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. Author manuscript; available in PMC: 2024 Apr 1.
Published in final edited form as: Exp Neurol. 2023 Jan 18;362:114325. doi: 10.1016/j.expneurol.2023.114325

Ethanol exposure disrupted the formation of radial glial processes and impaired the generation and migration of outer radial glial cells in forebrain organoids derived from human embryonic stem cells

Lanhai Lü 1,2,3, Fuqiang Yuan 1,2, Huadong Fan 1,2, Yihong Li 1,2, Jie Liu 1,2, Wenke Feng 1,2,3, Huang-ge Zhang 4,5, Shao-yu Chen 1,2
PMCID: PMC9992138  NIHMSID: NIHMS1867144  PMID: 36669750

Abstract

Radial glial cells (RGCs) play a pivotal role in cerebral cortical development by functioning as a source of new neurons and by supporting the migration of newborn neurons. These functions are primarily dependent on the apical-basolateral structures of radial glial processes. This study aims to investigate the effects of ethanol exposure on the development of radial glial processes and the generation, migration, and transformation of outer radial glial cells (oRGCs). For this purpose, forebrain organoids were developed from human embryonic stem cells. These forebrain organoids contain abundant neural progenitor cells (SOX2+), express high levels of neural epithelial markers β-catenin and PKCλ, and dorsal forebrain marker PAX6, and display well-organized cortical architectures containing abundant apical and basal RGCs, intermediate progenitors (IPCs), and neurons. Exposure of forebrain organoids to ethanol resulted in a significant increase in apoptosis in Nestin-positive radial glial cells. Ethanol exposure also remarkably decreased the levels of radial glial process-associated proteins, including Nestin, GFAP, and Vimentin, in radial glial cells and distinctly impaired the integrity and morphologies of radial glial processes. In addition, the ethanol-induced impairment of the radial glial processes is associated with decreased migration and proliferation of radial glial cells, reduction in the generation of HOPX+ oRGCs, and the accelerated transformation of oRGCs into astrocytes. These results demonstrate that ethanol exposure can disrupt cerebral cortex development by impairing the formation of radial glial processes and the generation, migration, and transformation of oRGCs.

Keywords: Ethanol, forebrain organoids, outer radial glial cells, cortex, fetal alcohol spectrum disorders

1. Introduction

Alcohol is one of the most common teratogens (Georgieff et al. 2018). Prenatal alcohol exposure can cause a broad spectrum of abnormalities, such as severe growth delay, facial dysmorphology, and brain damage that results in cognitive or behavioral deficits (Georgieff et al. 2018). Among the defects induced by prenatal ethanol exposure is microcephaly, a neurodevelopmental disorder characterized by markedly reduced brain size. Ethanol-induced impairment in neural stem cells, notably radial glial cells (RGCs), and subsequent defects in cortical development may contribute to microcephaly induced by prenatal ethanol exposure.

RGCs are essential for cortical neurogenesis. They can serve as neural progenitor cells and provide supportive scaffoldings for neuronal migration toward the cortical regions. According to their localization and morphologies, RGCs subdivide into two subpopulations: ventricular radial glial cells (vRGCs, also called apical radial glial cells, aRGCs) and outer radial glial cells (oRGCs, also called basal radial glial cells, bRGCs) (Ostrem et al. 2017; Penisson et al. 2019). RGCs retain some characteristics of neuroepithelial cells after they are produced, such as apical-basal polarity shown by the apical and basal process, and acquire some glial features, such as the expressions of glial markers glutamate aspartate transporter (GLAST) and glial fibrillary acidic protein (GFAP). In the human cortex, the subventricular zone (SVZ) is split into two regions by an inner fiber layer (IFL): an inner SVZ (iSVZ) and an outer SVZ (oSVZ). oRGCs, which originate from vRGCs, mainly reside in the oSVZ. oRGCs share some molecular features of vRGCs, including the expressions of radial glial markers, such as Nestin (NES), vimentin (VIM), and GFAP, polarized morphologies, and progeny (Ostrem et al. 2017). vRGCs and oRGCs show unique morphological characteristics. While vRGCs are bipolar cells with an apical process contacting the ventricular surface and a basal fiber reaching the pial surface, oRGCs possess a basal process similar to that of vRGCs but with or without an apical process (Nowakowski et al. 2016). Despite the differences in morphologies, both vRGCs and oRGCs can support the migration of newborn neurons through the basal process to reach the appropriate positions within the cortical plate (Nowakowski et al. 2016). Therefore, the structures of radial glial processes are essential for proper neuronal migration and the formation of cortical layers (Poluch and Juliano 2007). A number of studies confirmed that impairments of radial glial fiber contribute to several diseases, such as double cortex (Yamamoto et al. 2015), heterotopia (O’Neill et al. 2018), and autism (Pearson et al. 2020). Several studies have also demonstrated that prenatal alcohol exposure can impair radial glial cells and cortical development (Robertson et al. 2016; Yang et al. 2012; Ozturk et al. 2017; Zhang et al. 2014; Aronne et al. 2008; Sogut et al. 2017; Peng et al. 2004). However, due to the limited access to human embryonic tissues and ethical concerns, most studies that investigated the role of radial glial cells in ethanol-induced neurotoxicity have been conducted in animal models, human postmortem fetal brains, or retrospective surveys of autopsies.

