Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a , Schematic summarizing factors and gene markers involved in human cortical area specification. b , Overview of the protocol used to treat neural organoids with patterning factors from 3 to 5 div. Diff, differentiation medium ± vitamin A. c , Percentage of organoids with detectable SP8 > GFP expression after treatment with rostralizing factors (left) or EMX1>mNeonGreen expression after treatment with caudalizing factors (right). CHRDL1, chordin like 1; FST, follistatin; SB, SB-431542 dual SMAD inhibitor; CER1, cerberus 1; CHIR, GSK-3β inhibitor CHIR99021; WNT1, Wnt family member 1. Data indicate the mean ± s.d. from three lines per treatment ( n = 15 organoids per condition and line; except n = 16 for FGF8 high and CHIR low; n = 20, n = 19 and n = 17 for Chir high, and n = 18, n = 19 and n = 17 for Wnt1 treatment for the three lines). P values from one-way analysis of variance (ANOVA; Tukey’s multiple-comparisons test) for comparisons to untreated conditions are provided. d , Experimental procedure for assembloid generation with the SP8 > GFP line and OrEBs. D, day after EB formation; Diff, differentiation medium ± vitamin A; ULA, ultra-low attachment plate . e , RT–qPCR analysis of FGF8 target genes expressed in the SP8 > GFP EBs severed from OrEB after 1 day of co-culture. Data are the log of expression over TBP , shown as the mean ± s.d. ( n = 6 EBs for 0%, n = 5 for 1%, n = 5 for 10% grown from three independent clones). P values are the results of one-way ANOVA. f , GFP intensity of the organoids from g . Whiskers are the minima to maxima, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate the median ( n = 7 organoids for 0%, n = 11 for 1%, n = 19 for 10%, grown from three independent clones). P values are the results of one-way ANOVA. g , Images of organoids generated with the SP8 > GFP transgenic line and co-culture with OrEBs at 60 div (scale bars, 500 µm). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.1. a.u., arbitrary units.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Expressing, Quantitative RT-PCR, Co-Culture Assay, Clone Assay, Generated, Transgenic Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) Schematic diagram of CAG > FGF8 reporter (top). Genotyping of the selected clones (bottom) shows the three heterozygotes selected. The PCR was designed to amplify a wild type AAVS1 (WT) amplicon, a left homology arm amplicon (LA), a right homology arm amplicon (RA), and an inner part (IP). b ) RT-qPCR analysis of pluripotency markers and differentiation gene T/BRA in the selected CAG > FGF8 expressing clones and parental wild-type cells. Values are mean ± SD (n = 4 biological replicates for wt; n = 3 biological replicates, one for each CAG > FGF8 clone). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons test) among the different lines are: OCT4 wt vs. OCT4 CAG > FGF8, p = 0.9166; NANOG wt vs. NANOG CAG > FGF8, p = 0.9841; SOX2 wt vs. SOX2 CAG > FGF8, p = 0.9997; TBRA wt vs. TBRA CAG > FGF8, p > 0.9999. c ) Box plots showing RT-qPCR analysis of FGF8 and WNT1 genes in the selected clones of the CAG > FGF8 line compared to wt. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3); n = 6 from 3 independent clones. One-way ANOVA analysis is p < 0.0001 for FGF8 expression and p > 0.9999 for WNT1 expression. d ) Quantification of FGF8 protein levels in cell lysates (CELLS) and supernatants (SUP) measured by ELISA assay. Values are mean ± SD, n = 6 from 3 independent clones; P-values from two-sided unpaired t-tests are: CELLS wt vs CELLS CAG > FGF8, p = 0.0004; SUP wt vs SUP CAG > FGF8, p = 0.0823.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Clone Assay, Amplification, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) Representative images of 4 div embryoid bodies co-cultured with OrEBs containing cells expressing FGF8 and tdTomato mixed at different percentage (0%, 1%, 10%), immunostained for the FGF8 downstream target ETV1 (green) (scale bars 50 µm, the SP8 > GFP organoid is outlined with a dashed contour). The experiment has been repeated 4 times with organoids derived from three independent clones. b ) Immunostaining of 60 div organoids generated with the SP8 > GFP transgenic line and co-cultured with OrEBs in Fig. , stained for the rostral marker LMO4 (scale bars 500 µm in the main panels, 50 µm for the insets). The SP8 > GFP organoid is outlined with a dashed contour. c ) Box plots showing the fraction of LMO4+ cells normalized to total DAPI+ cells in 0%, 1% and 10% FGF8 conditions at 60 div. