TY - JOUR
AU - Han, Dong Wook
AB - Abstract Recent studies have demonstrated the generation of midbrain-like organoids (MOs) from human pluripotent stem cells. However, the low efficiency of MO generation and the relatively immature and heterogeneous structures of the MOs hinder the translation of these organoids from the bench to the clinic. Here we describe the robust generation of MOs with homogeneous distribution of midbrain dopaminergic (mDA) neurons. Our MOs contain not only mDA neurons but also other neuronal subtypes as well as functional glial cells, including astrocytes and oligodendrocytes. Furthermore, our MOs exhibit mDA neuron-specific cell death upon treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, indicating that MOs could be a proper human model system for studying the in vivo pathology of Parkinson's disease (PD). Our optimized conditions for producing homogeneous and mature MOs might provide an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD. Significance statement Although recent studies have demonstrated the generation of human pluripotent stem cell-derived midbrain-like organoids (MOs) that display structural and functional features of midbrain, a few issues, including the low generation efficiency and reproducibility of MOs, heterogeneous and immature structures of MOs, and physiologically irrelevant cellular composition of MOs, hinder the clinical translation of organoid technology. This article describes a novel strategy for the robust generation of homogeneous MOs using specific combination of dual SMAD inhibitors and in vitro WNT gradient. MOs generated by the optimized protocol with in vivo-like cellular composition are as structurally and functionally mature as the developing midbrain. DAC3.0 MOs with functional glial cells facilitate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-based in vitro disease modeling of PD. This study describes the novel strategy for the robust generation of homogeneous midbrain-like organoids (MOs) using specific combination of dual SMAD inhibitors and in vitro WNT gradient. DAC3.0 MOs generated by our optimized protocol with in vivo-like cellular composition are as structurally and functionally mature as the developing midbrain. DAC3.0 MOs with functional glial cells including astrocytes facilitate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-based in vitro disease modeling of PD, suggesting its potential usefulness for an advanced patient-specific platform for in vitro disease modeling as well as for drug screening for PD. Open in new tabDownload slide Open in new tabDownload slide differentiation, embryonic stem cells (ESCs), neural differentiation, Parkinson's disease INTRODUCTION Induced pluripotent stem cell (iPSC) technology1,, 2 represents a novel approach to patient-specific disease modeling and drug screening.3 Many previous studies have used patient-specific iPSCs for reproducing in vivo pathophysiology of various diseases in a dish, including neurodevelopmental disorders,4 neurodegenerative diseases,5 cardiovascular diseases,6 liver diseases,7 and others.8 However, current two-dimensional (2D) differentiation protocols typically produce singular cell types, but not multiple cell types, from pluripotent stem cells (PSCs).09,, 10 Previous studies on in vitro disease modeling were based on 2D differentiation systems and could not fully recapitulate in vivo disease pathology,10,, 11 as organs consist of a highly orchestrated three-dimensional (3D) structure comprising multiple cell types, each of which has a unique function. Indeed, previous iPSC-based in vitro modeling studies of Parkinson's disease (PD) using 2D differentiation technology described either no PD-associated pathophysiology12,, 13 (no phenotype and non-PD phenotype) or only early-stage symptoms of PD14-16 such as a neuronal differentiation defect, abnormal autophagy, and accumulation of α-synuclein, without recapitulating the end-stage pathophysiology of PD such as massive midbrain dopaminergic (mDA) neuron–selective cell death.17 Recent advances in the stem cell field have successfully demonstrated the generation of miniaturized 3D organ-like structures, so-called organoids, from human PSCs. By taking advantage of the self-organization capacity of PSCs, together with specific differentiation signals, organoids representing distinct organs,18 such as lung,19 liver,20 kidney,21 stomach,22 pancreas,23 intestine,24 and colon,25 could be generated. The generation of brain organoids that correspond to different parts of brain, such as cerebral cortex,26-30 optic cups,31 hippocampus,32 pituitary gland,33 and cerebellum,34 has been also described. Moreover, recent studies have also demonstrated the generation of midbrain-like organoids (MOs) from either human PSCs35,, 36 or predifferentiated neuroepithelial stem cells from human iPSCs.37 In contrast to cerebral organoids (COs),28,, 35 MOs express markers typical for human midbrain35 and produce neuromelanin-like granules,36,, 37 which display similar structures to those from human substantia nigra,38 suggesting that MOs have the potential for application in disease modeling and drug screening for midbrain-specific neurodegenerative diseases such as PD. However, a few issues need clear resolution before translating organoid technology to the clinic. Firstly, the efficiency of organoid generation is typically very low,39 resulting in organoid technology of low reproducibility. Secondly, nearly all kinds of organoids, including MOs, exhibit heterogeneous structures,10,, 40 potentially compromising the reliability of organoid-based drug screening and disease modeling data. Thirdly, not all cellular components could be reproduced in organoids even after long-term maturation, yielding organoids