With the development of stem cell biology and in vitro 3D culture techniques, brain organoids provide us with a powerful in vitro tool to study early human brain development (Lancaster et al. 2013; Qian et al. 2018; Qian et al. 2016). Using self-organizing and differentiation potentials, hiPSCs or hESCs cells can aggregate into 3D discrete and interdependent complex structures reminiscent of early to the mid-gestational human developing cortex. With the applications of patterning growth factors and the defined starting number of stem cells, brain organoids become more homologous and highly producible brain tissues. Forebrain organoids are brain region-specific organoids that can recapitulate critical features of human forebrain development, including neural progenitor proliferation, cell division, migration, and subsequent histological features (Qian et al. 2018; Qian et al. 2016). Forebrain organoids contain several organized cortical structures, including the ventricular zone (VZ), subventricular zone (SVZ), and cortical plate (CP), recapitulating the developing human cortex (Qian et al. 2018). Distinctly, forebrain organoids using SpinΩ bioreactors exhibit a well-developed oSVZ-like region containing abundant oRGCs, which is a distinct feature of the developing human cortex (Qian et al. 2018; Qian et al. 2016). Hence, forebrain organoids provide a reliable 3D platform for studying the early developing human cortex, notably radial glial cells, and modeling several neurological disorders, such as microcephaly (Lancaster et al. 2013; Qian et al. 2016), Lissencephaly (Bershteyn et al. 2017), and Miller-Dieker Syndrome (Iefremova et al. 2017).

In this study, forebrain organoids generated from human embryonic stem cells using SpinΩ bioreactors were used to investigate the effects of ethanol exposure on the development of radial glial processes and the generation, migration, and transformation of oRGCs. We found that exposure of forebrain organoids to ethanol resulted in a significant increase in apoptosis in radial glial cells and remarkably decreased the levels of radial glial process-associated proteins in RGCs, and distinctly impaired the integrity and morphologies of radial glial processes. In addition, the ethanol-induced impairment of the radial glial processes is associated with decreased migration and proliferation of RGCs, reduced generation of oRGCs, and the accelerated transformation of oRGCs into astrocytes. These results demonstrate that ethanol exposure can disrupt cerebral cortex development by impairing the formation of radial glial processes and the generation, migration, and transformation of oRGCs.

2. Materials and methods

2.1. Maintenance of human embryonic stem cells (hESCs)

Human embryonic stem cells (hESCs, W09 cell line) were purchased from WiCell Research Institute Inc (Madison, WI, USA). hESCs were maintained in mTeSR™1 medium (85850, STEMCELL Technologies, MA, USA) using Matrigel-coated six-well plates (BD354277, Corning, NY, USA). The colonies were fed daily with fresh mTeSR™1 medium and maintained at 37°C in humidified air with 5% CO2. hESCs were passaged every five to six days and split at the ratio of 1:3 to 1:5 for subculture. For the subculture of hESCs, cells were incubated with a gentle cell dissociation reagent (07174, STEMCELL Technologies, MA, USA) for 5 min and manually scraped using a 5 ml pipette to generate cell aggregates. The aggregates were collected and centrifuged. After removing the supernatant, cells were resuspended in fresh mTeSR™1 medium and replated onto Matrigel−coated six-well plates.

2.2. Generation of forebrain organoids from hESCs

The generation of forebrain organoids was performed as described by Qian et al. (Qian et al. 2018; Qian et al. 2016) with modifications. On day 0, hESCs were dissociated with a gentle cell dissociation reagent (07174, STEMCELL Technologies, MA, USA) to generate single cells. The cells were centrifuged at 300g for 5 min. and then resuspended in fresh embryoid bodies (EBs) formation medium [DMEM/F12, 20% KnockOut Serum Replacement (10828028, KOSR, Thermo Fisher Scientific, CA, USA), 1× GlutaMAX (35050061, Thermo Fisher Scientific, CA, USA), 1×MEM-NEAA (MEM Non-Essential Amino Acids Solution, 11140050, Thermo Fisher Scientific, CA, USA), 1×2-Mercaptoethanol (β-ME, O3446I Fisher Scientific, MA, USA), 1×Penicillin/Streptomycin (15140122, Thermo Fisher Scientific, CA, USA) plus 2 μM Dorsomorphin (P5499, Sigma-Aldrich, MO, USA) and 2 μM A 83–01 (2939, Tocris Bioscience, MN, USA) and 10 μM Y-27632 (72234, STEMCELL Technologies, MA, USA)]. 9,000 cells were plated into each well of Ultra-low attachment 96 well microplate (89089–826, VWR, GA, USA) in 100 μl EBs formation medium. After 48 hrs, half of the medium from each well was replaced with fresh EBs formation medium without Y-27632. On day 5 – 6, half of the medium was replaced with a freshly prepared neural induction medium consisting of DMEM: F12, 1× N2 Supplement (17502048, Thermo Fisher Scientific, CA, USA), 10 μg/ml Heparin (375095, Sigma, CA, USA), 1× Penicillin/Streptomycin (15140122, Thermo Fisher Scientific, CA, USA), 1× Non-essential Amino Acids (11140–050, Thermo Fisher Scientific, CA, USA), 1× Glutamax (10565–042, Thermo Fisher Scientific, CA, USA), 4 ng/ml WNT-3A (5036-WN-010, R&D Systems, MN, USA), 1 μM CHIR99021 (72054, STEMCELL Technologies, MA, USA), and 1 μM SB-431542 (1614, Tocris Bioscience, MN, USA). On day 7, organoids were embedded in Matrigel (354234, Corning™, NY, USA) and cultured for additional 6 days. On day 14, 10 – 20 matrigel embedded organoids were transferred to each well of a 12-well spinning bioreactor (SpinΩ) containing the differentiation medium consisting of DMEM: F12, 1× N2 and B27 supplements plus vitamin A (17504044, Thermo Fisher Scientific, CA, USA), 1× Penicillin/Streptomycin, 1× 2-Mercaptoenthanol, 1× Non-essential Amino Acids, 2.5 μg/ml Insulin (I0516, Sigma-Aldrich, MO, USA). On day 71, the differentiation medium was replaced with the maturation medium consisting of Neurobasal medium (21103049, Thermo Fisher Scientific, CA, USA), 1× B27 Supplement plus vitamin A, 1× Penicillin/Streptomycin, 1× 2-Mercaptoenthanol, 0.2 mM L-Ascorbic acid (A92902, Sigma-Aldrich, MO, USA), 20 ng/ml BDNF (78005, STEMCELL Technologies, MA, USA), 20 ng/ml GDNF (78058, STEMCELL Technologies, MA, USA), 1 ng/ml TGFβ (50036, Abcam, CA, USA), and 0.5 mM cAMP (Adenosine 3′,5′-cyclic monophosphate sodium salt monohydrate, A6885, Sigma-Aldrich, MO, USA). Some organoids were cultured in the maturation medium until day 84. All media were changed every other day.

