Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson’s Disease

Abstract

SummaryStem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long-term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson’s disease through gene expression analysis of >30 batches of grafted human embryonic stem cell (hESC)-derived progenitors. We found that many of the commonly used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a good manufacturing practice (GMP) differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

“…Recently, Kirkeby et al . found that EN1+ progenitors transplanted in animal models resulted in grafts rich in DA yield and density52. The fact that both FOXA2 and EN1 are robustly maintained in 3D differentiation, but not as effectively in 2D, may indicate that the former are primed for long-term survival.…”

Towards stem cell based therapies for Parkinson's disease

“…Developing GMPcompliant cell-manufacturing processes not only entails the use of appropriate starting cell lines and media components, but also requires monitoring and reduction of variability in cell fate at each step -so that a uniform cell product can be produced with each differentiation round. In such efforts in my own laboratory, we observed significant batch-to-batch variation in how many DA neurons are ultimately produced from FOXA2/LMX1A-expressing progenitors after transplantation and functional maturation in vivo (Kirkeby et al, 2017a). This observation was puzzling as, based on what was known about development at the time, these progenitors should all form mature midbrain DA neurons.…”

“…However, a parallel study based on single cell sequencing of the midbrain Lmx1a-expressing progenitors during mouse development revealed that FoxA2 and Lmx1a (as well as most other ventral midbrain markers commonly used to detect the DA progenitor population prior to grafting) are also co-expressed in early diencephalic progenitors that form the subthalamic nucleus (STN), and therefore do not exclusively identify the midbrain DA neuron domain (Kee et al, 2017). Thus, cells expressing FOXA2/LMX1A can give rise to two types of neurons, and the relative proportion of these can now be tracked and controlled using the predictive markers identified that are specifically expressed in the DA domain (Kee et al, 2017;Kirkeby et al, 2017a). This allowed us to better control stem cell differentiation in vitro by adding FGF8 to sufficiently caudalize the cells such that DA (and not STN cells) are formed with a high yield and purity (Kirkeby et al, 2017a).…”

“…This allowed us to better control stem cell differentiation in vitro by adding FGF8 to sufficiently caudalize the cells such that DA (and not STN cells) are formed with a high yield and purity (Kirkeby et al, 2017a). Thus, similar to the studies elucidating the role of the floor plate during development (Bonilla et al, 2008;Fasano et al, 2010;Kawasaki et al, 2000), refined understanding of developmental biology, this time achieved by the high resolution of cell diversity achieved via single cell sequencing, was essential for precise control of stem cell differentiation in vitro (Kee et al, 2017;Kirkeby et al, 2017a).…”

Neural Subtype Specification from Human Pluripotent Stem Cells

“…Interestingly, a recent study aiming at identifying markers to predict the yield of DA neurons in transplant showed that the commonly used DA neuron markers (FOXA2, LMX1A and CORIN) poorly correlate with DA neuron production in vivo whereas the markers of caudal ventral midbrain (FGF8, PAX5, EN2 and CNPY1 (Canopy FGF signaling regulator 1)) are associated with higher DA neuron yield. Addition of FGF8 later than day 9, but not earlier (before day 9), maintains the FOXA2/LMX1A/B expression in progenitors and increases the generation of DA neurons (Kirkeby et al, 2016). This result suggests that FGF8 promotes DA neuron program by patterning the progenitors to the caudal fate at a late stage.…”