Modeling human cortical development in vitro using induced pluripotent stem cells Jessica Mariania,b, Maria Vittoria Simoninia,b, Dean Palejeva,b, Livia Tomasinia,b, Gianfilippo Coppolaa,b, Anna M. Szekelya,c,d, Tamas L. Horvathe, and Flora M. Vaccarinoa,b,f,1 aProgram in Neurodevelopment and Regeneration, bChild Study Center, and Departments of cGenetics, dNeurology, eComparative Medicine, and fNeurobiology, Yale University School of Medicine, New Haven, CT 06520, USA Edited* by Pasko Rakic, Yale University, New Haven, CT, and approved June 11, 2012 (received for review February 21, 2012) Human induced pluripotent stem cells (hiPSCs) are emerging as described method for neural differentiation that allows formation a tool for understanding human brain development at cellular, of serum-free, floating embryoid body-like, quick aggregates molecular, and genomic levels. Here we show that hiPSCs grown (SFEBq) (16) we derived neurally differentiated SFEBq-like in suspension in the presence of rostral neuralizing factors can multilayered structures from hiPSC. We also performed detailed generate 3D structures containing polarized radial glia, interme- immunophenotyping of these cultures, analyzed their tran- diate progenitors, and a spectrum of layer-specific cortical neurons scriptome, and compared it with transcriptomes of hESCs, hiPSCs, reminiscent of their organization in vivo. The hiPSC-derived mul- human neuronal progenitors, and developing human brain tissue. tilayered structures express a gene expression profile typical of These combined analyses demonstrate that hiPSC-derived multi- layered structures display a gene expression profile typical of the the embryonic telencephalon but not that of other CNS regions. embryonic telencephalon, and that neurons within these structures Their transcriptome is highly enriched in transcription factors encompass both lower and upper cortical layer fates. controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the Results early human cerebral cortical wall at 8–10 wk after conception. Characterization of hiPSC Lines. We used two hiPSC lines: PGP1-1 Thus, hiPSC are capable of enacting a transcriptional program (17) and i03-01#9, a line derived in our laboratory. Both lines specifying human telencephalic (pallial) development. This model were established from healthy control adult male skin fibroblasts will allow the study of human brain development as well as dis- through retroviral reprogramming vectors expressing OCT4, NEUROSCIENCE orders of the human cerebral cortex. SOX2, KLF4, and c-MYC (18). These hiPSC lines exhibited typical hESC morphology and expressed canonical pluripotency human embryonic stem cell | embryo | differentiation | cortical layer markers by immunocytochemistry as well as NANOG, DNMT3B, and other endogenous mRNAs that characterize pluripotent merging data highlight the complexity and dynamic nature of cells (Fig. S1 A and B). Pairwise correlations of global gene ex- Egene expression in the central nervous system (CNS) and the pression data obtained by microarrays (Dataset S1) showed high divergence between human and other mammalian species, which is similarity in gene expression between PGP1-1 and the hESC line especially pronounced in the developing brain (1–4). Exploring H1 (Pearson’s r > 0.978), i03-01#9 and H1 (Pearson’s r > 0.975), ’ > such differences may reveal the genetic underpinnings of the larger and PGP1-1 and i03-01#9 (Pearson s r 0.979). size and complex architecture of the human brain and elucidate the We then tested the hiPSC lines with Pluritest (http://www. fi molecular and cellular substrates of higher cognitive functions, as pluritest.org), an approach that classi es samples according to fi well as of our vulnerability to neurodevelopmental and neurode- more than 450 genome-wide transcriptional pro les, including generative disorders. To understand the genetic programs that 223 hESC and 41 hiPSC lines from multiple laboratories (19). drive cell specification and differentiation in the human brain, it is The i03-01#9 and PGP1-1 displayed Pluritest scores consistent important to develop model systems that recapitulate dynamic with normal human pluripotent cells and clustered together with the H1 hESC line (green arrowhead, Fig. S1D). Taken together, aspects of neural development, in addition to making inferences fi fi from commonly used models of lower mammalian species. these results con rm that PGP1-1 and i03-01#9 ful ll the Recapitulating human neural development in vitro using human established criteria for completely reprogrammed iPSC lines. induced pluripotent stem cells (hiPSCs) can provide our first un- derstanding of how genetic variation and disease-causing muta- Excitatory Neuronal Precursor Cells Are Generated Within Multilayered tions influence neural development. Human iPSCs generated from Structures Derived from hiPSCs. Undifferentiated PGP1-1 and i03- 01#9 colonies were dissociated into single cells and cultured in reprogrammed cells can be differentiated into any