Modeling human cortical development in vitro using induced pluripotent stem cells

Characterization of hiPSC Lines.

We used two hiPSC lines: PGP1-1 (17) and i03-01#9, a line derived in our laboratory. Both lines were established from healthy control adult male skin fibroblasts through retroviral reprogramming vectors expressing OCT4, SOX2, KLF4, and c-MYC (18). These hiPSC lines exhibited typical hESC morphology and expressed canonical pluripotency markers by immunocytochemistry as well as NANOG, DNMT3B, and other endogenous mRNAs that characterize pluripotent cells (Fig. S1 A and B). Pairwise correlations of global gene expression data obtained by microarrays (Dataset S1) showed high similarity in gene expression between PGP1-1 and the hESC line H1 (Pearson’s r > 0.978), i03-01#9 and H1 (Pearson’s r > 0.975), and PGP1-1 and i03-01#9 (Pearson’s r > 0.979).

We then tested the hiPSC lines with Pluritest (http://www.pluritest.org), an approach that classifies samples according to more than 450 genome-wide transcriptional profiles, including 223 hESC and 41 hiPSC lines from multiple laboratories (19). The i03-01#9 and PGP1-1 displayed Pluritest scores consistent with normal human pluripotent cells and clustered together with the H1 hESC line (green arrowhead, Fig. S1D). Taken together, these results confirm that PGP1-1 and i03-01#9 fulfill the established criteria for completely reprogrammed iPSC lines.

Excitatory Neuronal Precursor Cells Are Generated Within Multilayered Structures Derived from hiPSCs.

Undifferentiated PGP1-1 and i03-01#9 colonies were dissociated into single cells and cultured in suspension in the presence of FGF2 (5–20 ng/mL) and inhibitors of the bone morphogenetic protein (BMP), Wnt/β-catenin, and TGF-β/activin/nodal pathways to induce forebrain fate. The resulting 3D aggregates were then transferred onto a coated surface and maintained without any additional factors until day 45–70. On day 25, cells within the SFEBq-like structures expressed early neuroepithelial markers, including BLBP, N-CADHERIN, PAX6, and NESTIN (Fig. S1C).

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). Superficially 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 (20). The proliferative markers Ki67 and phosphohistone H3 (pH3) were expressed by both radial glial cells and the surrounding intermediate precursors, and pH3 was expressed at the luminal (apical) surface, suggesting that radial glia undergo interkinetic nuclear migration (Fig. 1 M–O). More mature neurons (βIIITUBULIN⁺, MAP2⁺) also expressing T-box brain 1 (TBR1) (21), presumably progeny of TBR2⁺ precursors (20), were found superficially (basally) to the TBR2⁺ layer (Fig. 1 B, D, G, and P), reproducing the typical architecture of the developing mammalian cortex (22). In contrast, we found an almost complete absence of cells expressing ZIC1, a TF expressed in the 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.

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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 μm in A–D and M; 5 μm in E–G and I; 10 μm in H, J–L, N, and O.)

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 tended 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 percentage of cells expressing GAD-67 was 6.9 ± 1.0 and 7.4 ± 0.7 in two different preparations.

Neuronal Connectivity in the Multilayered Structures Derived from hiPSCs.

At day 70 of differentiation in vitro, the majority of polarized radial glial cells disappeared from the aggregates, together with the PAX6 and BLBP protein expression. Electron microscopy revealed the presence of immature synaptic boutons containing vesicles (Fig. 2 A–D, arrows), which contacted large neuronal cells via thickenings resembling immature synapses, in that the postsynaptic membrane specializations and associated structures did not appear fully developed. Immunostaining for the presynaptic markers synaptophysin (SYP) and synapsin I (SYN I), as well as postsynaptic density protein 95 (PSD-95), revealed extensive synaptic differentiation (Fig. 2 E, F, P, and Q). Immunoelectron microscopy demonstrated SYP immunoprecipitate within boutons containing synaptic vesicles (Fig. 2 G–O), which contacted dendrites and spines via synapses (Fig. 2 J–O, arrowheads). We also observed extensive immunostaining for the vesicular glutamate transporter vGLUT1 at both 50 and 70 d in culture, consistent with glutamatergic synapses differentiation (Fig. S4 I–K). By day 70, astroglial cells differentiated throughout the culture, as demonstrated by the presence of GFAP/S100β immunostained cells, particularly in the outer region (Fig. S2 D–I).

