Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons

Abstract

Recent advances in 3D culture systems have led to the generation of brain organoids that resemble different human brain regions; however, a 3D organoid model of the midbrain containing functional midbrain dopaminergic (mDA) neurons has not been reported. We developed a method to differentiate human pluripotent stem cells into a large multicellular organoid-like structure that contains distinct layers of neuronal cells expressing characteristic markers of human midbrain. Importantly, we detected electrically active and functionally mature mDA neurons and dopamine production in our 3D midbrain-like organoids (MLOs). In contrast to human mDA neurons generated using 2D methods or MLOs generated from mouse embryonic stem cells, our human MLOs produced neuromelanin-like granules that were structurally similar to those isolated from human substantia nigra tissues. Thus our MLOs bearing features of the human midbrain may provide a tractable in vitro system to study the human midbrain and its related diseases.

Figure 1. Generation and characterization of hMLOs from hPSCs

(A) Schematic diagrams illustrating the overall strategy to generate hMLOs. DIC images illustrate the typical morphology of cells at each stage. SBNC: SB431542, Noggin, and CHIR99021; SF: SHH-C25II and FGF8; BGAC: BDNF, GDNF, ascorbic acid, and db-cAMP. Scale bars=500 μm. (B) Left: Cryosection of an hMLO at day35 stained for Ki67 and MAP2. Right: A zoom-in view of the white box. White scale bar = 200μm. Yellow scale bar = 10μm. (C) Quantification of the percentage of Ki67⁺ and MAP2⁺ cells at day 25 and 35 hMLOs by FACS analysis. n=3, *p<0.05, Student’s t-test. (D) Immunostaining of EdU, OTX2, and aPKC at the apical region of a neuroepithelium (NE). Scale bar = 20μm. (E) Percentages of OTX2⁺ and OTX⁺/EdU⁺ cells at the apical region of NE; mean ± s.e.m., n=7. (F) Schematic of the laminar structure in the hMLOs (bas.: basal; ap.: apical; MZ: mantle zone; IZ: intermediate zone; VZ: ventral zone). (G) Cryosection of a day 35 hMLO stained for MAP2 and MASH1. Scale bar = 50μm. (H) EdU labeling and OTX2 immunostaining of a day 35 hMLO. Scale bar = 20μm. (I) Cryosection of a day 35 hMLO stained for NURR1 and MASH1. Scale bar = 20μm. (J) Cryosection of an hMLO at days 4, 14, and 24 stained for FOXA2 (floor plate progenitors) and OTX2 (midbrain intermediate progenitors). Scale bars = 50μm. (K) Immunostaing of FOXA2 and TH of hMLO at day 45. Scale bars = 50μm. The quantifications are shown in (L); mean ± s.e.m., n=3. (M) Cryosection of a day 45 hMLO labeled for LMX1A and TH. Scale bars = 50μm. The quantification is shown in (N); mean ± s.e.m., n=3. (O) Immunostaining of MZ cells at day 60 with DAT and TH antibodies, zoom-in view to illustrate some cells double positive for DAT and TH. The quantifications are shown in (P); mean ± s.e.m., n=3. Scale bar = 20μm. See also Figure S1.

Figure 2. Transcriptional characterization of hMLOs

(A) Heatmap showing differentially expressed genes between 2D-DA neurons and hMLOs, sorted by fold change (related to Table S1). (B) Heatmap and clustering of expression data from 2D-DA neurons, hMLOs, and prenatal midbrain. The correlation of normalised gene expression using differentially expressed genes between 2D-DA neurons and hMLOs was used to estimate the distance between samples. (C) Venn Diagram indicating the overlap of genes that show up- or downregulation in hMLOs and prenatal midbrain compared to 2D-DA neurons. (significance was estimated using Fisher’s exact test). (D) Heatmap showing genes that are differentially expressed between 2D-DA neurons and hMLOs and human prenatal midbrain, sorted by fold change (related to Table S2). (E) Example genes expressed in prenatal midbrain and hMLOs, but not in 2D-DA neurons, and (F) example genes commonly expressed in prenatal midbrain, hMLOs, and 2D-DA neurons (Black: normalized read count, blue: split reads that map to two exons. Shown is the average across all samples). See also Figure S2.