with physiologically irrelevant cellular composition.10 For example, MOs generated from PSCs contain no or a paucity of glial cells.35,, 36 Finally, yet most importantly, organoids representing distinct organs typically display relatively immature structures compared with their in vivo counterparts,40 although they conserve some key structural characteristics. In the current study, we developed a robust protocol for the generation of MOs. For this, we first screened small molecules for dual SMAD inhibition in order to achieve efficient specification into mesencephalon and, subsequently, into midbrain. Among the distinct small molecule combinations, the combination of dorsomorphin, A83-01, and CHIR99021 facilitated the robust production of MOs, with global enrichment of mDA neurons. Moreover, we also showed that the WNT signal is a key determinant of the regional identity of MOs, and by fine mapping the WNT gradient, we generated functionally and electrophysiologically mature MOs that produce dopamine and neuromelanin-like granules. Our MOs contain not only mDA neurons but also multiple neuronal subtypes, depicting a neuronal organization that is similar to the in vivo scenario. More importantly, we showed that these MOs contain functional glial cells, a finding that has not been described previously. Finally, the MOs containing functional astrocytes underwent massive cell death upon treatment with the astrocyte-mediated dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our novel and robust protocol for generating MOs might hold potential usefulness for understanding the early development of the midbrain in vivo and for modeling PD in vitro. MATERIALS AND METHODS Generation of brain organoids To generate brain organoids, 1 × 104 single cell-dissociated human embryonic stem cells (hESCs) were plated on each well of ultra-low-attachment U-bottom 96-well plates (Corning). At 24 hours after plating, brain organoid generation medium (BGM) (see Appendix S1) was added to embryoid bodies (EBs). The medium was replaced every other day. Brain organoids were embedded into growth factor-reduced Matrigel (Corning) droplets. Matrigel-embedded brain organoids were transferred to 6-cm petri dishes containing BGM with proper supplements for MOs and COs for inducing basal-apical lamination. For maturation, brain organoids were cultured on an orbital shaker (Stuart) with proper maturation medium (see Appendix S1). RESULTS Robust mesencephalon specification by specific dual SMAD inhibitors Although previous studies35,, 36 have successfully demonstrated the generation of MOs, the efficiency of MO generation and issues regarding the structural and functional heterogeneity of these MOs have not been addressed. The utility of MO technology for in vitro disease modeling and drug screening for midbrain-specific neurodegenerative diseases depends on an optimized protocol for robustly generating a structurally and functionally homogeneous population of MOs. First, we sought to optimize mesencephalon specification, the first step in the generation of MOs (Figure 1A). For this, first we treated hESCs with distinct combinations of dual SMAD inhibitors for inducing early neuroectodermal commitment41 and CHIR99021 for regional specification into mesencephalon.42,, 43 1 × 104 single-cell dissociated hESCs were plated on U-bottom 96-well plates with 0.8 μM CHIR9902136 and distinct combinations of dual SMAD inhibitors, which had been used in previous organoid studies: that is, 200 ng/mL Noggin + 10 μM SB431542 (NS),36,, 44 2 μM dorsomorphin + 2 μM A83-01 (DA),35 100 nM LDN + 10 μM SB431542 (LS),35 and 10 μM dorsomorphin + 10 μM SB431542 (DS)30 for achieving efficient mesencephalon specification (Figure 1A). Figure 1 Open in new tabDownload slide Robust specification into mesencephalon by specific dual SMAD inhibitors. A, Schematic illustration of the procedure for generating MOs. The generation of MOs can be achieved by a sequential protocol that includes mesencephalon specification, mesencephalic floor plate induction, basal-apical lamination, and maturation. B, The number and quality of EBs generated from distinct combinations of dual SMAD inhibitors. Data are presented as mean ± SD from three independent experiments. C, Expression of pluripotency, neuroectoderm, mesendoderm, and apoptosis markers was analyzed by qPCR using EBs grown under distinct dual SMAD inhibition conditions (7 days). Expression levels are normalized to those of undifferentiated hESCs. *P < .05. D, Morphology and average diameter of MOs (2WM) generated with distinct combinations of dual SMAD inhibitors. Data are presented as mean ± SD from three independent experiments. Scale bar, 500 μm. E, Representative confocal images of NS-, DA-, and LS-MOs (2WM) expressing TH, MAP2, and ASCL1. Scale bar, 50 μm. F, Expression of forebrain- and midbrain-specific marker genes was analyzed by qPCR in a time-course manner, on days 7, 14, and 21 after differentiation. Expression levels are normalized to those of undifferentiated hESCs. Data are presented as mean ± SD of triplicate values. *P < .05. 