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Fig. 2.

Evidence for synapses in hiPSC-derived multilayered structures at 50 and 70 d in vitro. (A–D) Day-70 multilayered structures analyzed by EM show examples of direct apposition between vesicles-containing putative axon terminals (black arrows) and neuronal cell bodies. (E and F) Synaptophysin/neurofilament 200 double immunostaining at day 70 showing numerous synaptophysin-labeled boutons decorating the neurites. (G–O) Immunoelectron microscopy showing synaptophysin-immunoprecipitate within putative axon terminals contacting cell bodies (soma) (G–I), dendrites (d) (J–L), and spines (s) (M–O). White arrowheads, synaptic thickening; m, mitochondia; a, autophagosome. (P and Q) Day-50 multilayered structures stained for SYN I and βIIITUBULIN (P) or PSD-95 and MAP2 (Q). White arrows in P point to SYN I-positive boutons. (Scale bars, 2 μm in A and C; 0.5 μm in B and D; 10 μm in E, P, and Q; 5 μm in F; 1 μm in G–O.)

Global Gene Expression Analysis Reveals the Implementation of a Forebrain Neuronal Gene Expression Program.

To ascertain whether these hiPSC-derived structures underwent a region-specific differentiation program, we assessed their gene expression profiles by microarrays. Whole-genome expression data analyzed by the Pluritest demonstrated progressively lower pluripotency scores and higher novelty scores, suggesting ongoing neuronal differentiation (Fig. S1D). Gene expression was compared between 3D multilayered structures at day 50 in vitro and their corresponding undifferentiated hiPSC lines PGP1-1 (one preparation) and i03-01#9 (four preparations). Hierarchical clustering of the samples demonstrated a highly consistent pattern of gene expression across all lines and preparations (Figs. S5 and S6). The combined dataset, including all hiPSC lines and different 3D preparations analyzed together, revealed that 3,363 genes were differentially expressed in day-50 multilayered structures compared with the undifferentiated hiPSC stage (P value ≤0.05 together with a fold change of ±2.0), of which 1,629 genes increased and 1,734 genes decreased in abundance. The top down-regulated molecules (between −9,110- and −1,384-fold) were LIN28A, DPPA4, RAB17, CDH1/E-CADHERIN, OCT4/POU5F1, ZSCAN10, and GAL, reflecting genes commonly expressed in hESCs and hiPSCs and epithelial-specific genes (boxed areas in Fig. S5A). In addition to OCT4, the expression of the other reprogramming factors (SOX2, KLF4, and c-MYC) as well as of other genes characterizing a pluripotent state (NANOG, DNMT3B, and JARID2) was also down-regulated (Dataset S1). Conversely, mRNAs encoding for known effectors of neuronal differentiation increased in abundance, including MAP2, βIII TUBULIN, ephrins and their receptors, semaphorins, and neural cell adhesion molecules (Table S1). Genes involved in neurotransmission were also up-regulated, including SNAP25, SNAP29, SNAP91, SYN I, SYP, Bassoon, PSD-95, and glutamate ionotropic and metabotropic receptors (Table S1).

