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, 25 (5), 784-791

Human 3D Cellular Model of Hypoxic Brain Injury of Prematurity

Affiliations

Human 3D Cellular Model of Hypoxic Brain Injury of Prematurity

Anca M Pașca et al. Nat Med.

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.

Conflict of interest statement

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).

Figures

Extended Data Fig. 1
Extended Data Fig. 1. Oxygen tension and cleaved caspase-3 in hCS following hypoxia.
(a) Oxygen tension (pO2, mmHg) measurements inside hCS from Fig. 1b, shown as a function of depth (data for each hCS was normalized as function its radius) (n= 6 hCS for 21% O2 and n= 7 hCS for <1% O2; from 3 hiPS cell lines). Shaded area indicates s.e.m. (b, c) Representative western blots and quantification of cleaved Cas3 (c-Cas3) in hCS after 24 hours and 48 hours of exposure to <1% O2 and at 72 hours after reoxygenation (one-way ANOVA, F3, 6= 1.56; P= 0.29); normalized to β-actin (n= 3 differentiated hiPS cell lines with two hCSs per condition; each line is shown in a different color). Western blots were cropped to show the relevant bands; molecular weight (MW) markers are indicated on the left (in kDa). Source Data 2 for uncropped western blots and Supplementary Table 2 for quantifications. Data are mean ± s.e.m. Individual values are indicated by dots.
Extended Data Fig. 2
Extended Data Fig. 2. RNA-seq clustering and qPCR validation.
(a) Hierarchical clustering of RNA-seq data showing clustering of samples based on exposure to oxygen concentration. Samples (n= 24) from hCS differentiated from 3 hiPS cell lines were collected at 24 hours or 48 hours after<1% O2, as well as 72 hours after re-oxygenation. We clustered based on all DE 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), 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), CENPF (two-tailed paired t-test, ****P< 0.0001), which were identified in the RNA-seq (n= 4 hiPS cell lines differentiated; each line is shown in a different color; expression normalized to the RPL13a housekeeping gene). Data are mean ±  s.e.m. Individual values are indicated by dots.
Extended Data Fig. 3
Extended Data Fig. 3. Immunocytochemistry quantifications in hCS following hypoxia.
(a) Quantification of the density of Hoechst+ cells in proliferative areas in hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across individual hCS proliferative zones from 3 hiPS cell lines per condition (n= 21 areas for 21% O2 versus n= 18 areas for <1% O2; two-tailed Mann-Whitney test, P= 0.09), and (Right) as average across different hiPS cell lines (n= 3 hiPS cell line, two-tailed Wilcoxon test, P = 0.50). (b) Quantification of the proportion of PAX6+ cells in whole-cryosections of hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across whole-section of hCS from 4 hiPS cell lines per condition (n= 25 sections for 21% O2 versus n= 23 sections for <1% O2; two-tailed unpaired t-test, P= 0.44), or (Right) as average across different hiPS cell lines (n= 4 hiPS cell lines; right: two-tailed Wilcoxon-test, P= 0.25). (c) Quantification of the proportion of TBR2+ cells in whole-cryosections of hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across whole-section of hCS from 4 hiPS cell lines per condition (n= 25 sections for 21% O2 versus n= 27 sections for <1% O2; two-tailed Mann-Whitney U test, ****P< 0.0001), or (Right) as average across different hiPS cell lines (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 proportion of cells co-expressing SOX2 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across individual hCS cryo-sections from 4 hiPS cell lines per condition (n= 10 sections for 21% O2, n= 12 sections for <1% O2; two-tailed unpaired t-test, P= 0.82; from 4 hiPS cell lines), or (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; right: two-tailed paired t-test, P= 0.06; each line is shown in a different color). Data are mean ± s.e.m., individual values are indicated by dots.
Extended Data Fig. 4
Extended Data Fig. 4. WGCNA analysis and qPCR validation.
(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 low O2 exposure (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 x 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), XBP1s (two-tailed paired t-test, *P= 0.04), which were identified in the RNA-seq (n= 4 hiPS cell lines; each line is shown in a different color; expression normalized to the RPL13a housekeeping gene). Data are mean ± s.e.m., individual values are indicated by dots.
