Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells

Abstract

Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

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Figure 1: Rapid induction of cortical neurons from hPSCs using a combinatorial small-molecule protocol.
Figure 2: Temporal and phenotypic characterization of hPSC-derived neurons by day 13 of differentiation.
Figure 3: Generation of neurons constituting multiple cortical layers upon long-term culture.
Figure 4: Accelerated induction yields hPSC-derived cortical neurons with mature electrophysiological properties in vitro.
Figure 5: Extensive axonal projections and integration of hPSC-derived neuron using P1S5D induction grafted into the neonatal mouse brain as assessed by iDISCO-based21 whole-mount brain imaging.
Figure 6: Summary of rapid cortical neuron induction paradigm.

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Acknowledgements

We would like to thank G. Wahl (Salk Institute) and R. Jaenisch (Whitehead Institute) for sharing plasmids, F. Zhang (MIT) for sharing the TALE-toolbox, and G. Ciceri and G. Cederquist (MSKCC) for their valuable input on experimental design and feedback on the manuscript. We thank M. Sheldon (Rutgers University) for sharing protocol for fibroblast preparation. This work was supported in part through grants from the Starr Foundation (L.S. and A.H.B.) and grants NS084334 and R01NS072381(L.S.) and by NYSTEM contracts C030137 (S.S., & L.S.) and C028128 (A.H.B.) and private funds from the Rockefeller University. The Molecular Cytogenetics Core Facility at MSKCC as well as other MSKCC facilities and investigators are supported by the NIH Cancer Center support grant P30 CA008748. Some of the images were obtained using instrumentation at The Rockefeller University Bio-Imaging Resource Center. The SKI Stem Cell Research Facility is supported by NYSTEM grants C029153 and C024175 and The Starr Foundation. X.-J.Z. and B.Z. were supported by NYSTEM fellowships (C026879).

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Contributions

Y.Q.: conception and study design, hESC manipulation, differentiation and characterization, in vitro and in vivo analyses and data interpretation and writing of manuscript. X.-J.Z.: electrophysiological recordings, in vivo transplantation, data analysis, interpretation and writing of manuscript. N.R. and Z.W.: iDISCO analysis of grafted animals, data analysis, interpretation and writing of manuscript. T.A. and Z.S.: iPSC differentiation studies, in vitro functional and electrophysiological analyses. M.Z.O. and A.H.B.: generation of the CUX2-tdTomato reporter line and writing of the manuscript. J.T. and B.Z.: generation of PAX6 and SIX1 reporter lines, data analysis. F.F. and N.Z.: neural crest differentiation protocols and data analysis. Y.G.: transplantation studies. R.A.: iDISCO analysis. M.K. and J.G.: iPSC differentiation studies, data interpretation. M.T.: iPSC induction and characterization, data analysis. M.T.-L.: design and interpretation of iDISCO studies, writing of manuscript.S.-H.S.: conception and study, data analysis and interpretation, writing of manuscript. L.S.: conception and study design, data analysis and interpretation, writing of manuscript.

Corresponding author

Correspondence to Lorenz Studer.

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Competing interests

The Memorial Sloan-Kettering Cancer Center has filed a provisional patent application (US PRO 62/287821) on the methods described in the manuscript.

Integrated supplementary information

Supplementary Figure 1 Dosage-dependent response of proliferation and viability upon P/S/D treatment.

(a) Percentage of mitotic cells expressing phospho-histone 3 (pH3) among total cells at one day after P/S/D treatment (day 3 of differentiation). (b) Percentage of apoptotic cells expressing cleaved caspase 3 (CC3) among total cells at one day after P/S/D treatment (day 3 of differentiation). The conditions highlighted in the dashed line boxes in a,b are the P1 dosage groups aligned by ascending order of S concentration. For a,b, N = 4 randomly selected photo frames from each of the 2 independent batches of cell cultures. Statistical analysis was carried out using the Dunnett’s multiple comparison test to compare each dosage with LSB+X at day 3 of differentiation. Only those comparisons that are significantly different from LSB+X are marked on the graph. (c) Summary of grouped results in a following the order of increasing concentration of P, or increasing concentration of S (d). (e) Summary of grouped results in b following the order of increasing concentration of P, or increasing concentration of S (f). For c-f, statistical analysis was carried out using the Dunnett’s multiple comparison test to compare each dosage of P with the no P group (c,e) and each dosage of S with the no S dosage groups (d,f). (g) Nomenclature for the various dosage groups shown in c-f. Black dots represent values from quantification of individual photos frames. Error bars represent s. e. m. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001.

Supplementary Figure 2 Gating of flow cytometry and impact of various small-molecule manipulations on FOXG1 expression.