2.3. Ethanol treatment

For ethanol treatment, forebrain organoids on day14 were exposed to 50 mM ethanol for 24 hrs. For the assessment of the short-term effects of ethanol, the organoids were collected immediately following the ethanol exposure, washed three times with 1× cold Dulbecco’s phosphate-buffered saline (DPBS), and fixed by 4% paraformaldehyde (PFA) in 1× DPBS buffer for further analysis. For the assessment of the long-term effects of ethanol, the forebrain organoids from the same ethanol-exposed batch were washed three times with a freshly prepared organoid differentiation medium and then transferred into SpinΩ bioreactors for cultures in the organoid differentiation medium or maturation medium. Samples were collected at indicated time points for analysis.

2.4. Tissue preparation and immunostaining

Forebrain organoids collected at indicated time points were washed three times with 1× cold Dulbecco’s DPBS, then fixed in 4% PFA in DBPS overnight at 4°C. Then organoids were washed with 1× DPBS three times and incubated overnight in a 30% sucrose-DPBS solution. After sedimentation, organoids were embedded in Tissue-Tek® OCT Compound (27050, Ted Pella, Inc.CA, U.S.A.), frozen at −20°C, sectioned at 20 μm with a cryostat (Leica CM3050S, Leica Microsystems Inc, IL, USA), and collected onto Superfrost Plus slides (22–037-246, Fisher Scientific, MA, USA). For immunohistochemistry, sections were blocked and permeabilized in 0.25% Triton X-100 and 10% donkey and goat serum in 1× DPBS. Sections were then incubated with the following primary antibodies, respectively, in 0.1% Triton X-100, 10% donkey and goat serum: rabbit anti-SOX2 (1:200, 23064, Cell Signaling Technology, MA, USA), mouse anti-TUJ1 (1:200, MMS-435P, Covance, NJ, USA), mouse anti-PAX6 (1:200, MA1–109, Thermo Fisher Scientific, MA, USA), mouse anti-Nestin (1:200, MA1–110, Thermo Fisher Scientific, MA, USA), rabbit anti-Nestin (1:200, ab92391, Abcam, MA, USA), rabbit anti-TBR2 (1:200, AB9618, Abcam, MA, USA), rat anti-CTIP2 (1:100, ab18465, Abcam, MA, USA), rabbit anti-β-Catenin (1:200, 9562, Cell Signaling Technology, MA, USA), mouse anti-PKCλ (1:200, 610207, BD Biosciences, CA, USA), rabbit anti-Hopx (1:200, 230544, Abcam, MA, USA), mouse anti-Hop (E-1) (HOPX, 1:200, sc-398703, Santa Cruz Biotechnology, CA, USA), mouse anti-S100β (1:200, S2532, Sigma-Aldrich, MO, USA), mouse anti-Ki-67 (1:200, MAB4190, Sigma-Aldrich, MO, USA), rabbit anti-cleaved Caspase-3 (1:200, 9661, Cell Signaling Technology, MA, USA), mouse anti-GFAP (1:200, G3893, Sigma-Aldrich, MO, USA). The sections were then incubated with the following secondary antibodies: Alexa Fluor 488, 594, or 647 -conjugated donkey anti-goat, -rabbit, -rat, or -mouse IgG (all used at 1:200 dilution, Invitrogen, CA, USA), respectively. Nuclei were stained by incubating the sections with 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:2000, CAS28718–90-3, Santa Cruz Biotechnology, CA, USA) in 1× DPBS for 10 min. After three washes with 1× DPBS, sections were mounted in anti-fade fluorescence mounting medium (ab104135, Abcam, MA, USA) and sealed by coverGrip™ coverslip sealant (Biotium, 23005). Images were captured by a Nikon A1R laser scanning confocal microscope (NY, USA) and analyzed by ImageJ 1.6.0 (NIH, USA).

2.5. BrdU pulse-chase labeling analysis

For BrdU (5-bromo-2’-deoxyuridine) pulse-chase labeling analysis, forebrain organoids from control and ethanol-treated groups were incubated with 10 μM BrdU (ab142567, Abcam, CA, USA) diluted with the forebrain organoid induction medium for 2 h. The organoid differentiation medium containing BrdU was removed, and forebrain organoids were washed three times with a fresh differentiation medium and then cultured in a fresh differentiation medium. The forebrain organoids were collected on days 2 or 7, fixed in 4% paraformaldehyde, cryoprotected by 30% sucrose-PBS solution overnight, and embedded in OCT Compound (Sakura Finetek, CA, USA). Frozen sections were cut into 15 μm, and BrdU labeling was detected by double immunostaining using the mouse anti-BrdU antibody (ab8152, Abcam, CA, USA) and rabbit anti-Nestin antibody (1:200, ab92391, Abcam, CA, USA) or rabbit anti-HOPX antibody (PA5–72855, Thermo Fisher Scientific, MA USA) as described above.

