Toward a 3D model of human brain development for studying gene/environment interactions

This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology.

performed at high doses that are not relevant for human exposure scenarios, since human exposure often occurs at low doses over pro longed time periods. Nonhuman animal testing also does not refl ect inter-individual diff erences among the human population. Moreover, the relevance of behavioral and histological data from rodents for human health has been questioned [6]. Consequently, current guidelines often do not provide suffi cient information to facilitate regulatory decision-making.

Modeling the complexity of the central nervous system in vitro
3D models are far superior for recapitulating the complex directional growth and connections that underlie neurodevelopment [7]. A two-dimensional model constrains cellular morphology, preventing complex synaptic interactions. In contrast, a 3D model enables neurons and astrocytes to assume a more natural shape and extend processes to synapses and neighboring cells.
Given the importance of cell-to-cell interactions in the brain, our laboratories have begun characterizing a 3D rat primary aggregating brain cell culture model, granted by the US Food and Drug Administration (#U01FD004230), in order to map pathways of DNT. We and others have used a variety of techniques to show the presence of all relevant cell types in these cultures [8][9][10][11][12]. Moreover, synaptogenesis and myelination can be observed. Consequently the model has the potential for detecting chemicals interfering with these processes; for example, by blocking the release of neurotrophic factors or interfering with cell adhesion. We are currently using emerging technologies to study neurotoxicity in these cultures [9,10]. For example, we have identifi ed metabolic profi les that can distinguish compounds with diff erent target organ toxicity (brain, kidney and liver). Th e DNT consensus process identifi ed our rat aggregating culture model among the most representative models for DNT studies [13][14][15].
To increase the usefulness of the model, we are adapting our model for studying cells of human origin. Th e in vitro data from the rat and the human model can then be compared to identify possible species diff erences to better predict the potential for human toxicity. Such an innovative approach is expected to provide more precise information for human risk assessment, and regulatory decision-making, than the current extrapolations and predictions based on animal models. In the fi eld of drug development, about 92% of substances fail during clinical trials due to eff ects in humans that were not identifi ed in preclinical animal tests [16]. Th e use of human cell models is therefore crucial and has the advantage of eliminating interspecies confounds.
Th e use of stem cells has the potential to create new humanized models for toxicity testing. Stem cells have the capacity for self-renewal and can diff erentiate into all cell types of the CNS, such as neurons, astrocytes and oligodendrocytes [17]. Th e main sources of generating stem cells with the potential to diff erentiate into neural lineages are pluripotent embryonic stem cells [18][19][20], human umbilical cord blood-derived neural stem cells [21], multipotent somatic stem cells derived from bone marrow [22,23] or other tissues including the CNS [24] as well as iPSCs [25].
Generating stem cells from any one source will have advantages and disadvantages. In this project we chose to use iPSCs, which overcome many limitations of other sources; for example, ethical issues, limited accessibility, and restricted genetic backgrounds. In addition, iPSCs seem to be more stable, with a higher neuronal diff erentiation effi ciency than, for example, somatic stem cells [26,27]. However, generating iPSCs is challenging. First, the percentage of reprogrammed cells after induction of pluripotency genes is often low. Moreover, many diff erentiation protocols still require further optimization because the procedure to obtain mature neurons and especially glial cells is very time consuming and the reproducibility and effi ciency of diff erentiation can be low. In this project we are evaluating diff erent protocols and culturing techniques to obtain a reproducible model with increased effi ciency of targeted diff erentiation. To establish a human aggregating model, we will combine astrocytes and neurons derived from iPSCs [28,29]. Methods have been described for diff erentiation of iPSCs to astrocytes and diff erent types of neurons [28,[30][31][32]. iPSC lines from diff erent individuals will provide a testing model with the ability to predict substance sensitivity in diff erent genetic backgrounds. Moreover, the development of neurons and glia from iPSCs is believed to recapitulate many stages of brain development in utero [32]. Each stage of neurodevelopment is unique and displays diff erent sensitivities to diverse xenobiotics. A DNT in vitro model should cover many of these stages.

Induced pluripotent stem cell diff erentiation
We have optimized protocols for diff erentiating human iPSCs into neural progenitor cells (NPCs) and neuronal and astroglia lineages ( Figure 1). First, the generation of NPCs involves a stepwise neural diff erentiation protocol through embryoid body formation as previously described [25,33]. iPSCs are detached after treatment with collagenase (1 mg/ml) for 1 hour and plated onto nontreated polystyrene plates in human embryonic stem cell medium in the absence of fi broblast growth factor but in the presence of heparin with the medium exchanged daily. After 4 days in this medium the cells are switched to human NPC medium (DMEM/F12, neurobasal, heparin, N2), exchanged every 2 days. After 2 weeks in suspension cultures, embryoid bodies are collected and mechanically dissociated into smaller cell clusters. Th ese cells are plated onto poly-l-ornithine and laminincoated plates and passaged mechanically as adherent cultures (Figure 1a) in human NPC expanding medium (KnockOut DMEM/F12, Glutamax, endothelial growth factor and basic fi broblast growth factor), neural supplement and penicillin-streptomycin. To determine the purity of the culture, we stain cultures for neural progenitor markers nestin and Sox-2 ( Figure 1b).
In the second protocol, for neuronal diff erentiation of NPCs, we plate early-passage NPCs on poly-l-ornithine and laminin-coated plates, with neuronal culture medium (neuro basal medium supplemented with 2 mM lglutamine, B27) for 1 week to generate immature neurons (Figure 1b). Immature neurons are then cultured in neuronal medium supplemented with brain-derived neuro trophic factor (10 ng/ml) and glial cell-derived neurotrophic factor (10 ng/ml) for 4 to 8 weeks to obtain mature neurons.
In the fi nal protocol, NPCs at a later passage are induced to diff erentiate into astrocytes by withdrawal of fi broblast growth factor-2 and addition of ciliary neurotrophic factor and 10% fetal bovine serum for 2 to 4 weeks. To determine the purity of cultures, we immunostain diff erentiated cells for glial fi brillary acidic protein and S100β (astrocytic markers), DCX and microtubuleassociated protein-2ab (neuronal markers), O4 and glutathione transferase π (oligodendrocytic markers). Extensive dendritic growth is observed by staining for microtubule-associated protein-2, and synapses are indicated with antibodies against synapsin-1 (Figure 1b). Neural progenitors can diff erentiate to neurons representative of cortical layers (Figure 1c) and GABAergic and glutamergic neurons as indicated by the expression of markers specifi c for cortical layers (Tbr1, Brn2, and Ctip2), glutamate decarboxylase, and the vesicular glutamate transporter, respectively (data not shown).

