Engineering Brain Organoids to Probe Impaired Neurogenesis

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Engineering brain organoids to probe impaired neurogenesis induced by cadmium Fangchao Yin, Yujuan Zhu, Yaqing Wang, and Jianhua Qin ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00160 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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ACS Biomaterials Science & Engineering

Engineering brain organoids to probe impaired neurogenesis induced by cadmium Fangchao Yin,

†,‡,§,# Yujuan

Zhu, †,‡,§,# Yaqing Wang,†,‡,§ and Jianhua Qin*,†,‡,§

†Division of Biotechnology, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, China. ‡CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences,

Shanghai, China. §University of Chinese Academy of Sciences, Beijing, China. #These authors contributing equally to this work.

*Correspondence should be addressed to J.Q. ([email protected]).

Abstract Brain organoids derived from human induced pluripotent stem cells (hiPSCs) are three-dimensional in vitro models with near-physiological cellular composition and structural organization, which is representative of the developing human brain. It provides an ideal experimental system for the investigation of brain development and diseases. Prenatal exposure to the heavy metal-cadmium (Cd) poses a serious health threat particularly to the developing brain due to a long biological half-life of Cd in vivo. Although it is known that prolonged exposure to Cd will cause toxic effects because of its low rate of excretion from the body, the underlying mechanisms of Cd neurotoxicity remain not completely clear. Herein, we proposed a simple approach to engineer brain organoids on an array chip with octagon-shaped micropillars and explored neural dysfunctions of brain organoids under Cd exposure. hiPSCs-derived brain organoids with millimeter-size recapitulated spatial and temporal patterning events in the early developing brain, including gene expression programs and three-dimensional (3D) organization. With Cd exposure, brain organoids displayed induced cell

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apoptosis, skewed neural differentiation and varied brain regionalization, indicating the presence of the impaired neurogenesis in human fetal brain. This work provides a simple manner to generate brain organoids efficiently, and a powerful platform for the investigation of abnormal neurogenesis induced by many different toxic factors in vitro. Keywords: array chip, brain organoids, fetal exposure to cadmium, pluripotent stem cells 1. Introduction The rise of hiPSCs through the reprogramming of somatic cells has offered a great opportunity to direct the differentiation into functional derivatives of interest and even complex near-physiological 3D tissues in the dish1. These 3D tissues with millimeter-size displayed native morphogenetic and histological realism in vitro, termed as organoids2-3. So far, various organoids derived from iPSCs have been established resembling the lung, intestine, retina, kidney, liver and brain

4-11

, and these organoids could serve as valuable in vitro models for studying a large scale of

intricate diseases. Particularly, brain organoids with different brain regions recapitulated the developing human brain at early stages during gestation4-6, 12. Although reductionist in nature, such brain organoids allowed to investigate microcephaly, a disorder characterized by small brain size6, and Zika virus exposure, which arrests the development of human fetal brain4, 13. As an industrial metal, Cd has received considerable concern in environmental and occupational health14. Human exposure to the toxic metal Cd occurs primarily through inhalation and industrial by-products, such as contaminated cereals. Once absorbed, Cd rapidly accumulates in human body over time due to a low excretion rate15. Thus, prolonged exposure to Cd will adversely affect human tissues including kidney, liver, brain in a variety of ways, and increase the risk of cancer and all-cause mortality. In particular, women are more susceptible to Cd toxicity compared to men16-17, and Cd might affect female reproductive system and fetal development18-19. Cd was examined to cross human placenta20-21, which is not a complete barrier, and finally accumulated in fetal tissues20, 22

. To our knowledge, Cd has been shown to be teratogenic on developing embryos in various

species23, and there is also increasing evidence for the association between maternal Cd exposure ACS Paragon Plus Environment