2.6. EdU cell proliferation assay

Forebrain organoids were cultured in the differentiation medium containing 10 μM 5-ethynyl-2′-deoxyuridine (EdU) with or without 50 mM ethanol for 24 hrs. After the forebrain organoids were washed with the fresh differentiation medium, forebrain organoids were cultured in the fresh differentiation medium until day 56 for analysis of the birth dating of outer radial glial cells. EdU was detected by using Click-iT™ Plus EdU Alexa Fluor™ Imaging Kit (C10339, Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. For the analysis of birthdated HOPX+ outer radial glial cells from EdU+ radial glial cells, the immunostaining of HOPX was performed using mouse anti-Hop (E-1) antibody (HOPX, 1:200, sc-398703, Santa Cruz Biotechnology, CA, USA). DAPI (1:2000, CAS28718–90-3, Santa Cruz Biotechnology, CA, USA) was used to stain nuclei to determine the total cells.

2.7. Statistical analysis

Statistical analyses were performed by using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). At least five forebrain organoids were included in each group. All experiments were replicated 3– 5 times. All data were expressed as means ± SEM of at least three separate experiments. Student’s t-test was used to compare the difference between groups. Differences between groups were considered significant at p < 0.05, p < 0.01 or p < 0.001.

3. Results

3.1. Generation and characterizations of hESCs-derived forebrain organoids

As described in the Methods section, to generate forebrain organoids, we first generated EBs from hESCs. As shown in Fig. 1A, the EBs generated from hESCs exhibited uniform sizes. On day 7, the EBs embedded in Matrigel were transferred into the low attachment microplate for the induction of neuroepithelium and the generation of forebrain organoids. Forebrain organoids were then transferred into SpinΩ bioreactors on day 14 and cultured for organoid differentiation and maturation. Immunofluorescence analysis of the expression of markers for forebrain neural progenitor and adherens junctions of neuroepithelial cells revealed that the organoids on day 14 contained many pseudostratified ventricular zone (VZ)-like structures, which are reminiscent of the human neuroepithelial tube. The stratified neuroepithelium-like structures were populated by a nearly pure population of SOX2+ NESTIN+ neural progenitor cells (Fig. 1B). We also found that those neural progenitors expressed high levels of markers for the adherens junctions of neuroepithelial cells, β-CATENIN and PKCλ (Fig. 1C). In addition, forebrain-specific progenitor marker PAX6 was expressed highly in forebrain organoids on days 14 and 28 (Fig. 1D). These results indicated that the forebrain organoids derived from hESCs displayed well-defined polarized neuroepithelial tube-like structures and contained abundant neural progenitor cells and neuroepithelial cells before subsequent differentiation.

Figure 1. Generations and characterizations of hESCs-derived forebrain organoids.

Figure 1.

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(A) Schematic diagram of the protocol for the generation of forebrain organoids from hESCs. Representative stages of forebrain organoids are shown as bright-field images. Scale bar = 500 μm. (B) Representative images of immunostaining of neural progenitor markers SOX2 and NESTIN in day 14 forebrain organoids that contain neuroepithelium-like structures with a nearly pure population of SOX2+NESTIN+ NPCs. Scale bar =50 μm. (C) Representative images of immunostaining of the markers for adherens junctions of neuroepithelial cells β-catenin and PKCλ. Neural progenitor cells in individual neuroepithelium-like structures expressed high adherens junctions markers β-catenin and PKCλ, and displayed well-defined polarized morphologies resembling neuroepithelial tube-like structures in day 14 forebrain organoids. Scale bar = 50 μm. (D) Representative images of immunostaining of neural progenitor marker SOX2 and forebrain marker PAX6 in day 14 and day 18 forebrain organoids. Nuclei were stained by DAPI (blue). Scale bar = 50 μm.

3.2. Forebrain organoids recapitulate cortical organizations and the development of outer radial glial cells

We used immunostaining and morphological analysis to assess the development of the cortical layers and radial glial cells in forebrain organoids. To characterize the developmental dynamics, we performed the immunostainings of forebrain organoids on days 28, 56, and 84. Our data showed that forebrain organoids exhibited well-defined VZ-like structures with densely packed SOX2+ neural progenitor cells on days 28 and 56 and VZ-like structures with less dense SOX2+ neural progenitor cells on day 84. In addition to containing abundant SOX2+ neural progenitor cells, forebrain organoids on day 28 also had a mixture of TBR2+ intermediate progenitor cells (IPs) and CTIP2+ deep-layer neurons that dominate outside VZ-like regions, reminiscent of the pre-plate (PP) in the early human forebrain (Fig. 2A). On day 56, the SOX2+ neural progenitors were organized into two distinct proliferation compartments: the ventricular zone (VZ) and the subventricular zone (SVZ), which were mainly composed of SOX2+ neural progenitor cells and TBR2+ intermediate progenitors (IPs) that could give rise to the deep-layer subcortical projection CTIP2+ neurons in the cortical plate (CP) (Fig. 2B). On day 84, there was a thin gap within the SVZ region that splits the SVZ region into two compartments: the inner subventricular zone (iSVZ), which contains SOX2+ neural progenitor cells and TBR2+ IPs, and the enlarged outer subventricular zone (oSVZ), which comprised a thick layer composed of SOX2+ radial glial cells, TBR2+ IPs, and CTIP2+ neurons (Fig. 2C). We also found that oRGCs were enormously expanded from day 28 to day 56, as evidenced by the increased number of HOPX+ oRGCs above the VZ regions (Fig. 2D). In addition, there were many vGLUT+ and GABA+ neurons (Fig. 2E) and s100β+ and GFAP+ astrocytes in forebrain organoids on day 84, indicating that forebrain organoids contained diverse neuronal and astrocytic cell types (Fig. 2F–G). These results indicate that our forebrain organoids display well-organized cortical architectures containing abundant radial glial cells, intermediate progenitors, and neurons and recapitulate the key features of early human forebrain development (Fig. 2H). These forebrain organoids provide us with a novel model to investigate the effects of ethanol on the proliferation, differentiation, and migration of human oRGCs.