2WM, 2 weeks of midbrain maturation; EBs, embryoid bodies; MOs, midbrain-like organoids; TH, tyrosine hydroxylase Figure 1 Open in new tabDownload slide Robust specification into mesencephalon by specific dual SMAD inhibitors. A, Schematic illustration of the procedure for generating MOs. The generation of MOs can be achieved by a sequential protocol that includes mesencephalon specification, mesencephalic floor plate induction, basal-apical lamination, and maturation. B, The number and quality of EBs generated from distinct combinations of dual SMAD inhibitors. Data are presented as mean ± SD from three independent experiments. C, Expression of pluripotency, neuroectoderm, mesendoderm, and apoptosis markers was analyzed by qPCR using EBs grown under distinct dual SMAD inhibition conditions (7 days). Expression levels are normalized to those of undifferentiated hESCs. *P < .05. D, Morphology and average diameter of MOs (2WM) generated with distinct combinations of dual SMAD inhibitors. Data are presented as mean ± SD from three independent experiments. Scale bar, 500 μm. E, Representative confocal images of NS-, DA-, and LS-MOs (2WM) expressing TH, MAP2, and ASCL1. Scale bar, 50 μm. F, Expression of forebrain- and midbrain-specific marker genes was analyzed by qPCR in a time-course manner, on days 7, 14, and 21 after differentiation. Expression levels are normalized to those of undifferentiated hESCs. Data are presented as mean ± SD of triplicate values. *P < .05. 2WM, 2 weeks of midbrain maturation; EBs, embryoid bodies; MOs, midbrain-like organoids; TH, tyrosine hydroxylase The EBs generated under all conditions, except for DS, were uniformly of high quality; few EBs were produced with DS and they were of relatively poor quality (Figure 1B; Figure S1A). As expected, all dual SMAD inhibition conditions robustly activated neuroectodermal markers, with complete suppression of pluripotency and mesendodermal markers (Figure 1C). Unexpectedly, under the DS condition, mesendodermal markers were also highly activated (Figure 1C). After the mesencephalon specification step, we induced the ventralization of mesencephalon-specified early EBs (day 4) to achieve further specification into the mesencephalic floor plate. This was done by treating EBs with both FGF8, a midbrain-hindbrain boundary-derived morphogen, and SAG, a sonic hedgehog agonist, for 5 days (Figure 1A). Then, we embedded midbrain-specified organoids (hereafter referred to as MOs) into Matrigel droplets to induce basal-apical lamination, followed by MO maturation (Figure 1A). After 1 week of midbrain maturation (1WM), we observed MOs of similar number and size for all dual SMAD inhibition conditions, except for DS (Figure S1B, C). As there were practically no viable MOs with DS (Figure S1D), we compared only the NS, DA, and LS conditions for the remainder of the analysis. After 2WM, we observed MOs of similar size expressing both tyrosine hydroxylase (TH), a marker for mDA neurons, and ASCL1, a marker for their progenitor population, under all conditions (Figure 1D,E; Figure S1E). Our qPCR analysis showed that midbrain markers were gradually increased in MOs generated under both the DA and LS conditions (hereafter DA-MOs and LS-MOs, respectively) in a time-dependent manner, compared with MOs generated with NS (NS-MOs) (Figure 1F). Taking these results together, we were able to optimize the conditions for mesencephalon specification of brain organoids, by comparing multiple conditions for dual SMAD inhibition for the robust generation of MOs that contain TH-positive mDA neurons. Generation of homogeneous midbrain organoids by specific dual SMAD inhibition Distinct combinations of dual SMAD inhibitors robustly led brain organoids to undergo early neuroectodermal commitment as well as mesencephalon specification (Figure 1C,F). However, after 1WM, we observed that MOs from different conditions exhibited distinct cellular responses, that is, highly specific activation of midbrain markers in DA-MOs and LS-MOs but residual expression of forebrain markers in NS-MOs (Figure 1F). These different responses in gene expression pattern prompted us to investigate the effect of dual SMAD inhibition on the structural heterogeneity of MOs. For this, we examined the numbers and distribution patterns of TH-positive cells in MOs produced under distinct conditions (NS-, DA-, and LS-MOs) after 4WM. As a result, NS-MOs and LS-MOs exhibited heterogeneous distribution of TH-positive mDA neurons (Figure 2A). In contrast, strong expression of TH was evenly observed in DA-MOs (Figure 2A). Notably, more than 85.7% ± 15.8% of MAP2-positive neurons in DA-MOs were TH-positive, whereas MOs under the other conditions showed polarized distributions of fewer TH-positive neurons (47.5% ± 14.0% in NS-MOs and 31.5% ± 11.3% in LS-MOs) (Figure 2B), indicating that more homogeneous MOs with more TH-positive mDA neurons were produced with DA. We also observed the relatively more abundant and homogeneous distribution of LMX1A, a marker for midbrain-specific progenitors and ASCL1, a marker for mDA neuronal progenitors in DA-MOs compared with NS- and LS-MOs (Figure S2). Similarly, both LMX1A and TH were homogeneously expressed in DA-MOs but weakly and heterogeneously expressed in both NS-MOs and LS-MOs (Figure 2C). Surprisingly, TBR1, a marker for cerebral cortex, was still highly expressed in both NS-MOs and LS-MOs but not expressed in DA-MOs (Figure 2D). Similar gene expression patterns were also observed in our RNA-sequencing (RNA-seq) data using day 7 EBs. In general, MOs produced with each combination showed activation of neuronal genes (Figure S1F,G). However, both NS- and LS-MOs exhibited high activation of cerebral cortex markers as well as non-neuronal genes (Figure 2E; Figure S1H). In contrast, DA-MOs exhibited strong suppression of those markers (Figure 2E; Figure S1G). Finally, a whole-mount imaging of MOs produced under these different conditions again confirms our finding that MOs containing a high number and homogeneous distribution of TH-positive mDA neurons were produced with DA (Figure 2F). Taken together, our data indicate that dual SMAD inhibition by DA during the early phase of MO generation not only induces robust specification into mesencephalic floor plate but also produces structurally homogeneous MOs. Figure 2 Open in new tabDownload slide Structural homogeneity of DA-MOs. A, Confocal images showing distribution of mDA neurons