tissue, including – the CNS, while maintaining the genetic background of the in- suspension in the presence of FGF2 (5 20 ng/mL) and inhibitors of the bone morphogenetic protein (BMP), Wnt/β-catenin, and dividual of origin. These critical features have been exploited to β model monogenic forms of neurodevelopmental disorders, such as TGF- /activin/nodal pathways to induce forebrain fate. The resulting 3D aggregates were then transferred onto a coated surface Rett and Timothy syndromes, and even psychiatric disorders with – complex inheritance, such as schizophrenia (5–7). and maintained without any additional factors until day 45 70. On The brain and spinal cord develop according to distinct dif- day 25, cells within the SFEBq-like structures expressed early ferentiation programs from the earliest stages of CNS develop- neuroepithelial markers, including BLBP, N-CADHERIN, PAX6, ment (i.e., at the progenitor stage during gastrulation) (8, 9). and NESTIN (Fig. S1C). Regional differences in gene expression within stem and pro- genitor cells appear at the onset of the formation of both mouse (10, 11) and human CNS, as shown by recent studies of the human Author contributions: J.M., A.M.S., and F.M.V. designed research; J.M., M.V.S., L.T., and transcriptome using postmortem tissue (4). T.L.H. performed research; J.M., M.V.S., D.P., L.T., G.C., and F.M.V. analyzed data; and J.M., M.V.S., A.M.S., and F.M.V. wrote the paper. Neural cells are thought to differentiate by “default” into an fl anterior, forebrain-like fate (12), and indeed monolayer neuronal The authors declare no con ict of interest. cultures derived from hiPSCs and human embryonic stem cells *This Direct Submission article had a prearranged editor. (hESCs) can express some forebrain markers (6, 13, 14). However, Freely available online through the PNAS open access option. they have not yet been shown to recapitulate the transcriptional 1To whom correspondence should be addressed. E-mail: fl[email protected]. program that gives rise to the mammalian telencephalon, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their regional specification remains unclear (15). Using a recently 1073/pnas.1202944109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1202944109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 hindbrain (Fig. 1 P and Q). Additionally, cells did not express ISLET1 and showed barely detectable levels of OLIG2 (Fig. 1R), TFs both expressed by more posterior and ventral regions of the brain and in the spinal cord. We next investigated the potential of these 3D multilayered aggregates to generate inhibitory neurons. On day 50 of neuronal differentiation, we found presence of GABAergic precursor cells (DLX1/2+, MASH1+) as well as more differentiated GAD-67+ inhibitory neurons (Fig. S3). Strongly positive DLX+ cells ten- ded to be rounded and devoid of GAD-67+ processes (Fig. S3B, arrowheads), whereas cells weakly stained for DLX acquired long GAD-67+ processes (Fig. S3 A and C, arrows) and seemed to be migrating within the structure. Importantly, cells positive for inhibitory markers did not express the excitatory neuronal marker TBR1, suggesting that excitatory and inhibitory neuronal lineages are distinct and coexist in the preparation. The GABAergic component was always segregated from excitatory cells, in the center (Fig. S3A) or at the periphery (Fig. S3 D–F). The overall Fig. 1. Radial glia and excitatory progenitors differentiate from hiPSCs at day 50 in vitro. Day-50 forebrain-like structures contain radial glial cells immunoreactive for NESTIN, PAX6, and BLBP (A, B, and E), and βIIITUBULIN+ and TBR1+ neurons (B, D, and G). The radial glia express SOX1 and SOX2 (H–L), are mitotically active, as shown by Ki67 expression (M–O, red), and are polarized, displaying N-CADHERIN+ apical end-feet (C and F) as well as apical mitoses (M–O, arrowheads). A layer of TBR2+ intermediate progenitors (C and F) surrounds the radial glial layer and displays pH3+ basal mitoses (M–O, arrows). The neurons express MAP2 but rarely ZIC1 (P and Q) and do not express ISLET1 and OLIG2 (R). (Scale bars, 200 μminA–D and M;5μminE–G and I;10μminH, J–L, N, and O.) Immunofluorescence analysis of the 3D structures at day 45– 50 in serial cryosections revealed neural tube-like substructures within each aggregate, composed of radially arranged cells with apico-basal polarity, organized around a central lumen (Fig. 1). The radially arranged cells were PAX6+, NESTIN+, BLBP+, and GFAP+ and displayed N-CADHERIN immunoreactivity along their apical edge (Fig. 1 A–C, E, and F and Fig. S2 A–C). These radial glia-like progenitors expressed the neuroepithelial transcription factors (TFs) SOX1 and SOX2 (Fig. 1 H–L). Su- perficially to the radial glial layer were numerous neuronal precursor cells expressing T-box brain 2 (TBR2) (Fig. 1 C and F), a TF expressed by intermediate neuronal progenitors in the subventricular zone (SVZ) of the mammalian cerebral cortex Fig. 2. Evidence for synapses in hiPSC-derived multilayered structures at (20).