(a) Gating of intracellular flow cytometry of TUJ1+ neuron population at day 13 for the various culture protocols used (blue: TUJ1 stain. red: isotype control). (b) Immunocytochemistry for FOXG1/TUJ1 co-expression at day 13. (c) Quantification of FOXG1 transcript expression level by qRT-PCR under P1S5D conditions but after systematic removal of one of the small molecules each. N = 3 independent batches of cell cultures. Scale bars: 100 μm. Error bars represent s. e. m.

Supplementary Figure 3 Characterization of additional fate markers at day 13 of differentiation.

(a) Expression of layer VI marker TLE4 in post-mitotic neurons, and CUX2 expression in progenitors at day 13 of differentiation in both P1S5D and P8S10D induction. (b) Quantification of mRNA expression at day 13 of markers other than TBR1 representing different brain areas and fates. N = 3 independent batches of cell cultures. Cortical progenitor marker OTX2, ZNF521, BRN2 and COUPTF1, layer V cortical neuron marker CTIP2, and the vesicular glutamate transporter VGLUT1 are upregulated compared to LSB+X. (c) Expression of BRN3A+ and ISL1+ cells in both P1S5D and P8S10D cultures at day 13. (d) Quantification of BRN3A+ cells at day 13. Scale bars: 50 μm. Error bars represent s. e. m.

Supplementary Figure 4 Rapid cortical neuronal induction in hiPSC lines.

(a) Validation of P1S5D and (b) P8S10D protocols on hiPSC lines by immunocytochemistry of TBR1/TUJ1 expression at day 13. (c) Quantification of neuronal induction efficiency at day 13 by intracellular flow cytometry for various hiPSC lines tested. N = 3 independent batches of cell cultures for line 1.1, 1.2, 1.3, and N = 1 for line 7.1, 7.2 and 7.4. Scale bars: 50 μm. Error bars represent s. e. m.

Supplementary Figure 5 Molecular characterization of long-term culture beyond day 13.

(a) Quantification of mRNAs expression in P1S5D and P8S10D treated culture (Fig. 3a) compared to LSB+X treated cultures. RGS4: cortical layer II-III,V marker. CHX10: retinal marker. GFAP, AQP4: astrocyte marker. OLIG2: oligodendrocyte precursor marker. For long-term culture of LSB+X cells, cells were maintained in N2 medium without re-adding small-molecule inhibitors. N = 3 independent batches of cell cultures. Error bars represent s. e. m.

Supplementary Figure 6 Generation of the CUX2-CreERT2 conditional reporter hPSC line and early generation of CUX2+ neurons in both P1S5D and P8S10D treated cells.

(a) Design of the homology donor targeting the CUX2 first exon. The selection cassette was excised upon expression of Flp recombinase after transgenesis. The location of primer sequences to confirm targeting is shown. (b) Schematic illustration of the targeted alleles. The transgenic lines express CreERT2 from the CUX2 locus and the FLEX-tdTomato conditional reporter under the CAG promoter at the AAVS1 safe harbor locus. Expression of CUX2 in the presence of 4OHT induces recombination at the reporter locus and tdTomato expression. (c) PCR confirmation of targeted transgenesis. Lanes 1-2 confirm 5’ and 3’ CreERT2 insertions at the CUX2 genomic locus, respectively, while lanes 3-4 confirm 5’ and 3’ CAG-FLEX/tdTomato insertions at the AAVS1 genomic locus, respectively. (d) Genomic DNA sequencing at the CUX2 locus confirms successful targeting of one allele and a wild-type non-targeted allele. (e) tdTomato positive post-mitotic neurons at day 70 in culture after 4OHT induction (i). Typical pyramidal morphology and lengthy projections can be observed upon higher magnification (ii). (f) CUX2+ neurons with mature morphologies observed in P1S5D and P8S10D culture at day 33 of differentiation. (g) Pyramidal morphology at day 33 suggests cortical projection neuron identify of P1S5D and P8S10D neurons. Scale bars in e (i) represents 100 μm, while those in others represent 50 μm.

Supplementary Figure 7 Summary of electrophysiological parameters for P1S5D cultures in long-term culture maintained in the absence of small molecules.

(a) Illustration of the P1S5D+none treatment analyzed in b,c for increasing levels of maturation upon further differentiation. (c) Time course quantitative analysis of electrophysiological properties of P1S5D+none conditions through day 37. Note that as time proceeded, resting membrane potential became hyperpolarized, input resistance decreased, Na+ channel current increased, action potential threshold decreased and the maximum firing frequency increased. Statistics was carried out first using ordinary one-way ANOVA to determine if statistically significant differences exist among the means of each group: F=0.3222, P=0.8093, R2=0.0248 (REM); F=7.554, P=0.0023, R2=0.5862 (half-width); F=0.7654, P=0.5209, R2=0.0560 (rising Tau); F=4.88, P=0.006, R2=0.2891 (input resistance); F=7.364, P=0.0005, R2=0.3676 (frequency). Then the Dunnett’s multiple comparison test was used to compare mean values of each group to day 16. Only those comparisons that are significant were marked on the graph. Error bars represent s. e. m. * P<0.05, ** P<0.01, *** P<0.001.