Extended Data Fig. 5
Extended Data Fig. 5. Immunocytochemistry quantifications of UPR, cell cycle and proliferation in hCS.
(a) Quantification of the proportion of cells co-expressing ATF4/PAX6 in cryo-sections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS cryo-sections from 3 hiPS cell lines per condition (n= 7 sections for 21% O2 versus n= 7 sections for <1% O2 versus n= 7 sections for <1% O2 with 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 (Right) as average across different hiPS cell lines (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 hours to 1.2 μM of tunicamycin in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS proliferative zones from 3 hiPS cell lines per condition (n= 22 areas for 21% O2, n= 23 areas for tunicamycin, n= 24 areas for tunicamycin with ISRIB; one-way ANOVA, F2,66 = 1.57, P= 0.21; Dunnett’s multiple comparison test versus 21% O2, P= 0.37, P= 0.50), or (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; 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) Proportion of TBR2+ cells that co-express c-CAS3 in whole cryo-sections of hCS maintained in 21% O2 or exposed to <1% O2 for 48 hours. (two-tailed Mann-Whitney U test, P> 0.99; n= 8 cryo-section from 2 hiPS cell lines). (d) Quantification of the proportion of cells co-expressing p27 and PAX6 in cryosections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS cryo-sections from 4 hiPS cell lines per condition (n= 10 sections for 21% O2 versus n= 10 sections for <1% O2 versus n= 9 sections for <1% O2 with 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 (Right) as average across different hiPS cell lines (n= 4 hiPS cell line; 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 co-expressing TBR2, Ki67 and PH3 in cryosections of hCS. White arrows show example of cells that are TBR2+/PH3+ or TBR2+/Ki67+. Scale bar, 50 μm. (f) Quantification of the proportion of cells co-expressing Ki67 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across individual hCS cryo-sections from 3 hiPS cell lines per condition (n= 6 sections for 21% O2 versus n= 6 sections for <1% O2; two-tailed unpaired t-test, P= 0.91), or (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; two-tailed paired t-test, P= 0.91; each line is shown in a different color). (g) Quantification of the proportion of cells co-expressing PH3 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across individual hCS cryo-sections from 3 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 (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; two-tailed paired t-test, P= 0.56; each line is shown in a different color). Data are mean ± s.e.m., individual values are indicated by dots.
FIGURE 1.
FIGURE 1.. Human cellular model for studying changes in oxygen tension in hCS.
(a) Scheme illustrating the major stages in the generation of hCS from hiPS cells as described in Ref. At day 74–78 of in vitro differentiation, hCS are exposed for 48 hours at <1% O2 in a gas-controlled culture chamber, and then maintained for another 72 hours at 21% O2. Control hCS are maintained at 21% O2 throughout. (b) O2 tension levels (pO2, mmHg) measured with optical microsensor at the surface and inside hCS (100 and 500 μm) before (n= 6 hCS) and at 48 hours of exposure to <1% O2 (n= 7); hCS from 3 hiPS cell lines; Kruskal-Wallis test, P< 0.0001, Dunn’s multiple comparison test, **P= 0.002, **P= 0.008, **P= 0.01. (c, d) Representative western blots and quantification of HIF-1α protein expression in hCS after 48 hours of exposure to <1% O2 and after 72 hours of reoxygenation versus unexposed (21% O2); normalized to β-actin (n= 5 differentiated hiPS cell lines with at least 2 hCSs per condition; Friedman’s test, P= 0.02, Dunn’s multiple comparison test versus 21% O2, *P= 0.02 for 48 hours and P> 0.99 for 72 hours). Data are mean ±  s.e.m. Individual values are indicated by dots. Western blots were cropped to show the relevant bands; molecular weight (MW) markers are indicated on the right (in kD). See Source Data 1 for uncropped western blots and Supplementary Table 2 for quantifications. (e) Representative immunostaining of HIF-1α (yellow) in hCS exposed for 48 hours to <1% O2 versus 21% hCS. Experiment performed in 2 hiPS cell lines. Nuclei labeled by Hoechst staining. Scale bar, 50 μm. (f) Volcano plots showing the results of RNA-seq experiments after exposure to <1% O2 for 24 hours or 48 