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3); n = 8 organoids. P-values from one-way ANOVA (Tukey’s multiple comparisons tests) are: 0% vs 1%, p = 0.0019, 0% vs 10%, p < 0.0001, 1% vs 10%, p < 0.0001. d ) Immunostaining of organoids generated with the SP8 > GFP transgenic line and co-cultured with OrEBs as described in Fig. for 60 days, stained for the neural marker Sox1 and DAPI. Scale bars 500 µm. e ) Quantification of the number of Sox1+ cells over total (DAPI + ) cells. Data show mean ± SD (n = 2 assembloids for 0%, n = 4 assembloids for 1%, n = 3 assembloids for 10%). P-values for each comparison resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 0% vs 1%, p = 0.113; 0% vs 10%, p = 0.2171; 1% vs 10%, p = 0.8893. f ) RT-qPCR analysis of anterior ( PAX6, SIX3 ) and optic cup ( SNAI2, PAX3, OTX2, LHX2, RAX ) markers from SP8 > GFP organoids severed from the co-culture experiment at 60 div. Data is Log of expression over TBP, shown as mean ± SD; n = 6 for 0% condition, n = 5 for 1% and 10% condition, from 3 independent clones. P-values from one-way ANOVA (Tukey’s multiple comparisons tests) are: RAX 0% vs. RAX 1%, p = 0.0718; RAX 0% vs. RAX10%, p = 0.0003; RAX 1% vs. RAX 10%, p = 0.0263; all the other comparisons are p < 0.0001. g ) GFP intensity per segment (P, proximal; M, medial; D, distal) in the 1% FGF8 condition from experiments shown in panel b . Each segmented line represents an individual organoid. n = 7 from 3 clones; ns, p = 0.5783 one-way ANOVA. Ns, non-significant. a.u., arbitrary units.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Cell Culture, Expressing, Derivative Assay, Clone Assay, Immunostaining, Generated, Transgenic Assay, Staining, Marker, Comparison, Quantitative RT-PCR, Co-Culture Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a , Experimental procedure for elongated assembloids using mosaic OrEBs containing CAG>tdTOMATO and non-fluorescent CAG > FGF8 -expressing cells. Diff, differentiation medium ± vitamin A. b , Representative images of elongated cortical assembloids at 1 div in the PDMS molds (indicated by white arrows in (i); scale bar, 500 µm), at 7 div after removal from the molds and before Matrigel embedding (ii) or after embedding in large Matrigel droplets ((iii); scale bar, 5 mm), at 120 div in the six-well plate ((iv); scale bar, 5 mm). c , Position of the OrEB on elongated cortical assembloids length (as a percentage) at 15, 60 and 120 div. Values are the mean ± s.d. ( n = 9 organoids for 15 div, n = 11 for 60 div and 120 div grown from three independent clones). P values for comparisons among time points (one-way ANOVA Tukey’s multiple-comparisons test) are: 15 div versus 60 div, P = 0.9455; 15 div versus 120 div, P = 0.9781; 60 div versus 120 div, P > 0.9999. d , Images of elongated cortical assembloids generated with the SP8 > GFP transgenic line and mosaic OrEBs at 60 div (scale bars, 500 µm). Right, SP8 > GFP intensity per segment (P, M and D) in individual assembloids. Each segmented line represents an individual elongated cortical assembloid ( n > 2 from at least 2 clones). P values are the results of one-way ANOVA among segments per condition (0%, P = 0.9045; 1%, P < 0.0001 and 10%, P = 0.9828). e , f , Images of proximal and distal CPNE8 ( e ) or NR2F1 ( f ) stainings (scale bars, 50 µm). Bottom, fraction of CPNE8 + ( e ) or NR2F1 + cells ( f ) normalized to total (DAPI + ) cells in proximal and distal insets of controls (conCAs) and polCAs at 60 div. Whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate the median; CPNE8: n = 20 insets for P and D from 3 conCAs, n = 42 insets for P and n = 39 for D from 3 polCAs; NR2F1: n = 60 insets for P and D from 6 conCAs, n = 40 insets for P and D from 4 polCAs. P values from one-way ANOVA (Tukey’s multiple-comparisons test) are: P = 0.9988 in CPNE8 proximal conCA versus distal conCAs, P = 0.8509 in NR2F1 proximal conCAs versus distal conCAs, P < 0.0001 for other comparisons; NS, not significant. g , g ′, Images of 60 div polCA immunostained with tdTomato in red, DAPI in blue and NR2F1 in white ( g ) or intensity rainbow ( g ′). Scale bar, 500 µm.