Figure 3. Identification of neuromelanin in hMLOs

(A) Appearance of dark granules in hMLOs at day 112. Note that dark pigments were localized within the neuronal compartment (arrows) as well as the extracellular compartment (arrowheads). Scale bar = 200μm. (B) Fontana-Masson staining to reveal NM-like granules within an hMLO. Note the presence of NM-like granules in both intra- and extracellular compartments (blue and black arrowheads, respectively). Black scale bars = 100μm. A red scale bar = 20μm. B′ is an enlarged view of a region in B. B″ is an enlarged view of a region in B′. (C) Fontana-Masson staining of human postmortem midbrain tissue. Black scale bars = 1mm. A red scale bar = 200μm. C′ is an enlarged view of a region in C was from tiling multiple images of a large area. C″ is an enlarged view of a region in C′. (D) NM content measurement in hMLOs (n=3, respectively). (E) SEM image of isolated NM granules in a day 122 hMLO and (F) in human postmortem midbrain tissue (See also Figure S3D–F and Table S3). Scale bar = 200nm. (G) The formation of NM-like granules was accelerated by L-DOPA (50μM) and dopamine (50μM) treatments. Red scale bar = 2mm. Black scale bar =100μm. G′, G″, and G‴ are high-magnification images of the black rectangle. (H) NM-like granules were not observed in murine MLOs. Please note that both the hMLO and mMLOs contain TH-positive mDA neurons (bottom panels). Red scale bar = 2mm. Black scale bar= 500μm. White scale bar= 20μm. See also Figure S3.

Figure 4. Functional characterization of dopaminergic neurons from hMLOs

(A) Schematic diagram illustrating the experiment to investigate the electrophysiological activity of hMLOs in situ [recordings (Rec.) and electric stimulation (Stim.)]. (B) Representative traces showing the presence of voltage-dependent Na⁺ and K⁺ currents in neurons inside hMLOs. The blue box highlights Na⁺ channel-dependent inward currents. (C) Averaged current-voltage relationship (I/V) curves for the Na⁺ and K⁺ currents recorded (n=12 and 14 for days 33–50 and 65–84, respectively). (D) Representative traces of multiple APs (the lower panel) recorded from neurons inside day 35 hMLOs, evoked by current injection (the upper panel). (E) The number of APs generated in response to a particular current pulse amplitude, of neurons inside hMLOs (n=12 and 14 for days 33–50 and 65–84, respectively). (F) Spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) (shown in 1) recorded from a neuron inside hMLO at day 80. These sEPSCs and sIPSCs were blocked by CNQX (an AMPA-type glutamate receptor antagonist) and AP5 (an NMDA-type glutamate receptor antagonist) (shown in 2) and by picrotoxin (PTX, a GABAA blocker) (shown in 3), respectively. (G) Electrical stimulation-evoked synaptic response recorded in the day 50 hMLO. The red dot indicates onset of electrical stimulation. (H and I) Representative trace and frequency of spontaneous APs. (J) Example traces of rebound depolarization. Insets show enlarged view of respective traces. (K) Representative trace of pacemaker-like firing and the effect of Quinpirole on firing frequency, and its statistical analysis (L) (*p=0.021, paired t-test, n=3). (M) TH immunostaining of a neuron filled with biocytin during the recording, indicating that the recorded neuron expressed TH. Scale bar = 5 μm. (N) Dopamine measurement in hMLOs and hCOs by HPLC [hMLO 4w, 5w, 7w, and 9w (n=4); hMLO 13w, hCO 7w and 27w (n=3)]. See also Table S4.