Abstract

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.

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Data availability

Gene expression data are available in the Gene Expression Omnibus (GEO) under accession number GSE112137. The data that support the findings of this study are available on request from the corresponding author.

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Acknowledgements

We thank W.E. Benitz, D.K. Stevenson, V. K. Bhutani and U. Francke for valuable scientific advice and discussions. This work was supported by the US National Institute of Health (NIH) BRAINS Award (MH107800), the MQ Fellow Award, the NYSCF Robertson Stem Cell Investigator Award, the Stanford Neurosciences Institute’s Human Brain Organogenesis Program and the Brain Rejuvenation Project, Stanford Bio-X, the Kwan Research Fund and the California Institute of Regenerative Medicine (CIRM) (to S.P.P.); the UCSF Weill Institute for Neurosciences (startup funds to A.J.W.); NIH R01MH108659 and R01MH108659 (to T.D.P.); and the NIH K12-HD000850 (Pediatric Scientist Development Program), the Association of Medical School Pediatric Department Chairs (AMSPDC) and Stanford Maternal and Child Health Research Institute Fellowship (to A.M.P.).

Author information

Affiliations

  1. Department of Pediatrics, Division of Neonatology, Stanford University, Stanford, CA, USA

    • Anca M. Pașca
  2. Department of Psychiatry and Behavioral Sciences & Stanford Human Brain Organogenesis Program, Stanford University, Stanford, CA, USA

    • Jin-Young Park
    • , Hyun-Woo Shin
    • , Omer Revah
    • , Ruth O’Hara
    •  & Sergiu P. Pașca
  3. Department of Pharmacology and Biomedical Sciences, Seoul National University, Seoul, Republic of Korea

    • Hyun-Woo Shin
  4. Institute for Neurodegenerative Diseases and Department of Psychiatry, UCSF Weill Institute for Neurosciences, University of California, San Francisco, CA, USA

    • Qihao Qi
    • , Rebecca Krasnoff
    •  & A. Jeremy Willsey
  5. Department of Neurosurgery, Stanford University, Stanford, CA, USA

    • Theo D. Palmer

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Contributions

A.M.P. and J-Y. P. performed the neural differentiation and hypoxia experiments. A.M.P., J.-Y.P., H.-W.S., O.R., Q.Q., R.K., A.J.W., R.O. and T.D.P. carried out experiments, analyzed data or contributed critical reagents. A.M.P. and S.P.P. wrote the manuscript with input from all authors. S.P.P. supervised the work.

Competing interests

Stanford University has filed a provisional patent application that covers the generation of region-specific brain organoids from pluripotent stem cells (US Application Serial No. 15/158,408) (A.M.P. and S.P.P.).

Corresponding author

Correspondence to Sergiu P. Pașca.

Extended data

  1. Extended Data Fig 1 Oxygen tension and c-CAS3 in hCS following hypoxia.

    a, Oxygen tension (\(P{\mathrm{o}_2}\), mm Hg) measurements inside hCS from Fig. 1b, shown as a function of depth (values for each hCS was normalized as a function of its radius) (n = 6 hCS for 21% O2 and n = 7 hCS for <1% O2; from three hiPS cell lines). Shaded area indicates s.e.m. b,c, Representative western blots (b) and quantification (c) of c-CAS3 in hCS after 24 h and 48 h of exposure to <1% O2 and at 72 h after reoxygenation (one-way ANOVA, F3,6 = 1.56, P = 0.29); normalized to β-actin (n = 3 differentiated hiPS cell lines with two hCS per condition; each line is shown in a different color). Western blots were cropped to show the relevant bands; molecular weight markers are indicated on the left (in kDa). See Supplementary Table 2 for quantifications. Uncropped blots are available as source data. Data are the mean ± s.e.m. Individual values are indicated by dots. Source data