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Expressing, Clone Assay, Generated, Transgenic Assay

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a-b ) Representative images of elongated assembloids (8 div) properly embedded maintaining elongated shape (a) compared to elongated assembloids from the same batch without matrigel, shrinking without spatial constrain (b). c ) Length of elongated assembloids after different days in vitro (div, div1 n = 8, div7 n = 6, div30 n = 4, div60 n = 24, div120 n = 22 assembloids grown from 3 independent clones). P-values resulting from one-way ANOVA Tukey’s multiple comparisons tests) among the different lines are: div1 vs div7, p = 0.0045; div1 vs div30, p = 0.9999; div7 vs div30, p = 0.0366; div7 vs div60, p = 0.9998; div7 vs div120, p = 0.51; div30 vs div60, p = 0.0049; div60 vs div120, p = 0.1825; div1 vs div60, div1 vs div120, div30 vs div120, p < 0.0001. d ) RT-qPCR analysis of neural markers (SOX2, SOX1, SP8 and LMO4), ventral marker NXK2.1 and differentiation markers SNAI1 and T-BRA, in elongated assembloids (eOrg) compared to round organoids (rOrg), after treatment with high FGF8 as described in Fig. . Values are mean ± SD, n = 3 in three lines (hESC H9, H1 and iPSC 178/5). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: eORGSOX2 d10 vs. rORGSOX2 d10, p = 0.0024; eORGSOX2 d30 vs. rORGSOX2 d30, p = 0.3144; eORGSOX2 d60 vs. rORGSOX2 d60, p = 0.9434; eORGSOX1 d10 vs. rORGSOX1 d10, p = 0.0009; eORGSOX1 d30 vs. rORGSOX1 d30, p = 0.1573; eORGSOX1 d60 vs. rORGSOX1 d60, p = 0.7266; eORGSP8 d10 vs. rORGSP8 d10, p = 0.0006; eORGSP8 d30 vs. rORGSP8 d30, p = 0.1891; eORGSP8 d60 vs. rORGSP8 d60, p = 0.8004; eORGLMO4 d10 vs. rORGLMO4 d10, p = 0.0114; eORGLMO4 d30 vs. rORGLMO4 d30, p = 0.8378; eORGLMO4 d60 vs. rORGLMO4 d60, p = 0.9821; eORGNKX2.1 d10 vs. rORGNKX2.1 d10, p = 0.0008; eORGNKX2.1 d30 vs. rORGNKX2.1 d30, p = 0.1345; eORGNKX2.1 d60 vs. rORGNKX2.1 d60, p = 0.9036; eORGSNAI1 d10 vs. rORGSNAI1 d10, p = 0.0026; eORGSNAI1 d30 vs. rORGSNAI1 d30, p = 0.6125; eORGSNAI1 d60 vs. rORGSNAI1 d60, p = 0.96; eORGT-BRA d10 vs. rORGT-BRA d10, p = 0.6815; eORGT-BRA d30 vs. rORGT-BRA d30, p = 0.3026; eORGT-BRA d60 vs. rORGT-BRA d60, p = 0.1297. e ) Fraction of TUNEL+ cells normalized to total DAPI+ cells in elongated versus conventional round organoids (div, days in vitro ). N = 36 insets for 4 div elongated, n = 33 for 4 div Round, n = 122 for 7 div elongated, n = 37 for 7 div Round, n = 81 for 30 div elongated, n = 82 for 30 div Round organoids from 3 lines per condition. P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div eOrg vs. 4 div rOrg, p < 0.0001; 7 div eOrg vs. 7 div rOrg, p = 0.31; 30 div eOrg vs. 30 div rOrg, p = 0.0115. f ) Box plots showing the fraction of TUNEL+ cells normalized to total DAPI+ cells in proximal and distal insets of elongated assembloids (n = 18 insets for 4 div Proximal and Distal, n = 32 for 7 div Proximal, n = 33 for 7 div Distal, n = 40 for 30 div Proximal, n = 42 for 30 div Distal; organoids are grown from 3 lines per condition). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: 4 div proximal vs. 4 div distal, p = 0.6097; 7 div proximal vs. 7 div distal, p = 0.6559; 30 div proximal vs. 30 div distal, p = 0.9897. g ) Representative images of the OrEB localization throughout elongated assembloids growth quantified in Fig. (scale bars 500 µm). h ) Top, representative images of proximal and distal CTIP2 staining (scale bars 50 µm). Bottom, fraction of CTIP2+ cells normalized to total DAPI+ cells in proximal and distal insets of controls (conCA) and polCAs at 60 div (n = 50 insets for P and D conCAs, n = 40 insets for P and D polCAs from 3 organoids). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p = 0.8204; proximal polCA vs. distal polCA, p < 0.0001. i ) Top, representative images of proximal and distal LMO4 staining (scale bars 50 µm). Bottom, fraction of LMO4+ cells normalized to total DAPI+ cells in proximal and distal insets of control(tdt) and polCAs at 60 div (n = 61 insets for P and D from 6 conCAs, n = 51 insets for P and D from 4 polCA). P-values resulting from one-way ANOVA (Tukey’s multiple comparisons tests) are: proximal conCA vs. distal conCA, p > 0.9999; proximal polCA vs. distal polCA, p < 0.0001. ns, non-significant. All whiskers are min to max, boxes represent the 25th to 75th percentiles (Q1 to Q3) and lines indicate median. j-j’ ) Images of immunostained longitudinal sections of elongated assembloids at 60 div (scale bars 500 µm).

