Cell replacement therapies are becoming increasingly viable treatments for neurodegenerative diseases. As one key example, a major strategy to address Parkinson’s disease (PD) is to replenish dopaminergic (DA) neurons in the midbrain. To this end, human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are differentiated into DA neurons in vitro, followed by surgical implantation into the brain. Unfortunately, experiments using animal models and in clinical trials indicate that on average 99% of the cells die following surgical implantation. Considerable resources are consumed to generate enough cells for implantation, given that only 1% survive to become DA neurons that can potentially alleviate PD symptoms. On average 100,000 DA neurons are needed to effectively treat PD in humans, requiring 10 million cells to be implanted per patient. There is therefore a crucial need to devise strategies to efficiently generate a large number of DA neurons. We are using biomaterial cell production platforms to address these challenges.
In previous work, we generated 10-20 fold higher numbers of viable, pluripotent, hESCs in a fully defined, xeno-free 3D gel, compared to equivalent culture on a 2D surface. Here, we have used a 3D differentiation culture to generate large numbers of DA neurons using dual-SMAD inhibition, followed by Wnt and SHH activation and neurogenesis. Production of DA neurons in such a next-generation 3D format calls for extensive characterization to monitor neurogenesis and verify the midbrain DA fate of the resulting neurons. The fate of the hESCs was monitored using immunocytochemistry and qPCR at regular intervals during the patterning and differentiation stages, providing dynamic information on the expression of various markers of interest. Subsequently, the final dopaminergic fate of the neurons was verified with immunocytochemistry, qPCR and electrophysiology.
Differentiation in 3D cultures resulted in equivalent or better levels of relevant marker expression compared to neurons differentiated in a traditional 2D format. FOXA2/LMX1A co-expression demonstrated their midbrain lineage, and TH expression confirmed their dopaminergic identity. Using an equivalent level of resources as for a 2D culture, we were able to generate 20 fold higher numbers of DA neurons. Marker analysis with immunocytochemistry and qPCR provided dynamic information on the expression of various markers of interest. Interestingly, we discovered that some markers, including FOXA2, were expressed at a higher level from an earlier stage in 3D culture compared to differentiation on a 2D surface. Moreover, immunocytochemistry and qPCR showed robust expression of mature dopaminergic markers including DAT and Nurr1. Furthermore, the DA neurons were shown to be electrophysiologically active and able to generate action potentials.
In summary, 3D culture in functionalized biomaterials improves the yield of DA neurons generated from hESCs in vitro. Promising results from current studies warrant further optimization of differentiation in 3D hydrogels to generate enriched populations of DA neurons. Generating large numbers of quality DA neurons could prove useful for a variety of applications including surgical implantation in PD, understanding development of DA neurons, and evaluating PD disease models.