provides proof-of-concept that biopreserved 'living neural tissue' survives under hypothermic and simulated physioxic conditions, demonstrating their translational potential in the human brain.