Figure 2. Forebrain organoids recapitulate cortical organization and the development of outer radial glial cells.

Figure 2.

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(A–C) Representative images of immunostaining of the markers for neural progenitor cells, SOX2, intermediate progenitor cells, TBR2, and deep-layer neurons, CTIP2 in forebrain organoids on days 28 (A), 56 (B), and 84 (C). White dashed lines demarcate VZ, SVZ, and CP. Yellow dashed lines highlight the gap between iSVZ and oSVZ (C). Scale bar = 50 μm. PP, preplate; VZ, ventricular zone; CP, cortical plate; SVZ, subventricular zone; oSVZ, outer subventricular zone; iSVZ, inner subventricular zone. (D) Representative images of immunostaining of outer radial glial cell marker HOPX on day 28 and day 56 forebrain organoids. Scale bar = 50 μm. (E-G) Representative images of immunostaining of neuronal subtype-specific markers, including glutamatergic marker, VGLUT1 (Green) and GABAergic marker (Red) (E), and astrocytic marker S100β (F) and GFAP (G) in day 84 forebrain organoids. Scale bar = 50 μm. (H) Schematic images of cortical organization in the forebrain organoids from week 4 to week 12 are shown in (H). PP, preplate; VZ, ventricular zone; MZ, marginal zone; CP, cortical plate; SVZ, subventricular zone; oSVZ, outer subventricular zone and iSVZ, inner subventricular zone.

3.3. Exposure of forebrain organoids to ethanol resulted in apoptosis in radial glial cells

To determine the effects of ethanol on forebrain organoids, we first examined whether exposure of forebrain organoids to 50 mM ethanol can induce apoptosis. This ethanol concentration was chosen because our previous study has shown that ethanol at this concentration can induce apoptosis in human embryonic cells (Li et al. 2021) and that a peak maternal blood alcohol concentration of 85 to 105 mM can produce major malformation with the characteristics of FASD in mouse embryos (Dunty et al. 2001; Kotch and Sulik 1992; Sulik et al. 1981). In addition, this is an ethanol concentration that can be observed in chronic alcoholics (Adachi et al. 1991). We found that exposure of organoids on day 14 to 50 mM ethanol for 24 hours resulted in a significant increase in apoptosis, as shown by the dramatically elevated immunostaining of cleaved caspase-3 (Fig. 3A). In addition, nearly 25% of the total number of cells and 30% of nestin+ positive cells were cCaspase-3 positive (Fig. 3B, C), indicating that exposure of forebrain organoids to ethanol can induce apoptosis in nestin+ radial glial cells.

Figure 3. Apoptosis in Nestin+ radial glial cells in day 14 forebrain organoids exposed to ethanol.

Figure 3.

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(A) Representative images of immunostaining of the cleaved Caspase 3 (cCas3) and Nestin in control and ethanol-exposed forebrain organoids. Scale bar = 50 μm. (B, C) Quantitative analysis of the ratio of cCas3+ cells to total cells or to total Nestin+ radial glial cells. Data are expressed as the percentage of cCas3+ cells in total cells (B) or the percentage of cCas3+Nestin+ cells in Nestin+ cells (C) and represent the mean ± SEM of three separate experiments. *p < 0.05, **p < 0.01 vs. control.

3.4. Ethanol exposure impaired the formation of radial glial processes and resulted in the loss of cellular polarity of radial glial cells in forebrain organoids

To determine whether ethanol exposure can impair the formation of radial glial processes, the expression of three proteins, Nestin, Vimentin, and GFAP, which are the major components of radial glial processes, was examined by using immunochemical staining. Nestin is a type VI intermediate filament (IF) protein expressed explicitly in embryonic cortical neural progenitor cells (Xue and Yuan 2010). Vimentin is a type III intermediate filament protein expressed in the transient radial glial cells, and GFAP is a class-III intermediate filament and an astrocyte differentiation marker for radial glial cells (Bramanti et al. 2010). For this study, forebrain organoids were treated with 50 mM ethanol on day 14 for 24 hours. The organoids were collected for the analysis of the expression of these proteins on day 15 (14 + 1), day 28 (14+14), or day 56 (14+42). As shown in Fig. 4A–E, vRGCs at day15 expressed high levels of Nestin, Vimentin, and GFAP. Ethanol exposure significantly decreased the expression of Nestin, Vimentin, and GFAP in vRGCs on day 15. In addition, the reduced expression of Nestin, Vimentin and GFAP was accompanied by a disrupted orientation of the radial glial processes in ethanol-exposed forebrain organoids (Figure 4A–B). Ethanol treatment also remarkably decreased the expression of Nestin in RGCs on day 28 (Fig. 4F–G). As indicated in Fig. 4D, Nestin-positive RGCs displayed parallel radial processes in the control forebrain organoids on day 56. In contrast, Nestin-positive radial glial fibers became shorter or absent in the ethanol group (Fig. 4H). These results indicate that ethanol exposure can impair the formation of radial glial processes and result in the loss of cellular polarity in RGCs, which may impair the migration of neurons.

Figure 4. Ethanol exposure impaired the formation of radial glial processes and resulted in the loss of cellular polarity of radial glial cells in forebrain organoids.

Figure 4.

