Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation

Abstract

Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson’s disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.

Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids

“…We established VM organoids by adapting a commonly used protocol for the generation of forebrain organoids 27 with the addition of dual-SMAD inhibition combined with exposure to SHH, GSK3i, and FGF8 in the same temporal sequence used in 2D culture to promote neurogenic conversion of VM FP progenitors toward a DA neuron identity (Fig. 1a) 28,29 . We confirmed that in 3D cultures, this patterning regime also resulted in efficient neuralization and ventral midbrain differentiation as evaluated by expression of CNPY1, LMX1a, LMX1b, OTX1, CORIN, SHH, and FOXA2 (Fig.…”

“…5a). We also performed scRNAseq of one-month-old MLOs generated using the protocol described by Jo et al (29,112 cells analyzed from two replicates), which showed the presence of the same cell types at the same timepoints (Supplementary Fig. 5b).…”

“…We also more closely dissected the DA neuron cluster (14,606 cells), which led to the identification of three molecularly distinct subgroups of mature human DA neurons. We used ventralizing and caudalizing factors known to direct a VM identity in 2D culture 28,29 and to result in the formation of functional DA neurons after transplantation in preclinical rat models of PD 50,51 . A subset of these factors was previously used to generate similar midbrain-patterned organoids derived from hPSCs [15][16][17] and other studies report VM organoids derived from neural progenitors [19][20][21] .…”

Dopamine Neuron Diversity: Recent Advances and Current Challenges in Human Stem Cell Models and Single Cell Sequencing

“…In the last 20 years, the most widely used differentiation protocols have all been based on adherent 2D cellular cultures, and a variety of approaches driving stem cell commitment toward neuron differentiation are currently available [32,33]. Although a 2D configuration limits cell growth to a monolayer and is reductionist in nature, 2D cell culture has the great advantage of generating homogeneous populations of differentiated cells with high purity, and of enabling uniform accessibility to patterning factors and gas exchange [20,34].…”

“…Dual SMAD inhibitors blocking TGFb and BMP signaling, combined with the ventralizing factor SHH and the caudalizing factor CHIR99021 for WNT pathway activation, are used for efficient specification of VM progenitors expressing a unique combination of markers found only in the midbrain floor plate, including FOXA2, LMX1A, EN1, and CORIN [17,41]. hPSC-derived DA progenitors can further differentiate into functional DA neurons with electrophysiological properties, and evidence indicates that they can also survive and mature after intracerebral transplantation in a rat model of PD, giving rise to DA-rich grafts that are able to innervate the host brain, leading to functional recovery [20,33].…”