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and the abnormality at birth in humans, including reduced size at birth and preterm delivery24-28. Such prenatal exposure to Cd poses a health threat particularly to the fetal brain29, however, there are few studies to explain the relationships between prenatal Cd exposure and adverse effects in early developing brain. Given the inherent differences between species in brain development, architecture and function, it is highly desirable to develop in vitro models of human brain to explore the complicated mechanisms of neuronal dysfunctions caused by Cd. Herein, we presented a hiPSCs-derived brain organoid model on a pillar array, which is amenable to explore the pathogenesis of fetal brain development with Cd exposure in vitro (Fig. 1). An array of octagon-shaped micropillars allows for the reaggregation of dissociated hiPSCs, termed embryoid bodies (EBs). These EB aggregates were subsequently induced into neural identities in suspension, and finally differentiated into millimeter-sized 3D brain organoids with rapid expansion of polarized neuroepithelium in extracellular matrix. The growth of these organoids recapitulates key aspects of human brain development, including authentic cell types, well-defined organization, and orderly neural differentiation, reminiscent of the early developing brain. With Cd exposure, it has a lasting effect on neural differentiation, maturation and cellular death. Immunostaining and real-time PCR was carried out to investigate the impaired neurogenesis in Cd-treated brain organoids. 2. Experimental 2.1 Design and fabrication of microfluidic devices The microfluidic devices were fabricated in poly(dimethylsiloxane) (PDMS, Sylgard, Dow Corning Corp.) using soft lithography technique as described in our previous studies30-31. The microarray chips were made up of two layers, the bottom and upper layer. The bottom layer was patterned with octagon-shaped micropillars (side length, 600 and 800μm; depth,1 mm; gap, 50μm) (Fig. 2a). The top layer was a simple quartz ring to hold cell culture medium. Briefly, PDMS monomer was mixed with a curing agent (184 Silicone Elastomer, Dow Corning Corp.) at a ratio of 10:1 by mass, and ACS Paragon Plus Environment


degassed to remove air bubbles. The polymer was cured in an over for 40-60 min at 80 ℃. The PDMS layer was then gently peeled away from the mold. The upper and lower layers were glued together using the glue, a mixture of PDMS monomer and curing agent at a ratio of 50:1 by mass. A thin layer of PDMS glue was spin-coated onto a clean glass at first. Then the top layers were dipped in the glue and bound with the bottom layers after curing at 80 ℃ for 40-60min.

2.2 Maintenance of hiPSCs hiPSC line derived from skin fibroblasts32-33 were cultured in mTeSR1 medium (STEMCELL) on 6-well plates coated with Matrigel (BD Bioscience), and the medium was changed daily. When passaging, cells were digested into small clumps with Accutase (Sigma) and propagated at 1:5 ratio every 5 days. Then cells were cultured in mTeSR1 medium containing ROCK inhibitor Y27632 (10 µM, Selleck) for 1 hr, and maintained in mTeSR1 medium. The expression of pluripotent markers SOX2 and OCT4 was regularly checked by immunostaining. 2.3 Brain organoid development on the chip The details about the disaggregation of brain organoids were described in our previous report34-35. Specifically, dissociated hiPSCs formed cell aggregates, named as embryonic bodies (EBs), and gradually self-organized into millimeter-sized organoids in Matrigel without the addition of the extra factors. 2.4 Brain organoids with Cd exposure To explore neural differentiation, brain organoids were randomly selected to be exposed to 1 µM Cd in NDM from day 11 to 21, and control organoids remained untreated. The medium was changed every two days. 2.5 Dissociated cells from brain organoids with Cd exposure Individual cells were dissociated from brain organoids at 20 days of differentiation by digestion with Accutase for 5 min. Then these single cells were seeded on the plates coated with Matrigel and


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maintained in neural differentiation medium for another 4 weeks. 2.6 Tissue preparation and immunohistochemistry Brain organoids for immunostaining were prepared as described in our previous work34-35. Primary and secondary antibodies were listed in supporting information (Table S1). The fluorescent images were photographed using confocal microscope (Olympus). 2.7 TUNEL assay Cell death was examined by fluorescent TUNEL (TACS 2 TdT–Fluor in situ apoptosis detection kit, Trevigen) according to the manufacturer’s instructions.

2.8 Real-time PCR

The changes in specific gene expression were tested by real-time PCR, and the details were described in our previous studies34-35. Primers used here were listed in the Table S2. 2.9 Reproducibility of experiments All experiments were repeated 3-10 times and the number of samples was as indicated in the legends. Representative images were photographed from at least 3 independent experiments. 3. Results and discussion 3.1 Characterization of hiPSCs-derived brain organoids on chip The development of 3D brain organoids from hiPSCs usually involves three steps including EBs formation, neural induction, neural differentiation and expansion in Matrigel. As such, we designed and fabricated a microfluidic device with an array of octagon-shaped micropillars to produce brain organoids in a high throughput manner (Fig. 2a). Prior to assay, dissociated hiPSCs were seeded on micropillar chip to generate EBs. As shown in Fig. 2b, abundant EBs with clear border and consistent morphology were visible after culture for one day on micropillar arrays. The neural ectoderm of these EBs was further induced via the culture in neural induction medium. Following



