Single-Cell Analysis Reveals a Close Relationship between Differentiating Dopamine and Subthalamic Nucleus Neuronal Lineages

Abstract

Stem cell engineering and grafting of mesencephalic dopamine (mesDA) neurons is a promising strategy for brain repair in Parkinson's disease (PD). Refinement of differentiation protocols to optimize this approach will require deeper understanding of mesDA neuron development. Here, we studied this process using transcriptome-wide single-cell RNA sequencing of mouse neural progenitors expressing the mesDA neuron determinant Lmx1a. This approach resolved the differentiation of mesDA and neighboring neuronal lineages and revealed a remarkably close relationship between developing mesDA and subthalamic nucleus (STN) neurons, while also highlighting a distinct transcription factor set that can distinguish between them. While previous hESC mesDA differentiation protocols have relied on markers that are shared between the two lineages, we found that application of these highlighted markers can help to refine current stem cell engineering protocols, increasing the proportion of appropriately patterned mesDA progenitors. Our results, therefore, have important implications for cell replacement therapy in PD.

Towards stem cell based therapies for Parkinson's disease

“…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).…”

A novel floor plate boundary defined by adjacentEn1andDbx1microdomains distinguishes midbrain dopamine and hypothalamic neurons

“…While this paper was in revision, an interesting manuscript relevant to this topic was published (Kee et al, 2017).…”

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

“…In current VM differentiation protocols, mesencephalic floor plate markers LMX1A, FOXA2, and OTX2 are commonly used to confirm the mesDA identity of progenitors in vitro prior to grafting (Doi et al., 2014, Jaeger et al., 2011, Kirkeby et al., 2012, Kriks et al., 2011). However, a new study has revealed that these and several other commonly used VM markers are also co-expressed in diencephalic progenitors of the subthalamic nucleus (STN) and therefore do not exclusively identify the mesDA domain (Kee et al., 2016). …”