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(A-B) Representative images of immunostaining of radial glial fiber proteins Nestin (A), GFAP, and Vimentin (B) in ventricular radial glial cells of control forebrain organoids and forebrain organoids exposed to 50 mM ethanol at day 14 for 24 hrs. Scale bar = 20 μm. (C-E) Quantitative analysis of the fluorescence intensity of Nestin, GFAP, and Vimentin in the control and ethanol groups. Data are expressed as fluorescence intensity and represent the mean ± SEM of three separate experiments. * p < 0.05, **p < 0.01 vs. control. (F) Representative images of immunostaining of Nestin in radial glial cells of day 28 forebrain organoids treated with or without 50 mM ethanol at day 14. Scale bar = 10 μm. (G) Quantitative analysis of the fluorescence intensity of Nestin in radial glial cells of day 28 forebrain organoids treated with or without 50 mM ethanol at day 14. Data are expressed as fluorescence intensity and represent the mean ± SEM of three separate experiments. ***p < 0.001 vs. control. (H) Representative images of the morphologies of radial glial fibers in day 56 forebrain organoids treated with or without 50 mM ethanol at day 14. Ethanol exposure resulted in a significant loss of basal process in the outer radial glial cells of forebrain organoids. Scale bar = 20 μm.

3.5. Ethanol exposure impaired the migration and proliferation of outer radial glia cells in forebrain organoids

We next tested whether ethanol-induced aberrant development of radial glial processes can impair the migration and the proliferation of oRGCs in forebrain organoids exposed to ethanol. Using BrdU pulse-chase analysis, we found that exposure of forebrain organoids to 50 mM ethanol on day 14 significantly suppressed the migration of BrdU+ RGCs from the apical membrane to the cortical plate in forebrain organoids on day 21, indicated by the distribution of the BrdU+/Nestin+ cells (Fig. 5A). While many cells in control groups were doubly labeled by both Nestin and BrdU, only a few Nestin+ RGCs were labeled by BrdU in forebrain organoids 7 days following ethanol exposure (on day 21), which indicated that ethanol exposure also impaired the proliferation of Nestin+ RGCs (Fig. 5A). In addition, ethanol exposure remarkably impaired the migration and the proliferation of HOPX+ oRGCs in ethanol-exposed forebrain organoids (Figs. 5B–C). By using EdU incorporation assay, we also evaluated the birth dating and migration of HOPX+ oRGCs in forebrain organoids. As shown in Fig. 5D–F, ethanol exposure on day 14 significantly decreased the numbers of EdU+ cells and HOPX+ oRGCs in cortical layers on day 56. In addition, the numbers of birthdated HOPX+ oRGCs derived from EdU-labelled apical RGCs were significantly decreased as compared with that of the control group. These results revealed that ethanol exposure decreased the migration and the proliferation of oRGCs in forebrain organoids.

Figure 5. Ethanol exposure impaired the migration and proliferation of outer radial glia cells in forebrain organoids.

Figure 5.

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(A) Tracing of BrdU and Nestin-positive radial glial cells 7 days after the forebrain organoids were cultured with BrdU (10μM) alone or BrdU and 50 mM ethanol on day 14. Scale bar = 20 μm. (B) Tracing of HOPX and BrdU-positive outer radial glial cells 7 days after the forebrain organoids were cultured with BrdU alone or BrdU and 50 mM ethanol on day 14. Scale bar = 20 μm. (C) Quantification of the ratio of HOPX+BrdU+ cells to total BrdU+ cells in control and ethanol groups. Data are expressed as the percentage of HOPX+BrdU+ cells in BrdU+ cells and represent the mean ± SEM of three separate experiments. *p < 0.05 vs. control. (D) Birthdating and migration of HOPX+ outer radial glial cells from EdU-labelled apical radial glial cells 42 days after the forebrain organoids were cultured with EdU alone or EdU and 50 mM ethanol on day 14. Scale bar = 50 μm. (E-F) Quantification of the ratio of EdU+ cells to total cells and HOPX+EdU+ cells to total cells. Data are expressed as the percentage of EdU+ cells in total cells or the percentage of HOPX+EdU+ cells in total cells and represent the mean ± SEM of three separate experiments. *p < 0.05 vs. control.

3.6. Effects of ethanol exposure on the production of outer radial glia cells and the transformation of radial glia cells into astrocytes

We next tested whether ethanol exposure can reduce the production of oRGCs and impact the transformation of RGCs into astrocytes. Our results showed that ethanol exposure significantly decreased the number of oRGCs in the SVZ region and the ratio of oRGCs to total RGCs in cortical layers on day 56 forebrain organoids as compared to the control group (Fig. 6A–B). Exposure of forebrain organoids to ethanol on day 14 also resulted in significant increases in the number of GFAP+ and s100β+ astrocytes with characteristic star-shaped morphologies in day 84 forebrain organoids as compared to control (Fig. 6C – F), indicating that ethanol can enhance the terminal differentiation of radial glial cells into astrocytes, which may further deplete the radial glial cell pools and contribute to the abnormal cortical development.

Figure 6. Effects of ethanol exposure on the production of outer radial glial cells and the transformation of radial glial cells into astrocytes.

Figure 6.

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(A) Representative images of the distribution of HOPX+ outer radial glial cells in day 56 forebrain organoids that were treated with or without 50 mM ethanol on day 14. Scale bar = 50 μm. (B) Quantification of the ratio of HOPX+ outer radial glial cells to total cells or HOPX+ outer radial glial cells to total radial glial cells. Data are expressed as the percentage of HOPX+ outer radial glial cells in total cells or the percentage of HOPX+ outer radial glial cells in total radial glial cells and represent the mean ± SEM of three separate experiments. *p < 0.05, *** p <0.001 vs. control. (C) Representative images of immunostaining of the marker for astrocyte GFAP in day 84 forebrain organoids that were treated with or without 50 mM ethanol at day 14. Scale bar = 50 μm. (D) Quantification of the ratio of GFAP+ cells to total cells. Data are expressed as the percentage of GFAP+ cells in total cells and represent the mean ± SEM of three separate experiments. *p < 0.05 vs. control. (E) Representative images of immunostaining of the marker for astrocyte S100β in day 84 forebrain organoids that were treated with or without 50 mM ethanol at day 14. Scale bar = 50 μm. (F) Quantification of the ratio of S100β+ cells to total cells. Data are expressed as the percentage of S100β+ cells in total cells and represent the mean ± SEM of three separate experiments. ** p <0.01 vs. control.