the addition of the extracellular matrix (Matrigel), these neuroepithelial tissues, which were characterized of visible neuroepithelial buds protruded from cell aggregates, rapidly expanded into larger tissues with millimeter size. In addition to the morphology, neural induction in whole brain organoids was initially examined through the performance of real-time PCR for markers of pluripotency and neuronal identities (Fig. 2c). In contrast with the reduction in the pluripotent markers OCT4 and NANOG, neural markers SOX1 and PAX6 were significantly upregulated at day 10 compared to the control, indicating the effective neuronal induction in cell spheroids. Human brain development occurs in an orderly sequence in vivo, involving neuronal proliferation, differentiation and migration to generate discrete brain regions and layered cortex. Therefore, we examined neurogenesis of brain organoids in great detail based on the data above. According to the real-time monitor of brain organoids, large neuroepithelial buds were observed containing fluid-filled cavities, which might be reminiscent of brain ventricles with appropriate apicobasal orientation. As depicted in Fig. 3a, the apical marker CD133-positive cells located in the inner area of the cavities, revealing the presence of apical-basal polarity. The development of the central nervous system is stringently orchestrated based on such an epithelial characteristic of apicobasal orientation36. A pool of neural progenitor cells (NPCs) lining the ventricular zone serves as the source of neurogenesis during brain development. As expected, the immunostaining for NPC markers SOX2 and NESTIN displayed the adjacent expression to the apical surface of ventricular cavities (Fig. 3b and 3c). To determine whether discrete brain regions was present in these neuronal tissues, immunostaining was further carried out for markers of human fetal brain at early stages. The stained tissue displayed a high expression of PAX2 (Fig. 3b and 3c), which was tightly related to the early development of the forebrain, midbrain and hindbrain. From Fig. 3d, we could observe the discrete expression of forebrain marker PAX6 and hindbrain marker ISL1, indicating the rudimentary ACS Paragon Plus Environment

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development of varied brain regions in brain organoids. Neurons generated from the NPCs zones could migrate radially to form layered cerebral cortex in vivo37-38. Hence, the cortex structure in tissues was examined at 40 days of differentiation through the immunostaining. TBR1, as a transcription factor, is known to be specifically expressed in preplate during cortical development39, and our staining revealed the high expression of TBR1 along the outside of the brain organoids (Fig. 3e). CTIP2, another transcriptional regulatory protein, is tightly associated with cortical architecture40. The fluorescent images demonstrated that CTIP2 was expressed adjacent to TBR1-positive preplate. This result might suggest the rudimentary corticogenesis in early developed neural tissues. Collectively, our data revealed that the array chip allows for the efficient generation of brain organoids, and these large organoids resembled key features of human fetal brain in terms of cell components, tissue architecture and regionalization. 3.2 Induced cell death with Cd exposure According to results illustrated above, EB aggregates could be progressively induced into neural identities through the culture in neural induction medium (day 6-11), therefore, the tissues were treated with Cd at 11 days of differentiation in order to explore neurogenesis in detail. Apoptotic cell death is considered as an active and ongoing event in the control of cell populations during normal neural development in vivo, and it could be induced by a variety of environmental agents, such as toxic metals, leading to the loss of cell populations41. Evidence is emerging that apoptosis might be a key factor in the metal-induced impairment of neurogenesis in model systems42. Here, in an attempt to assess cell death in Cd-exposed brain organoids, TUNEL assay was performed for the detection of apoptotic DNA fragmentation in individual cells. As shown in Fig. 4a, cell death was significantly induced in Cd-treated tissues at 20 days of differentiation. Quantification of dead cells based on a series of images further confirmed the great cell death with Cd exposure (Fig. 4b). Even 10 days after the removal of Cd, the treated tissues still exhibited a