hours, as well as after 48 hours of <1% O2 followed by 72 hours of re-oxygenation (total time of 120 hours). Each dot represents a single gene, with genes significantly upregulated shown in red, genes significantly downregulated in blue, and non-significant genes in grey (determined based on FDR ≤ 0.05 and fold change ≥ 1.5). The size of the points corresponds to the difference in expression level between low-oxygen exposed hCS and unexposed hCS (difference of medians); n= 24 samples from hCS derived from 3 hiPS cell lines. (g) Overlap between hypoxia-related transcriptome changes in hCS and layer-specific transcriptome signatures in the developing human cortex at PCW21 as described in ref. Strong enrichment is observed only in SVZ (p value corrected for multiple comparisons). SG, subpial granular zone; MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; VZ, ventricular zone.
FIGURE 2.
FIGURE 2.. Proportion of TBR2+ cells in hCS exposed to low oxygen.
(a, b) Representative images of proliferative areas in hCS maintained in 21% O2 (upper) or exposed to <1% O2 for 48 hours (lower); scale bar 50 μm. The VZ–, SVZ– and CP–like areas are delineated by the pattern of expression of PAX6, TBR2, CTIP2 and the density and orientation of nuclei (labeled with Hoechst). Scale bar, 50 μm. (c) Quantification of the density of TBR2+ cells in hCS after 48 hours of exposure to <1% O2 and after 72 hours of reoxygenation versus unexposed (21% O2). (Left) Data shown as average across individual hCS proliferative zones from 3 hiPS cell lines per condition (n= 30 areas for 21% O2, n= 21 areas for <1% O2; n= 21 areas for re-oxygenation; one-way ANOVA, F2, 69= 13.64, P< 0.0001, Dunnett’s multiple comparison test versus 21% O2, ****P< 0.0001, ***P= 0.0003), or (Right) as average across different hiPS cell lines (n= 3 hiPS cell lines; one-way ANOVA, F2, 4= 8.38, P= 0.03, Dunnett’s multiple comparison test versus 21% O2, *P= 0.03, *P= 0.06; each line is shown in a different color). (d) Quantification of the density of PAX6 cells in hCS after exposure to 48 hours of <1% O2. (Left) Data shown as average across individual hCS proliferative zones from 3 hiPS cell lines per condition (n= 21 areas for 21% O2 versus n=18 areas for <1% O2; two-tailed Mann-Whitney U test, P= 0.44) or (Right) as average across different hiPS cell lines (n= 3 hiPS cell lines; Wilcoxon test, P> 0.99 for all comparisons; each line is shown in a different color). Data are mean ± s.e.m., individual values are indicated by dots.
FIGURE 3.
FIGURE 3.. Unfolded protein response pathway in hCS exposed to low oxygen.
(a) Dendrogram for the Weighted gene co-expression network analysis (WGCNA), which identified genes with similar expression profiles and grouped them into modules (represented by colors). (b) Eigengene expression profiles (‘average’ expression profile of all module genes) for the top WGCNA modules associated with exposure of hCS to low O2 for 48 hours. The blue module (upper) contains genes strongly enriched for annotation in biological pathways related to the hypoxia response (HIF-1α transcription factor network), while the turquoise module (lower) is enriched for genes involved in the unfolded protein response (UPR) pathway. (c) Quantification of the density of TBR2+ cells in 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 proliferative zones from four hiPS cell lines per condition (left, n = 50 areas for 21% O2 versus n = 42 areas for <1% O2 versus n = 32 areas for <1% O2 with ISRIB; one-way ANOVA test, F2,21 = 15.85, P < 0.0001; Dunnett’s multiple-comparison test versus 21% O2, ****P < 0.0001 for <1% O2, P = 0.30 for <1% O2 + ISRIB) or as averages across different hiPS cell lines (right, n = 4 hiPS cell lines; one-way ANOVA test, F2,6 = 13.43, P = 0.006; Dunnett’s multiple-comparison test versus 21% O2, **P = 0.004 for <1% O2, P = 0.45 for <1% O2 + ISRIB; each line is shown in a different color). (d) Representative images of cells co-expressing ATF4 and TBR2 in hCS exposed to <1% O2 for 48 hours. Yellow arrow show example of TBR2+/ATF4; white arrows show example of cells that are TBR2+/ATF4+. Scale bar, 5 μm. (e) Quantification of the proportion of cells co-expressing ATF4 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB, and after 72 hours of re-oxygenation. (Left) Data shown as average across individual hCS cryo-sections from 4 hiPS cell lines per condition (n= 36 sections for 21% O2, n= 25 sections for <1% O2, n= 48 sections