Preparation of human three-dimensional neural cell cultures
Th e 3D human neural model is prepared using similar techniques to those established for our 3D rat primary aggregating brain cell cultures [34]. Th ree approaches have so far been tested. First, cells are diff erentiated from NPC rosette aggregates (Figure 2a) directly in 3D (Figure 2d), inducing the diff erentiation using diff erentiation media as described above for 4 weeks. For the second approach, cells from human NPC adherent cultures (Figure 2b) are detached and plated in un coated six-well plates with a density of 2×10 6 human NPCs/well (Figure 2c) for 4 weeks. In the fi nal approach, human NPCs (Figure 3a) are diff erentiated two-dimensionally for 4 weeks (Figure 3b). After 4 weeks of diff erentiation, cells are detached and cultured in a 3D suspension as previously described with a density of 2×10 6 cells/well (Figure 3c). Cultures are maintained at 37ºC in an atmosphere of 10% CO 2 during constant gyratory movement.
Similar to our rat brain aggregates, the 3D human neuronal model is characterized by (RT-)PCR and immuno cytochemistry for selected markers that have been found to be involved in neuronal and glial diff erentiation in primary rat cultures [35,36] (Figures 2  and 3). In addition, neurotransmitter receptor activity will be assessed by measuring intracellular messengers including calcium and cAMP. Our results so far have shown higher expression of various neuronal diff erentiation markers in the aggregated cultures obtained from single cells than from the aggregates obtained directly from rosettes ( Figure 2). Moreover, aggregates from neurons diff erentiated for 4 weeks in two dimensions show increased expression of diff erent neuronal markers after the 3D formation, suggesting that the model continues to mature in 3D (Figure 3).

Genetic sensitivity to xenobiotics
A limitation to traditional animal model-based chemical and drug testing has been the inability to address the infl uence of (epi)genetic background and medication history on sensitivity. To investigate the impact of diff erent genotypes of neurons derived from iPSCs on chemical sensitivity, we will generate iPSCs from patients with neurodevelopmental disorders such as Rett syndrome, tuberous sclerosis complex and Down syndrome. We chose these disorders as a point of entry because of their potential sensitivities to substances that induce oxidative stress [37]. Greater than 90% of the cases of Rett syndrome are due to methyl CpG binding protein 2 and mouse models of Rett syndrome have shown increased oxidative damage in diff erent tissues [38]. Tuberous sclerosis complex is an autosomal dominant neurodevelopmental disorder caused by mutations in the TSC1 and TSC2 genes. In the developing child, intractable epilepsy, cognitive impairment, and autism have been reported [39]. Down syndrome is due to trisomy in chromosome 21 and is associated with impaired neurological maturation and early neurodegeneration. Several genes are expressed at higher levels due to the trisomy, which aff ects reactive oxygen species including superoxide dismutase.
iPSCs derived from fi broblasts of individuals with these disorders, as well as healthy individuals, will undergo neural diff erentiation and exposure to chemicals with predicted neurodevelopmental toxicity (Table 1). First, we will analyze the cellular response to the toxicants in the 3D neural model derived from healthy iPSC donors by measuring oxidative damage and morphological and functional endpoints such as immunohistochemistry, gene expression and calcium fl ux. Second, we will determine whether iPSC-derived neurons and astrocytes from patients with neurodevelopmental disorders exhibit dysfunctional developmental processes as predicted by disease-relevant mutations. Finally, we will examine whether these genetic mutations increase sensitivity to a selection of DNT chemicals, presented in Table 1.
Development of such a 3D human brain model will represent a versatile tool for more complex testing platforms and strategies as well as research into CNS physiology and pathology. However, several challenges have to be considered. Diff erentiation and culturing protocols need to be optimized to generate reproducible models consisting of the same cell types. In this project, three diff erent protocols are evaluated to generate the most promising 3D neuronal model. Diff erentiation of the NPCs as monolayer cultures before culturing in 3D will allow for better control over the cell fate of the progenitors upon aggregation. However, this approach may not be able to capture the early neurodevelopmental window, a sensitive stage of development that is of high importance. Similar challenges will probably be faced for the disease model. One cannot guarantee that iPSCderived cells from a disease-permissive genetic background will retain relevant epigenetic modifi cations or exhibit clear developmental phenotypes. Several epigenetic modifi cations in the donor tissue might be lost or modifi ed by reprogramming of the cells. In addition, the diff erentiation effi ciency of cells from diff erent donors may vary, which will complicate the evaluation of eff ects after chemical exposure. Many challenges remain to be overcome, but as the iPSC fi eld matures we will gain a better understanding of how to apply this technology to generate viable models of the human developing brain.