Differentiation from human pluripotent stem cells of cortical neurons of the superficial layers amenable to psychiatric disease modeling and high-throughput drug screening

Abstract

Cortical neurons of the superficial layers (II-IV) represent a pivotal neuronal population involved in the higher cognitive functions of the human and are particularly affected by psychiatric diseases with developmental manifestations such as schizophrenia and autism. Differentiation protocols of human pluripotent stem cells (PSC) into cortical neurons have been achieved, opening the way to in vitro modeling of neuropsychiatric diseases. However, these protocols commonly result in the asynchronous production of neurons typical for the different layers of the cortex within an extended period of culture, thus precluding the analysis of specific subtypes of neurons in a standardized manner. Addressing this issue, we have successfully captured a stable population of self-renewing late cortical progenitors (LCPs) that synchronously and massively differentiate into glutamatergic cortical neurons of the upper layers. The short time course of differentiation into neurons of these progenitors has made them amenable to high-throughput assays. This has allowed us to analyze the capability of LCPs at differentiating into post mitotic neurons as well as extending and branching neurites in response to a collection of selected bioactive molecules. LCPs and cortical neurons of the upper layers were successfully produced from patient-derived-induced PSC, indicating that this system enables functional studies of individual-specific cortical neurons ex vivo for disease modeling and therapeutic purposes.

Figure 1

Differentiation of human embryonic stem cells (hESC) into late cortical progenitors (LCPs). (a) Regional profiling of neuroepithelial cells (NEPs) 10 days after induction of neural commitment. Results represent mean +/− s.d. of three independent biological replicates. (b, c) Representative photomicrographs illustrating PAX6 (b) and BF1 (c) expression in hESC-derived neural rosettes. NEPs organized into nestin immunopositive neuroepithelial structures (arrow) surrounding a lumen (star) with an apical side expressing ZO-1 (d, e). Loss of expression of ZO-1 and maintenance of strong expression of nestin after the first passage (g). After several passages homogeneous neural cell population (h) that does not express ZO-1 but maintains nestin expression (i) and express PAX6 (j), Blbp (k) and Sox2 (l) but neither Tbr2 (m) nor Tuj-1 (n). (o) Proportion of cells expressing radial glia markers, and of proliferating neural progenitors (Ki67/Sox2 double-immunopositive cells). (p) Expression profile of progenitors amplified in EFB medium. Green bars indicated genes downregulated in neural progenitors compared with the parental hESC, genes upregulated are shown in red. Results represent mean +/− s.d. of three independent experiments. Scale bar =100 μm.

Figure 2

Production of superficial-layer neurons from late cortical progenitors (LCPs). (a, b) Cortical neurons produced by NEP differentiation 21 days after mitogen withdrawal. (c) HuC/D and (d) Tuj-1 immunopositive neurons produced by LCPs 21 days after mitogen withdrawal. (e–i) Immunocytochemical characterization of neurons produced by LCPs. (m) Automated quantification of the proportion of HuC/D and Tuj-1-positive neurons produced by LCPs after 21 days of differentiation. (n) Automated quantification of HuC/D-positive neurons expressing markers of a neuronal subtype or (o) of a cortical molecular layer. (p) Molecular profiling by RT-qPCR of hESC-derived early neuroepithelial cells (NEP), LCPs and differentiated neurons at different time points of differentiation. Results are expressed as mean±s.d. of three independent experiments. Scale bar=50 μm. TH, tyrosine hydroxylase.

Figure 3

Amenability of late cortical progenitors (LCP) terminal differentiation into a high-throughput screening format. (a) Automated quantification showing the kinetics of neural proliferation (Ki-67) and neuronal differentiation (HuC/D) of LCPs in 384-well plates. (b) Automated quantification of the kinetic of neuritic outgrowth (Tuj-1-positive neurites). Results represent mean +/− s.d. of each parameter per field of 109 000 μm² in at least 16 independent wells (representative of one of three independent experiments). (c, d) Response of LCPs to increasing doses of DAPT (c) or Wnt3a (d). Results are expressed as mean +/− s.d. (e) Representative photographs captured by the Arrayscan microscope.

Figure 4

High-content screening of a library of 130 bioactive chemical compounds. (a)Screening flowchart. (b) List of compounds that increased the proportion of HuCD-positive neurons in the culture by more than 15%. (c) Representative results captured by the Arrayscan microscope. (d) List of compounds that modified significantly neurite length and/or arborization. (e) Representative results captured by the Arrayscan microscope, including the tracking of neurites. P-values were calculated using the Dunnett's test after an analysis of variance, NS; non significant.

Figure 5

Differentiation of induced pluripotent stem cells (iPSC) into neurons of the superficial layers. (a) Representative immunocytochemistry of late cortical progenitors (LCP) and cortical neurons produced from four different iPSC lines. 4603 and 1869 iPSC lines were derived from control donors and autism spectrum disorder 1 (ASD1) and ASD2 from autistic children with SHANK-3 mutations. Scale bar=50 μm (b) Kinetic of differentiation of iPSC-derived cortical neurons. (c) Percentage of upper layers neurons produced after 17 days of differentiation of iPSC-derived LCP. For (b, c) results are expressed as mean +/− s.d. of three independent experiments.

Figure 6

Overview of the timelines of the differentiation protocol. Phase I (days 0–35) corresponds to the steps of differentiation of human pluripotent stem cells (hPSC) into early cortical progenitors, the subsequent phenotypic transition to late cortical progenitors (LCP) and further amplification to create a large cell bank. After this phase, neuronal differentiation into cortical projection neurons is achieved in 14–17 days (phase II). As neuronal differentiation can be started directly from the stock of frozen LCP, the phase 1 does not need to be completed at each experiment. Liquid N2; liquid nitrogen.