higher rate of cell death based on the TUNEL staining and mRNA expression of caspase 3 (Fig. 4c and 4d), indicating the long-term toxic effect of Cd in neural development. 3.3 Impaired neural differentiation with Cd exposure It is well-known that NPCs maintenance and timed differentiation are fundamental processes that underlie the development of human nervous system. Thus, neural differentiation was further assessed by immunostaining and mRNA expression of NPC and neuron markers in the control or Cd-treated brain organoids at different time points (Fig. 5a). As depicted in Fig. 5b, the tissues with Cd treatment exhibited a slight increase in TUJ1-positive neurons at 20 days, while no obvious changes in SOX2-positive NPCs compared to the control group in the absence of Cd. Accordingly, the analysis of fluorescence intensity and mRNA level also revealed the grown expression of neuron marker not NPC markers (Fig. 5c and 5d), suggestive of precocious neural differentiation in brain organoids exposed to Cd. Interestingly, the proportion of NPCs was reduced after the removal of Cd, and neurons was concomitantly decreased at 30 days of differentiation (Fig. 5e-g), which implied the long-lasting effects of Cd on neurogenesis. Moreover, the reduction of NPCs might account for the inverse variation in neurons within the treated tissues. Immature brain varies considerably by brain regions in the vulnerability to physiological and pathological stimuli43, highlighting the importance of context in understanding the development of distinct brain regions with Cd exposure. As such, brain development was explored in the system of brain organoids with exposure to Cd. Immunohistochemical staining was carried out for markers of the early developing brain PAX2 and forebrain PAX6 in whole brain organoids. With the treatment of Cd, neural tissues exhibited the slightly decreased expression of PAX2 and PAX6 at the protein and mRNA levels (Fig. 6a-c). In accordance with the above observations, both PAX2- and PAX6-postive neurons were greatly reduced even following the removal of Cd (Fig. 6d-f), implying the lasting effects of Cd in the development of specific brain areas. Together, these results indicated that Cd exposure might cause persistent and irreversible changes in neural differentiation including


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neurogenesis and brain regionalization in the early developing brain. 3.4 Abnormal neural maturation with Cd exposure hiPSCs could differentiate into neural stem cells and further mature into lineages of excitatory and inhibitory neurons, and this process actually recapitulated neurogenesis and neural maturation in human fetal brain. Data above demonstrated the adverse effects of Cd exposure to brain organoids, which resembled the fetal brain full of immature neuronal cells at the early stages of gestation. Therefore, we next investigated the neural maturation with Cd exposure. To this end, individual neural cells were cultured on the petri dish for neural maturation following dissociation from millimeter-size brain organoids by digestion. In accordance with the observations in 3D neural tissues, cell monolayers exhibited much less TUJ1-positive neurons with Cd exposure (Fig.7a-b), further proving the neurotoxicity of the metal Cd. Such a decrease in differentiated neurons might aggravate the reduction in the excitatory and inhibitory neurons (Fig.7c-d). As the excitatory–inhibitory networks are fundamental to proper brain development, the disturbance in neural maturation may provide new insights into the pathogenesis and therapeutics of neural dysfunctions. 4. Conclusions In this study, we engineered an in vitro model of hiPSCs-derived brain organoids on micropillar chip, and explored neural dysfunctions under Cd exposure. The device with an array of octagon-shaped micropillars allows for the simple generation of brain organoids in a high throughput manner. Pluripotent stem cells in micropillar arrays self-organized into millimeter-sized brain organoids, resembling cellular components and structural organization of the developing human brain. With Cd treatment, these brain organoids exhibited the increased cell death, impaired neurogenesis, skewed neural maturation, and disturbed brain regionalization. Such changes might be potential causes for various learning and behavioral deficits observed in various neurological diseases due to Cd exposure. These findings elucidated a series of alterations in biological events, and verified the feasibility



of such an in vitro model for the investigation of Cd-induced impairment of neurogenesis in the developing brain. Together, the engineered brain organoid-on-an-array chip incorporates microfabrication technology with stem cells biology, which allows to examine abnormal neurogenesis induced by many different toxic factors in vitro, and might contribute to our better understanding of the mechanisms underlying clinical features observed in postnatal neural disorders. Supporting Information Lists of antibodies and primer pairs. Acknowledgements This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA16020900, XDPB0305), National Nature Science Foundation of China (No. 91543121, 81573394, 31671038, 31600784, 81603075), Innovation Program of Science and Research from the DICP, CAS (DICP TMSR201601).

Conflict of interest

There are no conflicts of interest to declare. References

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Graphic for manuscript

Figure 1. Schematic view of the workflow in this study. 3D brain organoids derived from hiPSCs were generated on the array chip, and applied as a model to investigate neural impairments induced by Cd during early brain development.


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Figure 2. Simple generation of hiPSCs-derived brain organoids on an octagon-shaped pillar chip. a, Chip design and main steps involved in the generation of brain organoids on the array chip. b, Representative bright field images of neural tissues on the chip. Neuroepithelial buds (indicated by red arrows) rapidly protruded from larger cell spheroids after embedding in Matrigel on day 11. Scale bars: 400 µm (left), 200 µm (right). c, mRNA expression was examined for the pluripotency markers (OCT4A, NANOG) and neural markers (SOX1, PAX6) using real-time PCR in hiPSCs or cell aggregates at 10 days. The expression values were normalized to the β-actin expression level. n = 3 independent experiments. Data are shown as mean ± SD. *, P-value

Engineering Brain Organoids to Probe Impaired Neurogenesis

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