  2. Extended Data Fig 2 Gene expression changes in hCS following oxygen deprivation.

    a, Hierarchical clustering of RNA-seq data showing clustering of samples on the basis of exposure to oxygen concentration. Samples (n = 24) from hCS differentiated from three hiPS cell lines were collected after 24 h or 48 h exposure to <1% O2, as well as 72 h after reoxygenation. We clustered on the basis of all differentially expressed genes identified (1,754 unique genes). Clustering with all expressed genes results in a similar dendrogram (data not shown). b,c, Validation by qPCR of hypoxia-related genes PLOD2 (two-tailed paired t-test, **P = 0.006), PFKP (two-tailed paired t-test, **P = 0.008), PDK1 (two-tailed paired t-test, ***P = 0.0005) and IGFBP2 (two-tailed paired t-test, ***P = 0.0002), and cortical progenitor and cell cycle-related genes EOMES (also known as TBR2) (two-tailed paired t-test, **P = 0.006), EMX1 (two-tailed paired t-test, *P = 0.02), ASPM (two-tailed paired t-test, **P = 0.002) and CENPF (two-tailed paired t-test, ****P < 0.0001), which were identified in the RNA-seq analysis (n = 4 hiPS cell lines differentiated; each line is shown in a different color; expression normalized to the RPL13a housekeeping gene). Data are the mean ± s.e.m. Individual values are indicated by dots.

  3. Extended Data Fig 3 Immunocytochemistry quantifications in hCS following oxygen deprivation.

    a, Quantification of the density of Hoechst+ cells in proliferative areas in hCS after 48 h exposure to <1% O2. Data are shown as averages across individual hCS proliferative zones from three hiPS cell lines per condition (left, n = 21 areas for 21% O2 versus n = 18 areas for < 1% O2; two-tailed Mann–Whitney test, P = 0.09) and as averages across different hiPS cell lines (right, n = 3 hiPS cell lines, two-tailed Wilcoxon test, P = 0.50). b, Quantification of the percentage of PAX6+ cells in whole cryosections of hCS after 48 h exposure to <1% O2. Data are shown as averages across whole sections of hCS from four hiPS cell lines per condition (left, n = 25 sections for 21% O2 versus n = 23 sections for <1% O2; two-tailed unpaired t-test, P = 0.44) or as averages across different hiPS cell lines (right, n = 4 hiPS cell lines; two-tailed Wilcoxon test, P = 0.25). c, Quantification of the percentage of TBR2+ cells in whole cryosections of hCS after 48 h exposure to <1% O2. Data are shown as averages across whole sections of hCS from four hiPS cell lines per condition (left, n = 25 sections for 21% O2 versus n = 27 sections for <1% O2; two-tailed Mann–Whitney U test, ****P < 0.0001) or as averages across different hiPS cell lines (right, n = 4 hiPS cell lines; two-tailed paired t-test, *P = 0.03; each line is shown in a different color). d Quantification of the percentage of cells coexpressing SOX2 and TBR2 in cryosections of hCS after 48 h exposure to <1% O2. Data are shown as averages across individual hCS cryosections from four hiPS cell lines per condition (left, n = 10 sections for 21% O2 and n = 12 sections for <1% O2; two-tailed unpaired t-test, P = 0.82; from four hiPS cell lines) or as averages across different hiPS cell lines (right, n = 3 hiPS cell lines; two-tailed paired t-test, P = 0.06; each line is shown in a different color). Data are the mean ± s.e.m.; individual values are indicated by dots.

  4. Extended Data Fig 4 Transcriptome analyses in hCS following oxygen deprivation.

    a, Hierarchical clustering of WGCNA modules identified in the RNA-seq data. Clustering is based on the module eigengenes (average expression profile of all module genes). The turquoise and blue modules are very similar in overall eigengene expression pattern. b, Statistical significance for correlation of each module with exposure to low oxygen (bars are labeled by the color of the modules). The blue and turquoise modules are highly associated with exposure (FDR ≤ 0.05). c, Enrichment for pathways in the turquoise and blue modules (bars are labeled by the color of the modules in which they are enriched). Only pathways with Bonferroni-corrected FDR < 1 × 10−4 are shown. d, Validation by qPCR of the UPR-related genes PERK (two-tailed paired t-test, *P = 0.03), ATF3 (two-tailed paired t-test, *P = 0.03) and XBP1s (two-tailed paired t-test, *P = 0.04), which were identified in the RNA-seq analysis (n = 4 hiPS cell lines; each line is shown in a different color; expression normalized to the RPL13a housekeeping gene). Data are the mean ± s.e.m.; individual values are indicated by dots.

