The project advanced in a satisfactory manner towards our three main objectives:
1) Define adult human mDA neuron subtypes, including A9/SNc neurons. We have analyzed scRNA-seq data from adult substantia nigra samples and identified different subtypes of DA neurons (aim 1.1) (Siletti et al. 2023). We found transcriptional profiles ranging from a complete DA phenotype to TH+ neurons with a partial DA and GABAergic phenotypes. In another analysis we analyzed and annotated human embryonic midbrain samples (Braun et al, 2023) and revealed 20 distinct subtypes of TH+ neurons expressing well-known DA markers and unique profiles. This study revealed the generation of DA neurons from midbrain progenitors and the temporal sequence of transcription factors expressed in different cell types along this development. The development of mDA neurons was further investigated and we found Rgl1 to be their neurogenic progenitor (for mDA neurons) and identified the transcription factor BMAL1 as a novel regulator of mDA neurogenesis. (Asgrimsdottir et al, in press, 2026). We progressed in several directions. We characterized the differentiation of hPSCs into mDA neurons at the single cell level and show that differentiation follows key aspects of midbrain development and gives rise to high quality embryonic DA neurons (Nishimura et al, 2023). We also shortened the differentiation protocol and made it more cost-effective (Pantazis et al, 2022). We generated an inducible Cas9 line for a loss of function CRISPR screen to find transcription factor candidates important for lineage specification (FOXA2, LMX1A, EN1, ASCL1, NEUROG2, NR4A2, TH, DDC and SOX6). We also examined the contribution of extracellular modulators of mDA differentiation and show that the heparan sulfate (HS)-modifying enzymes SULF1 and SULF2 are key modulators of anterior-posterior and dorsal-ventral patterning during mDA neuron development in vitro (Tremolanti et al, manuscript under revision)
2) Develop a cell-replacement strategy for PD based on the transplantation of hPSC-derived mDA progenitors differentiating into A9/SNc neurons. In an in silico forward programming simulation we used data from the literature and identified a number of candidates which were also hand-curated in the targeted transcription factor sgRNA library that was used for the loss of function screen in aim 1.
Transplantation of human PSC derived mDA progenitors into hemiparkinsonian immunocompromised mice, which previously have not been utilized in intracranial grafting experiments, restored motor function. Progenitors were shown to mature into a large percentage of DA neurons expressing canonical markers for A9-subtype mDA neurons, which was further confirmed by a comparison of the transcriptional profiles to those of native adult human mDA cells showing a successful generation of in vivo differentiated cells resembling human SOX6 mDA neurons (Garcia Swinburn et al, submitted manuscript).
3) Develop a novel cell-replacement strategy for PD based on the in situ conversion of striatal astrocytes into induced A9/SNc mDA neurons by direct in vivo reprogramming. We improved a previous protocol which resulted in a 70% increase of TH + neurons. We also performed single cell RNA sequencing to compare the in vivo reprogrammed mDA neurons to human embryonic mDA neurons. This analysis is under way.
Our work resulted in a total of 6 publications, 1 manuscript in press, 1 manuscript under revision, 1 submitted manuscript, 2 manuscripts in preparation.
