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. 2017 Feb 1;26(3):552-566.
doi: 10.1093/hmg/ddw412.

Transcriptomic Profiling of Purified Patient-Derived Dopamine Neurons Identifies Convergent Perturbations and Therapeutics for Parkinson's Disease

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Free PMC article

Transcriptomic Profiling of Purified Patient-Derived Dopamine Neurons Identifies Convergent Perturbations and Therapeutics for Parkinson's Disease

Cynthia Sandor et al. Hum Mol Genet. .
Free PMC article

Abstract

While induced pluripotent stem cell (iPSC) technologies enable the study of inaccessible patient cell types, cellular heterogeneity can confound the comparison of gene expression profiles between iPSC-derived cell lines. Here, we purified iPSC-derived human dopaminergic neurons (DaNs) using the intracellular marker, tyrosine hydroxylase. Once purified, the transcriptomic profiles of iPSC-derived DaNs appear remarkably similar to profiles obtained from mature post-mortem DaNs. Comparison of the profiles of purified iPSC-derived DaNs derived from Parkinson's disease (PD) patients carrying LRRK2 G2019S variants to controls identified significant functional convergence amongst differentially-expressed (DE) genes. The PD LRRK2-G2019S associated profile was positively matched with expression changes induced by the Parkinsonian neurotoxin rotenone and opposed by those induced by clioquinol, a compound with demonstrated therapeutic efficacy in multiple PD models. No functional convergence amongst DE genes was observed following a similar comparison using non-purified iPSC-derived DaN-containing populations, with cellular heterogeneity appearing a greater confound than genotypic background.

Figures

Figure 1.
Figure 1.
Purification of iPSC dopaminergic neurons by flow cytometry. (A) Representative immunostains of neurons demonstrates successful differentiation of control and PD samples in DaNs. (B) Representative FACS plots of the DA neuron isolation for control. (C) PD LRKK2-G2019S samples. Vertical axis denotes live/dead stain and horizontal axis TH- and TH+ cells. IgG2a was used as an isotype control. (D) Successful purification of TH positive neurons: qRT-PCR for TH expression on RNA extracts from sorted and unsorted control and LRKK2-G2019S lines.
Figure 2.
Figure 2.
Transcriptomic evaluation of an iPSC-derived and purified model of dopaminergic neurons. (A) Principal component analyses performed from FPKM values of 17170 of 20157 protein coding genes for which the variance was different zero. x and y axis represent the principal component 1 and 2 explaining 34% and 15% of variance, respectively. (B) Expression level of 16 gene dopaminergic markers. The two vertical dotted lines represent the 50nd (gray) and 75nd (black) percentiles of expression level measure. (C) Comparisons of the transcriptional profiles of all eight iPSC-derived DaNs cell lines with the following publically-available transcriptional profiles: (i) RNA seq profiling generated from 53 human postmortem tissue profiles made available by the Genotype-Tissue Expression (GTEx) project (http://www.gtexportal.org/) (ii) RNA sequencing data profiling of up to sixteen cortical and subcortical structures across the full course of human brain development (http://www.brainspan.org/) (iii) microarray profiles of eight iPSC-derived unpurified DaNs cell lines including two controls lines (C1.1,C2), three lines carrying LRRK2-G2019S mutations (L1.1Mut, L2.3Mut, L2.2Mut) and three matching isogenic lines with engineered-corrections for LRRK2-G2019S mutation, isogenic line of L1.1Mut, L2.3Mut, L2.2Mut (L1.1GC2, L2.3GC, L2.2GC) (GSE43364, (23)) (iv) microarray profiles of two laser-captured human dopaminergic neuron dataset (GSE20141 & GSE24378). (v) RNA sequencing data profiling of 14 samples coming from of 7 iPSC derived DaN (two replicates by cell line) and FACs sorted by using a combination of surface markers (CD133, a stem/progenitor marker; CD56, a nerve cell adhesion molecule; CD15 and CD184, NSC markers; and CD24, a cell differentiation antigen) derived from following subjects: (1) man with a five-year history of PD (PD) and heterozygous for GBA-N370S variant, (2) his monozygotic twin brother without PD (Non-PD), (3) one sporadic PD patient (Sporadic-PD) and (4) four control subjects (C) (GSE62642) (25) (Materials and Methods). The brainspan dataset uses the following acronyms: URL upper (rostral) rhombic lip; VFC ventrolateral prefrontal cortex; DFC dorsolateral prefrontal cortex; LGE lateral ganglionic eminence; ITC inferolateral temporal cortex (area TEv); STC posterior (caudal) superior temporal cortex (area TAc); AMY amygdaloid complex; MFC anterior (rostral) cingulate (medial prefrontal) cortex; HIP hippocampus (hippocampal formation); CGE caudal ganglionic eminence; Ocx occipital neocortex; DTH dorsal thalamus; M1C-S1C primary motor-sensory cortex (samples); MGE medial ganglionic eminence; OFC orbital frontal cortex; PCx parietal neocortex; TCx temporal neocortex; M1C primary motor cortex (area M1); STR striatum; IPC posteroventral (inferior) parietal cortex; A1C primary auditory cortex (core); V1C primary visual cortex (striate cortex); S1C primary somatosensory cortex (area S1); CB cerebellum; MD mediodorsal nucleus of thalamus; CBC cerebellar cortex
Figure 3.
Figure 3.
Functional associations within general PLN of 94/168 DE genes. The colour of links between genes indicates the most informative dataset for the relationship between gene pairs (see legend). The Panel (A) shows the relation between the up (red) and down (blue) regulated genes (reference control). The Panel (B) lists differentially expressed genes whose orthologue’s disruption in the mouse yields the phenotype abnormal capabilities/coordination/movement ((MP:0002066). The Panels C and D show genes for which expression is increased (red) or decreased (blue) after rotenone and clioquinol respectively by using top 1000 of up and dow regulated genes of CMAP rank matrix of each instance (Materials and Methods).

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