expressing both MAP2 and TH in MOs grown under distinct conditions (NS-, DA-, and LS-MOs) after 4WM. Scale bar, 100 μm. B, Percentage of TH-positive mDA neurons. Data are presented as mean ± SD from three independent experiments. ***P < .001. C and D, Confocal images showing expression pattern of the midbrain markers LMX1A and TH (C) and cerebral cortex markers TBR1 and TUJ1 (D) in NS-, DA-, and LS-MOs (4WM). Scale bar, 100 μm. E, The Integrative Genomics Viewer (IGV), a basic RNA-seq processing tool displaying expression patterns of the cerebral cortex marker FOXG1 and the midbrain marker LMX1A in day 7 EBs. F, Multiple section images of whole-mount confocal microscopy showing the distribution of TH-positive mDA neurons in MOs grown under distinct conditions (4WM). Scale bar, 100 μm. 4WM, 4 weeks of midbrain maturation; EBs, embryoid bodies; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TH, tyrosine hydroxylase Figure 2 Open in new tabDownload slide Structural homogeneity of DA-MOs. A, Confocal images showing distribution of mDA neurons expressing both MAP2 and TH in MOs grown under distinct conditions (NS-, DA-, and LS-MOs) after 4WM. Scale bar, 100 μm. B, Percentage of TH-positive mDA neurons. Data are presented as mean ± SD from three independent experiments. ***P < .001. C and D, Confocal images showing expression pattern of the midbrain markers LMX1A and TH (C) and cerebral cortex markers TBR1 and TUJ1 (D) in NS-, DA-, and LS-MOs (4WM). Scale bar, 100 μm. E, The Integrative Genomics Viewer (IGV), a basic RNA-seq processing tool displaying expression patterns of the cerebral cortex marker FOXG1 and the midbrain marker LMX1A in day 7 EBs. F, Multiple section images of whole-mount confocal microscopy showing the distribution of TH-positive mDA neurons in MOs grown under distinct conditions (4WM). Scale bar, 100 μm. 4WM, 4 weeks of midbrain maturation; EBs, embryoid bodies; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TH, tyrosine hydroxylase WNT gradient is a key determinant for the regional identity of midbrain organoids The WNT signaling pathway is critical for inducing distinct regional identities of the developing brain.42,, 43 Previous trials for generating MOs had used specific concentrations of CHIR99021, either 0.8 μM36 or 3 μM.35 However, the WNT gradient had not been properly recapitulated in vitro for generating MOs, probably resulting in structurally and functionally heterogeneous brain organoids. Thus, as the second step in the generation of MOs, we sought to further fine-tune the regional identity of DA-MOs by screening for the ability of specific concentrations of CHIR99021 to generate homogeneous and mature MOs. For this, we tried to generate the in vitro WNT gradient by using different concentrations of CHIR99021 (0, 0.8, 1.5, 2, 2.5, or 3 μM) together with IWP2, a WNT inhibitor45,, 46 that eliminates endogenous WNT (Figure 3A). We found that there was a positive correlation between the concentration of CHIR99021 used and the size of DA-MOs generated (Figure S3A,B), with the higher concentrations of CHIR99021 leading to gradually lower levels of pro-apoptotic genes and hence larger MOs (Figure S3C). In general, a higher concentration of CHIR99021 led to the gradual increase in the expression of a subset of genes related to neuronal processes (Figure 3B,C; Figure S3D). The expression levels of cortex markers were decreased in a concentration-dependent manner (Figure 3D). In contrast, at higher concentrations of CHIR99021, midbrain markers were highly expressed, with maximum levels reached at 3 μM CHIR99021 (Figure 3E). Our RNA-seq analysis yielded similar changes at the transcriptional level. Compared with DA-MOs at a lower concentration of CHIR99021, DA-MOs at higher concentrations (2 and 3 μM) exhibited increased expression of midbrain markers and suppression of cerebral cortex markers (Figure 3F,G). We next performed whole-mount imaging and found that DA-MOs treated with higher concentrations of CHIR99021 displayed homogeneous distribution of mDA neurons with stronger expression of TH (Figure 3H). Notably, both 5 and 10 μM of CHIR99021 caused regional specification into more caudalized regions with decreased numbers of mDA neurons (Figure S3E-G). Collectively, our data indicate that the WNT gradient is a key determinant in establishing the regional identity of brain organoids and that a specific concentration of WNT (3 μM) facilitates the generation of homogeneous MOs. Figure 3 Open in new tabDownload slide WNT gradient is a key determinant of homogeneous MO generation. A, Schematic illustration depicting the procedure of generating homogeneous MOs by using an in vitro WNT gradient. B, Heatmap representing the global gene expression profile of MOs (3WM) generated by the in vitro WNT gradient. The red-blue color scale is the normalized expression value, denoted as the row Z-score. Red and blue colors indicate increased expression and decreased expression, respectively. Differentially expressed genes among each sample are categorized into five groups (class 1 to 5). C, GO enrichment analysis based on the gene set highly enriched in class 1. D and E, Expression of forebrain-, D, and midbrain-specific marker genes, E, was analyzed by qPCR in DA-MOs with different concentrations of CHIR99021 (2WM). Expression levels are normalized to those of nontreated DA-MOs. Data are presented as mean ± SD of triplicate values. *P < .05. F and G, Heatmaps representing the expression patterns of markers related to the midbrain, F, and forebrain, G, using DA-MOs produced by the in vitro WNT gradient (3WM). Color bar at the bottom indicates gene expression in log2 scale. Red and green colors represent higher and lower expression levels, respectively. H, The representative section