  5. Extended Data Fig 5 Quantifications in hCS following oxygen deprivation or tunicamycin exposure.

    a, Quantification of the percentage of cells coexpressing ATF4 and PAX6 in cryosections of hCS after 48 h exposure to <1% O2 in the presence or absence of 10 nM ISRIB. Data are shown as averages across individual hCS cryosections from three hiPS cell lines per condition (left, n = 7 sections for 21% O2 and n = 7 sections for <1% O2 versus n = 7 sections for <1% O2 + ISRIB; one-way ANOVA F2,18 = 1.37, P = 0.27; Dunnett’s multiple-comparison test versus 21% O2, P = 0.70, P = 0.54), or as averages across different hiPS cell lines (right, n = 3 hiPS cell lines; one-way ANOVA, F2,4 = 4.64, P = 0.09; Dunnett’s multiple-comparison test versus 21% O2, P = 0.48, P = 0.21; each line is shown in a different color). b, Quantification of the density of PAX6+ cells in hCS after exposure for 48 h to 1.2 μM tunicamycin in the presence or absence of 10 nM ISRIB. Data are shown as averages across individual hCS proliferative zones from three hiPS cell lines per condition (left, n = 22 areas for 21% O2, n = 23 areas for tunicamycin, n = 24 areas for tunicamycin + ISRIB; one-way ANOVA, F2,66 = 1.57, P = 0.21; Dunnett’s multiple-comparison test versus 21% O2, P = 0.37, P = 0.15), or as averages across different hiPS cell lines (right, n = 3 hiPS cell lines; Friedman’s test, P = 0.19; Dunnett’s multiple-comparison test versus 21% O2, P = 0.20, P = 0.08; each line is shown in a different color). c, Percentage of TBR2+ cells that coexpress c-CAS3 in whole cryosections of hCS maintained in 21% O2 or exposed to <1% O2 for 48 h. (two-tailed Mann–Whitney U test, P > 0.99; n = 8 cryosections from two hiPS cell lines). d, Quantification of the percentage of cells coexpressing p27 and PAX6 in cryosections of hCS after 48 h exposure to <1% O2 in the presence or absence of 10 nM ISRIB. Data are shown as averages across individual hCS cryosections from four hiPS cell lines per condition (left, n = 10 sections for 21% O2 versus n = 10 sections for 1% O2 and n = 9 sections for <1% O2 + ISRIB; one-way ANOVA, F2,26 = 0.10, P = 0.90 Dunnett’s multiple-comparison test versus 21% O2, P = 0.88, P = 0.90), or as averages across different hiPS cell lines (right, n = 4 hiPS cell lines; one-way ANOVA F2,6 = 0.30, P = 0.74; Dunnett’s multiple-comparison test versus 21% O2, P = 0.67, P = 0.94; each line is shown in a different color). e, Representative images of cells coexpressing TBR2, Ki67 and PH3 in cryosections of hCS. White arrows show examples of cells that are TBR2+PH3+ or TBR2+Ki67+. f, Quantification of the percentage of cells coexpressing Ki67 and TBR2 in cryosections of hCS after 48 h exposure to <1% O2. Data are shown as averages across individual hCS cryosections from three hiPS cell lines per condition (left, n = 6 sections for 21% O2 versus n = 6 sections for <1% O2; two-tailed unpaired t-test, P = 0.91) or as averages across different hiPS cell lines (n = 3 hiPS cell lines; two-tailed paired t-test, P = 0.91; each line is shown in a different color). g, Quantification of the percentage of cells coexpressing PH3 and TBR2 in cryosections of hCS after 48 h exposure to <1% O2. Data are shown as averages across individual hCS cryosections from three hiPS cell lines per condition (n = 6 sections for 21% O2 versus n = 6 sections for <1% O2; two-tailed unpaired t-test, P = 0.55) or as averages across different hiPS cell lines (right, n = 3 hiPS cell lines; two-tailed paired t-test, P = 0.56; each line is shown in a different color). Data are the mean ± s.e.m.; individual values are indicated by dots.

Supplementary information

  1. Supplementary Information

    Supplementary Tables 1, 2 and 4

  2. Reporting Summary

  3. Supplementary Table 3

    List of differentially expressed genes (RNA-seq)

Source data

  1. Source Data Fig. 1

    Uncropped western blots.

  2. Source Data Fig. 4

    Uncropped western blots.

  3. Source Data Extended Data Fig. 1

    Uncropped western blots

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https://doi.org/10.1038/s41591-019-0436-0