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Single-cell transcriptomics and in vitro lineage tracing reveals differential susceptibility of human iPSC-derived midbrain dopaminergic neurons in a cellular model of Parkinson’s disease.

ABSTRACT: Advances in stem cell technologies open up new avenues for modelling development and diseases. The success of these pursuits however rely on the use of cells most relevant to those targeted by the disease of interest, for example, midbrain dopaminergic neurons for Parkinson’s disease. In the present study, we report the generation of a human induced pluripotent stem cell (iPSC) line capable of purifying and tracing nascent midbrain dopaminergic progenitors and their differentiated progeny via the expression of a Blue Fluorescent Protein (BFP). This was achieved by CRISPR/Cas9 assisted knock-in of BFP and Cre into the safe harbour locus AAVS1 and an early midbrain dopaminergic lineage marker gene LMX1A, respectively. Immunocytochemical analysis and single cell RNA sequencing of iPSC-derived neural cultures confirms developmental recapitulation of the human fetal midbrain and high quality midbrain cells. By modelling Parkinson’s disease-related drug toxicity using 1-Methyl-4-phenylpyridinium (MPP+), we showed preferential reduction of BFP+ cells, a finding demonstrated independently by cell death assays and single cell transcriptomic analysis of MPP+ treated neural cultures. Together, these results highlight the importance of disease relevant cell type in stem cell modelling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE249360 | GEO | 2023/12/08

REPOSITORIES: GEO

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Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.

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Single-cell transcriptomics and in vitro lineage tracing reveals differential susceptibility of human iPSC-derived midbrain dopaminergic neurons in a cellular model of Parkinson’s disease.

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