images of whole mount confocal microscopy displaying global enrichment of TH-positive mDA neurons in DA-MOs with the in vitro WNT gradient (3WM). Scale bar, 200 μm. 3WM, 3 weeks of midbrain maturation; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TH, tyrosine hydroxylase Figure 3 Open in new tabDownload slide WNT gradient is a key determinant of homogeneous MO generation. A, Schematic illustration depicting the procedure of generating homogeneous MOs by using an in vitro WNT gradient. B, Heatmap representing the global gene expression profile of MOs (3WM) generated by the in vitro WNT gradient. The red-blue color scale is the normalized expression value, denoted as the row Z-score. Red and blue colors indicate increased expression and decreased expression, respectively. Differentially expressed genes among each sample are categorized into five groups (class 1 to 5). C, GO enrichment analysis based on the gene set highly enriched in class 1. D and E, Expression of forebrain-, D, and midbrain-specific marker genes, E, was analyzed by qPCR in DA-MOs with different concentrations of CHIR99021 (2WM). Expression levels are normalized to those of nontreated DA-MOs. Data are presented as mean ± SD of triplicate values. *P < .05. F and G, Heatmaps representing the expression patterns of markers related to the midbrain, F, and forebrain, G, using DA-MOs produced by the in vitro WNT gradient (3WM). Color bar at the bottom indicates gene expression in log2 scale. Red and green colors represent higher and lower expression levels, respectively. H, The representative section images of whole mount confocal microscopy displaying global enrichment of TH-positive mDA neurons in DA-MOs with the in vitro WNT gradient (3WM). Scale bar, 200 μm. 3WM, 3 weeks of midbrain maturation; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TH, tyrosine hydroxylase DAC3.0 MOs are structurally and functionally mature Throughout our optimization steps for the generation of MOs, we were able to generate MOs that homogeneously contain TH-positive mDA neurons using DA and 3 μM CHIR99021 (hereafter DAC3.0 MOs). To address the structural homogeneity of DAC3.0 MOs, we next analyzed the structure of these MOs by immunohistochemistry. DAC3.0 MOs after 1WM showed early-stage progenitors expressing either ASCL1 or LRTM1 (Figure 4A). DAC3.0 MOs after 4WM displayed a well-defined laminated structure containing a FOXA2+ (Figure 4A) ventricular zone, an ASCL1+ (Figure 4A)/LMX1A+ (Figure 4B) intermediate zone, and a TH+/MAP2+/DAT+ (Figure 4B) marginal zone, indicating that DAC3.0 MOs are structurally similar to the developing midbrain (Figure 4C; Figure S4A, Video S1). Gene expression profiling by RNA-seq showed enhanced expression levels of maturation-related genes, indicating the gradual maturation of DAC3.0 MOs (Figure S4B,C). DAC3.0 MOs were found to homogeneously contain TH-positive mDA neurons but not to express cerebral cortex markers FOXG1 and TBR1 (Figure S4D). Moreover, DAC3.0 MOs (8WM) were found to contain mature mDA neurons expressing NURR1, GIRK2, and CALB (Figure 4D; Figure S4E). Transmission electron microscopy (TEM) analysis showed a typical apical-basal differentiation pattern with abundant neuronal bundles in the marginal zone and cells tightly connected in the ventricular zone (Figure 4E). Taken together, our structural analysis indicates that DAC3.0 MOs are structurally mature and similar to the developing midbrain. Figure 4 Open in new tabDownload slide Structural and functional maturity of DAC3.0 MOs. A and B, Confocal images showing micro-anatomical structures of early-, A, (1WM) and late-stage, B, (4WM) DAC3.0 MOs. Scale bar, 50 μm. C, Illustration describing the layer structure of developing in vivo midbrain. D, Confocal images displaying markers of the mature mDA neurons NURR1, GIRK2, and Calbindin. Scale bar, 25 μm. E, TEM images of cross-sectional and longitudinal-sectional views of an axon bundle in the marginal zone and adherent junctions (arrows) in the ventricular zone of DAC3.0 MOs (2WM). Scale bar, 2 μm. F, Levels of dopamine produced by DAC3.0 MOs were measured by high-performance liquid chromatography (HPLC) 7 days after differentiation (12WM, and 20WM). *P < .05. G, Representative traces of multiple action potentials (Aps) recorded from neurons in DAC3.0 MOs (8WM), evoked by current injection. H, Accumulation of neuromelanin-like granules in DAC3.0 MOs was observed at 10WM. Pigmented cytoplasm of neurons (arrows) and neuromelanin-like aggregates (arrowheads) are shown in DAC3.0 MOs. Scale bar, 20 μm. I, Fontana-Masson staining of DAC3.0 MOs. Cerebral organoid was used as a negative control. Scale bar, 50 μm. J, TEM images displaying the fine structure of neuromelanin-like granules (arrowheads) in DAC3.0 MOs. Scale bar, 1 μm. 4WM, 4 weeks of midbrain maturation; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TEM, transmission electron microscopy Figure 4 Open in new tabDownload slide Structural and functional maturity of DAC3.0 MOs. A and B, Confocal images showing micro-anatomical structures of early-, A, (1WM) and late-stage, B, (4WM) DAC3.0 MOs. Scale bar, 50 μm. C, Illustration describing the layer structure of developing in vivo midbrain. D, Confocal images displaying markers of the mature mDA neurons NURR1, GIRK2, and Calbindin. Scale bar, 25 μm. E, TEM images of cross-sectional and longitudinal-sectional views of an axon bundle in the marginal zone and adherent junctions (arrows) in the ventricular zone of DAC3.0 MOs (2WM). Scale bar, 2 μm. F, Levels of dopamine produced by DAC3.0 MOs were measured by high-performance liquid chromatography (HPLC) 7 days after differentiation (12WM, and 20WM). *P < .05. G, Representative traces of multiple action potentials (Aps) recorded from neurons in