4. Discussions

Maternal alcohol abuse during pregnancy can damage the developing fetus and lead to fetal alcohol spectrum disorders (FASD), encompassing a range of developmental abnormalities such as growth deficits, midface abnormalities, and dysfunctions in the central nervous system (Welch-Carre 2005). A number of studies have demonstrated that prenatal alcohol exposure impaired cortical development (Robertson et al. 2016; Yang et al. 2012). Due to the limited resources of human samples and the consideration of ethnicity, most in vivo experiments investigated the molecular mechanisms involved in ethanol-induced cortical damages were performed in mice (Ozturk et al. 2017; Zhang et al. 2014), rats (Aronne et al. 2008; Sogut et al. 2017), and Xenopus (Peng et al. 2004). While these studies have contributed significantly to our understanding of the effects of alcohol exposure on cortical development, the limitations of these animal studies should be considered due to the fact that some essential features of the human cortex are not displayed in these animal models (Pinson et al. 2019). It is well-known that oRGCs are more abundant and proliferative in the human brain than in the mouse brain, and the mouse brain contains a minimal number of oRGCs, which cannot further develop into a specific oSVZ layer, making it very difficult to study the behavior of oRGCs using mouse models (Osumi and Kikkawa 2013; Zimmer-Bensch 2019). Therefore, it is essential to develop appropriate models that mimic the key features of the developing human brain and contain abundant aRGCs and oRGCs in VZ and oSVZ, respectively, to investigate the precise molecular and cellular mechanisms underlying the impaired human cortical development induced by prenatal alcohol exposure. With the rapid developments of cerebral organoids, the three-dimensional self-assembled multicellular structures are available to provide a complementary platform to investigate the precise molecular and cellular mechanisms underlying ethanol-induced neurotoxicity in humans. Currently, several brain regional-specific organoids have been developed from human iPSCs or hESCs, such as forebrain organoids (Cederquist et al. 2019; Krefft et al. 2018; Qian et al. 2016), midbrain organoids (Kim et al. 2019; Smits et al. 2019), cerebellar organoids (Balion et al. 2020; Ballabio et al. 2020) and hippocampal organoids (Todd et al. 2013). These brain organoids have been applied to model many diseases, such as Zika virus-induced microcephaly (Lancaster et al. 2013; Qian et al. 2016), Alzheimer’s disease (Gerakis and Hetz 2019; Ghatak et al. 2019), Parkinson’s disease (Chlebanowska et al. 2020), Autism diseases (Hali et al. 2020; Yang and Shcheglovitov 2020), Schizophrenia (Kathuria et al. 2020) and Miller-Dieker syndrome (Iefremova et al. 2017). They are also used to investigate the neurotoxic effects of a wide variety of compounds, including nicotine (Wang et al. 2018), tranylcypromine (Huang et al. 2017), cystatin B (Penna et al. 2019), as well as stimuli such as gamma-irradiation (Shakhbazau et al. 2019) and hypoxic insults (Boisvert et al. 2019; Daviaud et al. 2019). More recently, brain organoids have emerged as a novel model for investigating ethanol-induced neurotoxicity (Arzua et al., 2020; Zhu et al., 2017). Here, we developed forebrain organoids from hESCs using SpinΩ bioreactors to model the neurotoxic effects of prenatal ethanol exposure. We found that forebrain organoids derived from hESCs exhibited the early cellular and histological features of the human cerebral cortex. These forebrain organoids contained abundant neural progenitor cells, including vRGCs, oRGCs, intermediate progenitor cells, neuroblasts, newborn neurons at an early stage and mature neurons, and astrocytes after long-term cultures. These results indicate that our forebrain organoids display well-organized cortical architectures containing abundant RGCs, intermediate progenitors, and neurons and recapitulate the key features of early human forebrain development. After exposure to ethanol, these forebrain organoids exhibited increased apoptosis, decreased expression of Nestin, Vimentin and GFAP, abnormal morphologies of radial glial fibers, and loss of cell polarities. In addition, ethanol exposure also decreased the migration and differentiation of RGCs, but accelerated the transformation from oRGCs to astrocytes.

Neuroepithelial cells (NECs) have the potential to generate neural progenitor cells, including RGCs and intermediate progenitor cells (IPs) (Taverna et al. 2014). When neurogenesis starts, neuroepithelial cells downregulate some epithelial characteristics, particularly the tight junctions and apical-basal polarity, giving rise to RGCs that acquire new features such as expressing glial markers GFAP and GLAST (Gotz and Huttner 2005). Through the asymmetric division, neurons are directly or indirectly produced from RGCs (Anthony et al. 2004). Both neuroepithelial cells and aRGCs undergo interkinetic nuclear migration (INM) during cell division, which involves microtubules and actin filaments (LaMonica et al. 2013). Compared with NECs, aRGCs exhibit more elongated shapes. Their basolateral plasma membrane displayed two subcompartments: the apical and the basal processes (Arai and Taverna 2017). Experiments confirmed that the basal process plays a pivotal role as scaffolding for radial neuronal migration and helps neurons translocate from the birthplace to the final destination (Arai and Taverna 2017; Kosodo and Huttner 2009). In addition, the basal process also regulates mitotic division by maintaining basal lamina contact during M-phase (Kosodo and Huttner 2009). A recent study found that the reduced thickness of radial glial processes contributed to a remarkable reduction of cortical neurons and oRGCs (Fietz et al. 2020). In this study, we found that ethanol exposure significantly decreased the expressions of radial glial process-related proteins Nestin, Vimentin and GFAP. These results indicate that ethanol exposure impaired the formation of radial glial processes and resulted in the loss of cellular polarity in RGCs, which may impair the production of oRGCs and the migration of neurons,