for <1% O2 with ISRIB, n= 23 sections for re-oxygenation; Kruskal-Wallis test P< 0.0001, Dunn’s multiple comparison versus 21% O2, ****P< 0.0001, P> 0.99, **P= 0.001), or (Right) as average across different hiPS cell lines (n= 4 hiPS cell line; Friedman test P= 0.01, Dunnett’s multiple comparison test versus 21% O2, *P= 0.01, P> 0.99, P= 0.51; each line is shown in a different color). (f) Quantification of the density of TBR2+ cells in hCS after exposure for 48 hours to 1.2 μM of tunicamycin in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS proliferative zones from 3 hiPS cell lines per condition (n= 22 areas for 21% O2, n= 23 areas for tunicamycin, n= 24 areas for tunicamycin with ISRIB; one-way ANOVA, F2, 66= 12.69, P< 0.0001; Dunnett’s multiple comparison test versus 21% O2, ****P< 0.0001 for Tunicamycin; ***P=0.003 for Tunicamycin + ISRIB or (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; one-way ANOVA F2, 4= 11.85, P= 0.02; with Dunnett’s multiple comparison test versus 21% O2, ***P= 0.01 for Tunicamycin ; *P= 0.03 for Tunicamycin + ISRIB; each line is shown in a different color). (g) Representative images of cells co-expressing p27 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB. Yellow arrow show example of TBR2+/p27; white arrows show example of cells that are TBR2+/p27+. Scale bar, 25 μm. (h) Quantification of the proportion of cells co-expressing p27 and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS cryo-sections from 4 hiPS cell lines per condition (n= 27 sections for 21% O2 versus n= 28 sections for <1% O2 versus n= 36 sections for <1% O2 with ISRIB; Kruskal-Wallis test P< 0.0001, Dunn’s multiple comparison versus 21% O2, ****P<0.0001, P= 0.54), or (Right) as average across different hiPS cell lines (n= 4 hiPS cell line; one-way ANOVA F2, 6= 43.78, P= 0.0003, Dunnett’s multiple comparison test versus 21% O2, ***P= 0.0006, P= 0.43; each line is shown in a different color). (i) Quantification of the proportion of cells co-expressing CTIP2 (also known as BLC11B) and TBR2 in cryosections of hCS after exposure to 48 hours of <1% O2 in the presence or absence of 10 nM ISRIB. (Left) Data shown as average across individual hCS cryo-sections from 3 hiPS cell lines per condition (n= 30 sections for 21% O2, n= 27 sections for <1% O2, n= 34 sections for <1% O2 with ISRIB; one-way ANOVA, F2, 88= 12.31, P< 0.0001, Dunnett’s multiple comparison test versus 21% O2, ****P< 0.0001, P= 0.58), or (Right) as average across different hiPS cell lines (n= 3 hiPS cell line; one-way ANOVA F2, 6= 53.67, P= 0.0001, Dunnett’s multiple comparison test versus 21% O2, ***P= 0.0002, P= 0.96; each line is shown in a different color). Data are mean ± s.e.m., individual values are indicated by dots.
FIGURE 4.
FIGURE 4.. Validation in primary human cortical tissue in vitro.
(a) Scheme showing sectioning and gas-chamber exposure to low oxygen of human cortical tissue (~PCW20). (b) Macroscopic image of cortical tissue and sectioning. Dashed lines indicate approximate regions of sectioning for slice culture. (c, d) Representative western blots and quantification of HIF-1α protein expression in human cortical sections (PCW20) after 48 hours of exposure to <1% O2 with or without 10 nM ISRIB normalized to β-actin (n= 5 slices; one-way ANOVA F2, 8= 19.39, P= 0. 0009, Dunnett’s multiple comparison test versus 21% O2, **P= 0.003, ***P= 0.0007). Western blots were cropped to show the relevant bands; molecular weight (MW) markers are indicated on the left (in kDa). See Source Data 3 for uncropped western blots and Supplementary Table 2 for quantifications. (e) Image of proliferative zones (VZ, SVZ) in cortical primary tissue delineated by expression of PAX6, TBR2 and Hoechst. Scale bar, 100 μm. (f) Quantification of density of TBR2+ cells in cryosections of primary human fetal tissue exposed after exposure for 48 hours to <1% O2 in the presence or absence of 10 nM ISRIB (n= 4 sections; one-way ANOVA F2, 9= 5.32, P= 0.02, Dunnett’s multiple comparison test versus 21% O2, *P= 0.02, P= 0.69). (g) Quantification of density of PAX6+ cells in cryosections of primary human fetal tissue exposed after exposure for 48 hours to <1% O2 in the presence or absence of 10 nM ISRIB (n= 4 sections; one-way ANOVA F2, 9= 2.29, P= 0.15, Dunnett’s multiple comparison test versus 21% O2, P= 0.23, P= 0.12). Data are mean ± s.e.m. Individual values are indicated by dots.

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