DAC3.0 MOs (8WM), evoked by current injection. H, Accumulation of neuromelanin-like granules in DAC3.0 MOs was observed at 10WM. Pigmented cytoplasm of neurons (arrows) and neuromelanin-like aggregates (arrowheads) are shown in DAC3.0 MOs. Scale bar, 20 μm. I, Fontana-Masson staining of DAC3.0 MOs. Cerebral organoid was used as a negative control. Scale bar, 50 μm. J, TEM images displaying the fine structure of neuromelanin-like granules (arrowheads) in DAC3.0 MOs. Scale bar, 1 μm. 4WM, 4 weeks of midbrain maturation; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TEM, transmission electron microscopy To investigate the functionality of DAC3.0 MOs, we assessed the production of dopamine. DAC3.0 MOs began producing dopamine at 12WM, with the highest dopamine level found at 20WM (Figure 4F). Moreover, DAC3.0 MOs were found to be electrically active, as evidenced by the action potential elicited by neurons in DAC3.0 MOs (Figure 4G; Figure S4F,G). Notably, neuromelanin-like granules, which accumulate in the substantia nigra pars compacta in humans, were found to accumulate homogeneously in DAC3.0 MOs (Figure 4H). Fontana-Masson staining showed that neuromelanin-like granules accumulate in the outer layer of DAC3.0 MOs, an area enriched for functional mDA neurons, but not in COs that had maturated during the same periods (Figure 4I), suggesting that mDA neurons from DAC3.0 MOs might produce neuromelanin-like granules. TEM analysis revealed the presence of neuromelanin-like granules in DAC3.0 MOs (Figure 4J; Figure S4H). Collectively, our data indicate that DAC3.0 MOs are as structurally and functionally mature as the developing midbrain. DAC3.0 MOs display cell type composition similar to that of midbrain Midbrain contains not only mDA neurons, a functional cell type representative of the midbrain,47 but also several other cell types, such as different neuronal subtypes, astrocytes, and oligodendrocytes,48 which exert roles in maintaining the functional homeostasis of the midbrain.49-51 Thus, we next investigated the cellular composition of DAC3.0 MOs. We found that both GABAergic and mDA neurons are intimately colocated in most of the rosettes (Figure 5A), like in midbrain, where GABAergic neurons closely interact with mDA neurons in the substantia nigra, ventral tegmental area, and rostromedial tegmental nucleus.50 In contrast, DAC0 MOs (0 μM CHIR99021) and NS-MOs (0.8 μM CHIR99021) exhibited heterogeneous distribution of GABAergic neurons. Indeed, most of the rosettes in NS-MOs produced exclusively either TH- or GAD67-positive neurons (Figure S5A) and most of the rosettes in DAC0 MOs produced mainly GABAergic neurons (Figure S5B). Moreover, we found glutamatergic neurons in DAC3.0 MOs (Figure 5B; Figure S5C). Figure 5 Open in new tabDownload slide In vivo-like cell type composition of DAC3.0 MOs. A, Confocal images representing neural lobes containing TH-positive mDA neurons and GAD67-positive GABAergic neurons in DAC3.0 MOs (8WM). Scale bar, 100 μm. B, A magnified view of GABA-positive GABAergic neurons and GLUT-positive glutamatergic neurons in DAC3.0 MOs (8WM). Scale bar, 25 μm. C, Heatmaps representing the expression patterns of markers related to astrocytes and oligodendrocytes in DAC3.0 MOs (3WM, 8WM, and 20WM). Color bar at the left bottom indicates gene expression in log2 scale. Red and green colors represent higher and lower expression levels, respectively. D, Confocal images showing the global distribution of astrocytes expressing GFAP in DAC3.0 MOs (8WM). Scale bar, 100 μm. E, Percentage of GFAP-positive astrocytes in DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. F, Global distribution of oligodendrocytes expressing MBP in DAC3.0 MOs (8WM). Scale bar, 100 μm. G, Percentage of MBP-positive oligodendrocytes in DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. H, Representative image of astrocytes expressing GFAP/S100β (left panel) and morphology of single astrocyte (right panel). Scale bar, 25 μm. I, TEM images displaying direct connection between astrocyte containing glycogen granules and neuronal axon (Ax). Scale bar, 2 μm. J, Morphology of oligodendrocytes expressing MBP located close to TH-positive mDA neurons. Scale bar, 25 μm. K, TEM images showing myelinating oligodendrocytes in DAC3.0 MOs. Scale bar, 2 μm. 8WM, 8 weeks of midbrain maturation; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TEM, transmission electron microscopy Figure 5 Open in new tabDownload slide In vivo-like cell type composition of DAC3.0 MOs. A, Confocal images representing neural lobes containing TH-positive mDA neurons and GAD67-positive GABAergic neurons in DAC3.0 MOs (8WM). Scale bar, 100 μm. B, A magnified view of GABA-positive GABAergic neurons and GLUT-positive glutamatergic neurons in DAC3.0 MOs (8WM). Scale bar, 25 μm. C, Heatmaps representing the expression patterns of markers related to astrocytes and oligodendrocytes in DAC3.0 MOs (3WM, 8WM, and 20WM). Color bar at the left bottom indicates gene expression in log2 scale. Red and green colors represent higher and lower expression levels, respectively. D, Confocal images showing the global distribution of astrocytes expressing GFAP in DAC3.0 MOs (8WM). Scale bar, 100 μm. E, Percentage of GFAP-positive astrocytes in DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. F, Global distribution of oligodendrocytes expressing MBP in DAC3.0 MOs (8WM). Scale bar, 100 μm. G, Percentage of MBP-positive oligodendrocytes in DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. H, Representative image of astrocytes expressing GFAP/S100β (left panel) and morphology of single astrocyte (right panel). Scale bar, 25 μm. I, TEM images displaying direct connection between astrocyte containing glycogen granules and neuronal axon (Ax). Scale