Ingenuity pathway analysis for differentially expressed genes in day-50 3D structures revealed that the most significantly enriched biological function categories were Nervous System Development and Function, Cancer, Cellular Assembly and Organization, and Neurological Disease (Dataset S2). The most significant gene ontology terms under the Nervous System Development and Function category were Neurogenesis of Cerebral Cortex and Hippocampus, Guidance of Neurites, Brain Development, Hippocampal Development, Cortical Development, Neuronal and Glial Differentiation, and Synaptic Transmission, and most of these included up-regulated genes. In contrast, the Cancer category included predominantly down-regulated genes with known involvement in the control of cell cycle, suggesting that the majority of the neuronal cells at day 50 are likely already postmitotic (Dataset S2). Virtually all terms in the Cellular Assembly and Organization category involved neural cell functions, such as growth and fasciculation of neurites and synapse formation (Dataset S2). Significant Neurological Disease associations included genes implicated in disorders of the developing nervous system, such as schizophrenia, and neurodegenerative disorders, such as Alzheimer’s and Huntington disease (Dataset S2).

Consistent with the above characterization, the top DAVID gene ontology clusters were Neuron Differentiation/Morphogenesis, Synapse Formation, Forebrain/Pallium Development, and Regulation of Transcription (Dataset S3). Thus, differentially expressed genes in hiPSC-derived structures at day 50 in vitro converged toward telencephalic development, neurogenesis, neuronal process outgrowth, and synaptogenesis.

Comparison of gene expression in our hiPSC-derived cells with normal human neural progenitors (NHNP) derived from early human embryo of ∼8–19 gestational weeks and differentiated in vitro (23) revealed a significant similarity of gene expression patterns. Among the 4,427 genes significantly changed in NHNP between time 0 and 4 wk of neuronal differentiation in vitro (23) and the 3,363 genes significantly changed between the hiPSC and 50 d in vitro stage in our model, 1,100 genes were in common. The P value for such overlap is <10⁻¹² (calculated using numerical simulations and tail probability estimation).

Regional Specificity of Multilayered Aggregates Is Most Consistent with a Dorsal Telencephalic Fate.

To further understand whether our 3D multilayered structures were regionally specified as reflected by their pattern of gene expression, we took into account published datasets of telencephalic gene expression profiles (24, 25), literature data, and the Allen brain atlas of gene expression database. Overall the expression pattern of the hiPSC-derived multilayered structures at day 50 in vitro was more typical of dorsal forebrain than ventral forebrain (Fig. 3A) and excluded hindbrain and spinal cord fates. For instance, genes that confer hindbrain and spinal cord fates, such as HOX genes, GBX2, HB8, NKX2.2, OLIG2, EVX1/2, ZIC1/2, and GCM1, and transcripts specific of olfactory bulb development, such TBX21, were not expressed or down-regulated (Fig. 3A and Dataset S1). Among the top 10 genes with the highest degree of up-regulation in the day-50 neural structures were those involved in specification and patterning of the mammalian neocortex, including LHX2, LEF1, TBR2, PAX6, FEZF2, and RELN (Fig. 3A, Table 1, and Fig. S5B, boxed area). Very high levels of up-regulation were also noted for COUP-TF1, EMX2, and EMX1 and for other genes involved in cortical architecture and layer identity, such as TBR1, CTIP2, CUX1, CUX2, POU2F2/BRN2, and TLX (Table 1). Some genes involved in GABAergic differentiation were also expressed (including MASH1 and ARX), but the basal telencephalon differentiation program did not seem to be fully implemented (Fig. 3A and Fig. S3). This observation is intriguing, given previous findings suggesting that a lineage of human neocortical GABAergic progenitors expressing MASH1 originates from the dorsal forebrain (26).

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Fig. 3.

Regional and temporal specification of hiPSC-derived neuronal cells. (A) Fold differences in neuronal gene expression compared with undifferentiated hiPSCs for dorsally and ventrally enriched telencephalic genes. The gene lists were manually curated according to published data (24, 25, 35). Orange, genes that are statistically different at P < 0.05 and fold change ±2.0; gray, genes that do not fulfill the minimum P value criterion. (B) Number of developing human brain samples in Kang et al. (4) correlating with the day-50 multilayered structures within 95% confidence interval of the maximal correlation. Human brain samples are displayed according to developmental stages (Upper) or brain regions (Lower). PCW, postconceptional week; M, months; Y, years; FC, frontal, PC, parietal, TC, temporal, and OC, occipital cerebral cortical wall; HIP, hippocampal anlage; AMY, amygdala; DIE, diencephalon; URL, upper rhombic lip.