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: In Vitro, Clone Assay, Quantitative RT-PCR, Marker, TUNEL Assay, Staining, Control

Journal: Nature Methods

Article Title: A polarized FGF8 source specifies frontotemporal signatures in spatially oriented cell populations of cortical assembloids

doi: 10.1038/s41592-024-02412-5

Figure Lengend Snippet: a ) UMAP embedding for the scRNA-seq dataset containing cells derived from dissections of three segments of polCA and control counterparts annotated by stress levels based on Gruffi analysis. b ) Sankey plot showing similar mapping of cells from individual segments of both control and polCA to stressed cells according to Gruffi’s results. c ) Box plots showing the number of cells expressing FGF8 normalized by the total number of cells per segment for the three replicates. The box displays the median, the inter-quartile range, the minimum and maximum values for each segment. d ) Dot plot for the top five markers for each cluster, genes with logFC> 3 are shown. LogFCs colors are scaled by gene. e ) UMAP embedding plots colored by expression levels of markers of radial glia ( NES, SOX2, VIM ), cycling radial glia ( TOP2A, MKI67 ), Cajal-Retzious cells ( RELN ), neurons ( DCX, TUBB3 ), interneuron progenitor cells ( DLX6-AS1, GAD2 ), LGE-derived progenitors ( GSX2 ), MGE-derived progenitors ( NKX2.1 ), retinal progenitor cells ( RORB, VSX2 ), stress responsive cells ( GOLGA4 ), endothelial cells ( DCN, BGN, COL1A2 ), BMP responsive cells ( TTR, RSPO2, LMX1A, MSX1 ), cilium bearing cells ( PCP4, NPHP1 ), oligodendrocytes ( OLIG1, OLIG2 ), astrocytes ( GFAP, AQP4 ) and microglia ( AIF1, CD68 ).

Article Snippet: Levels of FGF8 protein in cell extracts and supernatants were measured using an ELISA kit (Cusabio CSB-E15861h) according to the manufacturer’s instruction.

Techniques: Derivative Assay, Control, Expressing