bar, 2 μm. J, Morphology of oligodendrocytes expressing MBP located close to TH-positive mDA neurons. Scale bar, 25 μm. K, TEM images showing myelinating oligodendrocytes in DAC3.0 MOs. Scale bar, 2 μm. 8WM, 8 weeks of midbrain maturation; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; mDA, midbrain dopaminergic; MOs, midbrain-like organoids; TEM, transmission electron microscopy Although a previous study had described the generation of functionally mature MOs, as shown by the MOs' mature electrophysiological ability and production of neuromelanin-like granules,36 the presence of mature glial cell types such as astrocytes and oligodendrocytes in MOs has been poorly described.52 First, we performed RNA-seq analysis and found that both astrocyte and oligodendrocyte markers were highly enriched in mature DAC3.0 MOs (Figure 5C; Figure S5E,F). Notably, neuronal genes were activated earlier in DAC3.0 MOs than were markers for both astrocytes and oligodendrocytes (Figure 5C; Figure S5D), as in the developing midbrain, where neurogenesis precedes gliogenesis.53 Moreover, we observed numerous glial cells in our mature DAC 3.0 MOs. After 8WM, we detected both astrocytes (Figure 5D,E) and oligodendrocytes (Figure 5F,G) expressing multiple cell type-specific markers (Figure S6A,B). DAC 3.0 MOs also exhibited morphology typical of both astrocytes (Figure 5H; Figure S6C) and oligodendrocytes (Figure 5J; Figure S6E). Our TEM analysis clearly showed functional astrocytes containing glycogen granules in contact with neuronal axons (Figure 5I; Figure S6D). Moreover, we found oligodendrocytes that show myelination, like their in vivo counterparts (Figure 5K; Figure S6F). Taken together, our data indicate that DAC3.0 MOs have a similar cellular composition to the midbrain, with multiple functional cells types. DAC3.0 MOs with functional astrocytes facilitate dopaminergic neurotoxin-based in vitro modeling of PD PD, a prevalent neurodegenerative disease,54 is caused primarily by the death of mDA neurons.55 However, the mechanisms underlying this selective cell death in the brain of patients with PD remain largely unknown. To replicate the neuropathology of PD, many previous studies had used animal models with a variety of dopaminergic toxins.56 Among them, MPTP is known to be the most reliable and frequently used toxin due to its ability to stably induce clinical symptoms that are indistinguishable from PD.57,, 58 After crossing the blood-brain barrier, MPTP is converted first into 1-methyl-4-phenyl-2,3-dihydropyridinium by monoamine oxidase B and then into 1-methyl-4-phenylpyridinium (MPP+) in astrocytes.58 The MPP+ released into the extracellular space could bind to the dopamine transporter and gain entry into mDA neurons.59 As the concentration of MPP+ in the mitochondria increases,60 oxygen-free radicals are produced and ATP synthesis decreases.61,, 62 Since astrocytes play a key role in the mode of action of MPTP,58,, 63 MPTP, a representative and reliable dopaminergic neurotoxin, has not been properly used in previous iPSC-based in vitro modeling studies of PD. Instead, previous studies64-66 have treated iPSC-derived mDA neurons with MPP+, the activated form of MPTP, to evaluate the toxic effect of MPTP, as neuron-to-glial cell interactions cannot be replicated by 2D differentiation technology. As DAC3.0 MOs contain a large number of glial cells, such as astrocytes, we next investigated whether DAC3.0 MOs could replicate the neuropathology of PD upon treatment with MPTP. To this end, we treated DAC3.0 MOs (8WM) with four different concentrations of MPTP (0, 10, 50, and 100 μM) for 48 hours and assessed MPTP-mediated cell death (Figure 6A). MPTP-treated DAC3.0 MOs exhibited massive cell death, as evidenced by the increased numbers of both Caspase-3- and TUNEL-positive cells (Figure 6B,C). Moreover, the number of apoptotic cells increased in a dose-dependent manner (Figure 6D,E), indicating that the cell death induced in DAC3.0 MOs is mediated solely by MPTP treatment. Notably, MPTP-mediated cell death is observed largely in TH-positive mDA neurons (Figure 6F; Figure S7A) but rarely in other cellular components, such as GABAergic neurons, astrocytes, and oligodendrocytes (Figure 6F; Figure S7B). Taken together, our data indicate that DAC3.0 MOs produced by our optimized protocol contain functional glial cells that facilitate the action of MPTP, a representative dopaminergic neurotoxin, which could be used for the in vitro modeling of PD. Figure 6 Open in new tabDownload slide MPTP-based in vitro modeling of Parkinson's disease using DAC3.0 MOs. A, Schematic illustration of MPTP assay using DAC3.0 MOs. B and C, Confocal images showing Caspase-3 expression, B, and the presence of TUNEL-labeled mDA neurons, C, in MPTP-treated DAC3.0 MOs (0, 10, 50, and 100 μM). Scale bar, 100 μm. D and E, Percentage of Caspase-3-positive, D, and TUNEL-positive, E, mDA neurons in MPTP-treated DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. *P < .05, **P < .01, ***P < .001. F, mDA neuron-specific cell death by MPTP treatment. TUNEL-positive cells were barely observed in GABAergic neurons (GABA), astrocytes (AQP4), and oligodendrocytes (PLP). Scale bar, 25 μm. mDA, midbrain dopaminergic; MOs, midbrain-like organoids; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Figure 6 Open in new tabDownload slide MPTP-based in vitro modeling of Parkinson's disease using DAC3.0 MOs. A, Schematic illustration of MPTP assay using DAC3.0 MOs. B and C, Confocal images showing Caspase-3 expression, B, and the presence of TUNEL-labeled mDA neurons, C, in MPTP-treated DAC3.0 MOs (0, 10, 50, and 100 μM). Scale bar, 100 μm. D and E, Percentage of Caspase-3-positive, D, and TUNEL-positive, E, mDA neurons in MPTP-treated DAC3.0 