Supplementary Figure 8 Co-culture of hPSC-derived neurons with astrocyte or astrocyte conditioned medium.

(a) Schematic illustration of long-term maintenance of P8S10D+D neurons with astrocyte co-culture or conditioned media. (b) Bright field images of P8S10D+D neurons co-cultured with astrocytes or conditioned media at day 25 and day 35. (c) Representative traces of action potential firings of P8S10D+D neurons co-cultured with astrocytes or conditioned media at day 25 and day 35, evoked by current injection from -30 to +100 pA. (d) Bright field images of P8S10D+D neurons co-cultured with astrocytes at day 90. N = 15, 23, 7, 5 cells recorded for cultures with astrocytes at day 25, 35, and cultures in conditioned medium at day 25, 35. (e) Quantitative analysis of passive membrane properties and action potential properties. (f) MAP2ab staining of P8S10D+D neurons with astrocytes co-culture shows increased complexity of dendrite branching with time in culture. (g) Sholl analysis at day 36 of P8S10D+D neurons co-cultured with astrocytes, compared with neurons cultured with conditioned medium alone. Scale bars: 50 μm. Error bars represent s. e. m. *** P<0.001.

Supplementary Figure 9 iDISCO based whole brain immunofluorescence analyses of P8S10D and LSB+XAV grafts at 1 month after transplantation.

(a) P8S10D grafted half brain, stained for GFP (whole view and details of the frontal cortical region). P8S10D grafts showed inconsistent survival after transplantation into neonatal mouse cortex. However, animals with surviving graft showed long fiber projections across cortical regions. GFP+ cells devoid of axons were abundantly detected outside of the graft (boxed region). (b) LSB+X grafted half brain, stained for GFP. Whole view (side and dorsal) and detail of the graft margin (boxed region). LSB+X grafts showed massive overgrowth in host brain resulting in tumor-like structures with very limited evidence of neuronal differentiation and maturation. Only a few short fiber tracts can be seen at the margin of the graft (boxed region). N = 2 animals for P8S10D condition, and 4 animals for LSB+XAV condition analyzed. Scale bars: 500 μm.

Supplementary Figure 10 Trajectories and morphologies of P1S5D grafted neurons at 1.5 months after transplantation.

(a) Landmarks of the adult mouse brain are identified by tissue autofluorescence. The image represents 100-μm thick maximum projection of optical sections taken at the center of an adult mouse brain after iDISCO processing, and shows the major myelinated tracts from 488 nm laser excitation via endogenous fluorescence. (b) Examples of axons from grafted neurons following major pathways. iDISCO treated 1.5 months old mouse brain grafted at birth, stained for GFP. In the hippocampus, grafted neurons that are present in CA3 (arrow) and are sending axons along the fimbria tract toward the septum. In the cortex, grafted neuron project fibers across the hemisphere following callosal axons. (c) Examples of axons from grafted neurons not following major pathways. Maximum projections, 100 μm thick, of whole iDISCO treated 1.5 months old mouse brains grafted at birth, stained for GFP. In the striatum, GFP+ axons descending from the grafted neurons in the cortex are seen mainly outside of the main descending tracts. In the cortex, large bundles of GFP+ axons are observed in aberrant position. (d) A few representative morphologies seen in the grafted brains: most human neurons are clustered in the graft core, and therefore their dendritic morphologies are masked. However, a few GFP+ neurons are found outside of the graft core and exhibit diverse morphologies as shown. N = 3. Scale bars: 1 mm (a), 500 μm (b,c), 200 μm (d).

Supplementary Figure 11 in vivo electrophysiological properties of P1S5D grafted neurons.

(a, b) Recordings of action potentials and firing patterns as well as sEPSCs from a GFP+ graft at P10 (a) and P30 (b), respectively, with unusually mature properties. (c,d) Most GFP+ cells in grafts exhibited more immature firing patterns as illustrated by representative action potentials in c and sEPSCs in d recorded at P15 and P45. N = 4 animals for P1S5D condition analyzed, and 1 animal for P8S10 condition analyzed.

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Qi, Y., Zhang, XJ., Renier, N. et al. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells. Nat Biotechnol 35, 154–163 (2017). https://doi.org/10.1038/nbt.3777

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