Human midbrain dopaminergic neuronal differentiation markers predict cell therapy outcomes in a Parkinson’s disease model

Abstract

Human pluripotent stem cell–based (hPSC-based) replacement therapy holds great promise for the treatment of Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we have characterized the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including the ventral midbrain, the isthmus, and the ventral hindbrain, resulting in a heterogenous donor cell population. We reconstructed the differentiation trajectory of the mDA lineage and identified calsyntenin 2 (CLSTN2) and protein tyrosine phosphatase receptor type O (PTPRO) as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate high enrichment of mDA neurons (up to 80%) following progenitor cell sorting and transplantation. Marker-sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker-sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.

Midbrain Dopaminergic Neuron Development is Regulated by Two Molecularly Distinct Subtypes of Radial Glia Cells

“…6C and Table S3). Moreover, GSEA identified novel neurogenesis-related genes in hFP-Rgl1 previously shown to enrich for mDA progenitors, including TPBG and PTPRO (Xu et al, 2022; Yoo et al, 2021).…”

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo

“…We show that ST introduces less bias in cell type composition compared to scRNA-seq. 35 , 36 This is most likely because there is no tissue dissociation before cDNA synthesis, leading to an equal likelihood of capture for most cell types. This is especially obvious when looking at the vulnerable cell types, such as neurons, which become dwarfed by the glial cell numbers in scRNA-seq.…”

“… 32 , 33 , 34 This approach, however, leaves the transplant’s final composition challenging to predict and control. 35 The final graft composition in vivo from hESC-derived dopaminergic transplants has been analyzed in bulk 36 and at single-cell resolution. 18 However, the quantitative results of the latter study were at odds with histological assessments since the sequencing study suggested that neurons make up the minority of the transplant.…”

Mitochondrial Dysfunction and Neuronal Anomalies inPOLGMutant Midbrain Organoids

“…Clusters were annotated based on the expression of known markers identified from several literatures. DA progenitors were annotated for their highly expressed markers such as NEUROG2, NHLH1 and SOX4 [29,30]. DA-0 was annotated based on the high expression of ErbB4 known to be expressed in DA neurons and low level of SYT1 a post-synaptic marker [31].…”