MOs. Data are presented as mean ± SD of triplicate values. *P < .05, **P < .01, ***P < .001. F, mDA neuron-specific cell death by MPTP treatment. TUNEL-positive cells were barely observed in GABAergic neurons (GABA), astrocytes (AQP4), and oligodendrocytes (PLP). Scale bar, 25 μm. mDA, midbrain dopaminergic; MOs, midbrain-like organoids; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine DISCUSSION Our understanding of human diseases depends mainly on pathological outputs of internal organs, without having a fundamental understanding of the underlying etiology. Emphasis has been placed on establishing in vitro disease modeling systems that could effectively reproduce the disease pathophysiology. iPSC technology has provided a novel concept for patient-specific disease modeling. However, current 2D differentiation protocols are insufficient for closely replicating in vivo situations because 2D conditions typically produce a singular differentiated target cell type without forming the in vivo-like tissue architecture composed of multiple cellular components. Recent organoid technology has opened up a new avenue for in vitro modeling of various diseases and understanding early development even at the organ level. As organoids theoretically contain multiple cell types with a similar structure to their in vivo counterparts, the potential exists to achieve more precise and reliable in vitro disease modeling for various diseases. Indeed, in many cases, the cause and progression of neurodegenerative diseases are mediated by multiple cellular components in organs.67,, 68 However, it has not been described whether organoids from patients could sufficiently mimic the in vivo disease pathology, which is caused by tight crosstalk between distinct cellular components. In the current study, we describe the generation of DAC3.0 MOs that exhibit structural and functional similarities to the midbrain, in vivo, as evidenced by their laminated structures (Figure 4A,B), homogeneous distribution of mature mDA neurons (Figures 3H and 4D), production of dopamine (Figure 4F), electrophysiological activity (Figure 4G), and production of neuromelanin-like granules (Figure 4H-J). Most importantly, DAC3.0 MOs display midbrain-like cellular makeup with multiple functional cells types (Figure 5; Figures S5 and S6), such as astrocytes and oligodendrocytes. Therefore, to address whether DAC3.0 MOs can recapitulate the phenotype of PD mediated by crosstalk between distinct cellular components, we treated these organoids with MPTP, a dopaminergic neurotoxin that causes the selective cell death of mDA neurons via functional astrocytes.58,, 63 Upon treatment of DAC3.0 MOs with MPTP, we observed that mDA neurons underwent cell death, indicating that brain organoid technology could even recapitulate in vivo pathophysiology mediated by cell-to-cell interactions—that is, between astrocytes and mDA neurons. Organoid technology presents an advanced concept of disease modeling and drug screening, as it could replicate the in vivo scenario at higher resolution compared with conventional 2D differentiation methods. However, the low generation efficiency, heterogeneous structures, and biased or limited cellular compositions of the resultant organoids have been critical hurdles for the clinical translation of organoid technology. To overcome these issues, we first tried to optimize the early mesencephalon specification step by comparing distinct combinations of dual SMAD inhibitors. Moreover, by creating an in vitro WNT gradient, we fine-mapped specific concentrations of WNT that facilitate the generation of structurally and functionally homogeneous MOs (DAC3.0 MOs). Further efforts for generating DAC3.0 MOs in a high-throughput drug screening platform are urgently required for the clinical translation of organoid technology. Our optimized protocol for robustly producing homogeneous MOs might be useful for the precise in vitro modeling of PD and the efficient screening of drug candidates for PD. CONCLUSION In the current study, we described a novel strategy for the robust generation of homogeneous MOs, namely DAC3.0 MOs using a specific combination of dual SMAD inhibitors and in vitro WNT gradient. DAC3.0 MOs exhibit structural and functional similarities to the midbrain, in vivo, as shown by their laminated structures, homogeneous distribution of mature mDA neurons, robust production of neuromelanin-like granules, and midbrain-like cellular makeup with functional glial cells. Moreover, DAC3.0 MOs can recapitulate in vivo pathophysiology of PD mediated by crosstalk between distinct cellular components. ACKNOWLEDGMENTS This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (NRF-2016R1A2B3011860, NRF-2017M3C7A1047640) and by a grant from the Next-Generation BioGreen 21 Program (PJ01322101), Rural Development Administration, Republic of Korea. CONFLICT OF INTEREST The authors declared no potential conflicts of interest. AUTHOR CONTRIBUTIONS T.H.K.: conception and design, collection and/or assembly of data; J.H.K., S.H., J. Kim, C.P., J.-H.L., H.K.R., J.E.N, J. 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes Funding information National Research Foundation of Korea, Grant/Award Numbers: NRF-2016R1A2B3011860, NRF-2017M3C7A1047640; Rural Development Administration, Grant/Award Number: PJ01322101 ©AlphaMed Press 2020 This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Generation of homogeneous midbrain organoids with in vivo-like cellular composition facilitates neurotoxin-based Parkinson's disease modeling
JO - Stem Cells
DO - 10.1002/stem.3163
DA - 2020-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/generation-of-homogeneous-midbrain-organoids-with-in-vivo-like-01aIMz8Hzj
SP - 727
EP - 740
VL - 38
IS - 6
DP - DeepDyve
ER -