Transcription Factors Expression Patterns Suggest Dynamic Acquisition of Cortical Layer Identities.

Using a panel of antibodies directed to TFs that control cortical layer fates during normal cortical development (22, 27⇓–29), we next investigated whether the implementation of the neocortical transcriptional program resulted in the specification of cortical excitatory neuron subtypes. We found that MAP2⁺ neurons coexpressed TFs that specify a wide spectrum of upper and lower cortical layer identity. These included TBR1, CTIP2, TLE4 (cortical layers V–VI) and BRN2 and SATB2 (cortical layers II–IV) (Fig. 4). The proportion of neurons for lower layers was 30–40%, whereas that for upper layers was 10–20% (Fig. 4S). Double-staining experiments showed that TBR1 and TLE4 (layers V–VI) colocalized with CTIP2 (layer V) in many neurons, but cells displayed predominant expression of either one factor or another (Fig. 4 C–F and J–M and Fig. S4 A–D). In contrast, cells of lower layers (TBR1, CTIP2, and TLE4) showed more segregated pattern of expression from those of upper layers (SATB2 and BRN2) (Fig. 4 G–I, N–R, and S and Fig. S4 E–H and L). In addition to SATB2 and BRN2 proteins, a number of TFs required for cortical upper layer formation were up-regulated at the mRNA level: CUX1 and CUX2 (fourfold), BRN2 (14-fold) (Fig. 3A and Table 1), PAX6 (970-fold), and TLX (43-fold) (Table 1). No appreciable differences in neuronal differentiation and substructural organization were observed between the two iPSC lines (compare Fig. 4 and Fig. S4) and concentrations of FGF2 (Fig. 4S).

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Fig. 4.

Specification of cortical layer fates in hiPSC-derived neurons. (A) Scheme of TF directing cortical layer fates. (B–R) Immunostaining for TFs in sections from 3D structures at day 50 in vitro. (B) Double immunostaining for MAP2 and CTIP2, showing CTIP2 nuclear localization in MAP2⁺ cells; (C–F) TBR1 and CTIP2; (G–I) TBR1 and SATB2; (J–M) TLE4 and CTIP2; (N–P) TLE4 and SATB2; and (Q and R) TBR1 and BRN2, showing differences in relative colocalization of these TFs in differentiating neurons. White arrowheads, cells stained for a single TF; white arrows, cells that colocalize two factors. Broken line in Q delimits the radial glial layer where BRN2 is also expressed but was excluded from quantification. (S) Stereological quantification of TF expression in 3D structures cultured with different amounts of FGF2 for the first 18 d. DAPI is in blue. (Scale bars, 200 μm in B and G; 100 μm in C–F and J; 25 μm in H and I; 20 μm in K–P.)

Correspondence with Patterns of Gene Expression in Developing Human Brain.

To verify the conclusion that the mRNA and protein expression patterns of our hiPSC-derived multilayered structures at day 50 are typical of the human dorsal forebrain, we performed a pairwise correlation analysis (details in SI Materials and Methods) between the global gene expression levels of our samples (including the hiPSC and the day-50 multilayered structures) and 1,340 postmortem human tissue samples at 15 different developmental stages, comprising the cerebellar cortex, mediodorsal nucleus of the thalamus, striatum, amygdala, hippocampus, and 11 areas of the neocortex (4). The day-50 multilayered aggregates from the i03-01#9 line showed the highest correlation coefficients with the human postmortem dataset, and correlated only (as estimated by the 95% confidence interval) with human cerebral cortex at 4–10 postconceptional weeks (PCW), with a prevalence for 8–10 PCW and frontal regions (Fig. 3B).