RGCs can receive specific signals that regulate cell fates via the basal process (Kosodo and Huttner 2009; Miyata et al. 2004). The basal process also works as molecular transporter, particularly for rapid directed RNA transport and local translation (Pilaz et al. 2016), and is a very dynamic compartment during cortical development (Yokota et al. 2010). Impairment of the radial glial scaffold is one of the main causes of abnormal neuronal migration and a double cortex formation (Yamamoto et al. 2015). Therefore, morphological changes in radial glial processes might influence the fate decision and the choice of proliferation or differentiation of RGCs during neurogenesis. It was found that RGCs exhibited classical radial glial morphology before 16.5 gestational weeks (GW) with apical and basal processes spanning the entire thickness of the telencephalic wall, of which this radial glial scaffold is defined as the continuous scaffold stage (Nowakowski et al. 2016), but displayed truncated morphologies at the late stage of neurogenesis (GW17 - GW24), with fibers terminating in the oSVZ region. This kind of radial glial scaffold was a discontinuous scaffold stage, and vRGCs turn into ‘truncated’ radial glia (tRG), which are no longer a significant source of neurons. After vRGCs differentiate into tRGs, oRGCs are the sole contributor to the radial glial fibers, which guide neurons to the cortical plate during supragranular layer neurogenesis (Nowakowski et al. 2016). Functional analysis showed that the basal process is crucial to maintaining the proliferative abilities of oRGCs (Fietz et al. 2010). Hence, the radial glial scaffolds of both vRGCs and oRGCs play a pivotal and distinct role in expanding cortical layers and neuronal migrations during brain development. The impairments of radial glial fiber development will contribute to the abnormal development of the cerebral cortex. The results from this study have shown that ethanol-exposed forebrain organoids exhibited increased cell apoptosis in Nestin+ RGCs and decreased expressions of radial glial process-associated proteins Nestin, Vimentin, and GFAP. The ethanol-induced reduced synthesis of radial glial intermediate filament proteins further caused abnormal structures of radial glial processes. Ethanol exposure changed the structure of the radial glial processes from parallel basal processes into round and short basal processes on day 56. In addition, ethanol exposure significantly inhibited the migration of Nestin+ RGCs and reduced the number of HOPX+ oRGCs These results demonstrated that ethanol exposure can impair the formation of radial glial scaffolding and decrease the migration and proliferation of oRGCs in forebrain organoids.

Astrocytes are the primary terminal differentiation of RGCs (Akdemir et al. 2020). Because ethanol exposure resulted in the loss of basal processes in oRGCs, we expected that ethanol exposure can accelerate the terminal transformations of oRGCs into astrocytes. We found that exposure of forebrain organoids to ethanol resulted in significant increases in the number of GFAP+ and s100β+ astrocytes with characteristic star-shaped morphologies in day 84 forebrain organoids as compared to control, indicating that ethanol can accelerate the transformation of oRGCs into astrocytes, which may further deplete the RGC pools and contribute to the abnormal cortical development. These findings are supported by a previous study that showed that exposure of brain organoids to ethanol resulted in an increased proportion of astrocytes compared to the control group (Zhu et al., 2017).

In conclusion, we have developed forebrain organoids from hESCs that display well-organized cortical architectures containing abundant RGCs, intermediate progenitors, and neurons and recapitulate the key features of early human forebrain development. Exposure of forebrain organoids to ethanol resulted in a significant increase in apoptosis in RGCs. Ethanol exposure also remarkably decreased the levels of radial glial process-associated proteins in RGCs and distinctly impaired the integrity and morphologies of radial glial processes. In addition, the ethanol-induced impairment of the radial glial processes is associated with decreased migration and proliferation of RGCs, reduction in the generation of HOPX+ oRGCs, and the accelerated transformation of oRGCs into astrocytes. These results demonstrate that ethanol exposure can disrupt cerebral cortex development by impairing the survival and proliferation of RGCs, the formation of radial glial processes, and the generation, migration, and transformation of oRGCs. These results provided new insight into the mechanisms underlying ethanol-induced abnormalities in fetal cortical development and FASD in humans.

Highlights.

  • Ethanol increased apoptosis in radial glial cells in human forebrain organoids.

  • Ethanol impaired the integrity and morphologies of radial glial processes.

  • Ethanol decreased the migration and proliferation of radial glial cells.

  • Ethanol reduced the generation of outer radial glial cells in forebrain organoids.

  • Ethanol accelerated the transformation of outer radial glial cells into astrocytes.

Acknowledgments

This work was supported by the National Institutes of Health Grants AA028435, AA024337 (S.-Y.C.), and AA023190 (W.F.) from the National Institute on Alcohol Abuse and Alcoholism. H-GZ was supported by a Research Career Scientist (RCS) award.

Abbreviations

RGCs

Radial glial cells

NEC

neuroepithelial cells

hESCs

human embryonic stem cells

PP

preplate

VZ

ventricular zone

CP

cortical plate

MZ

marginal zone

SVZ

subventricular zone

oSVZ

outer subventricular zone

iSVZ

inner subventricular zone

IFL

inner fiber layer

IPs

intermediate progenitors

DAPI

4′,6-diamidino-2-phenylindole

EBs

embryoid bodies

GDNF

glial cell-derived neurotrophic factor

TGF-β

transforming growth factor-beta

cAMP

Adenosine-3′,5′-cyclic monophosphate

GFAP

glial fibrillary acidic protein

GLAST

glutamate aspartate transporter

DPBS

Dulbecco’s phosphate-buffered saline

PFA

paraformaldehyde

BrdU

5-bromo-2’-deoxyuridine

INM

interkinetic nuclear migration

Footnotes

Declaration of Competing Interest

The authors declare no competing financial interests.

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