Dissecting infant leukemia developmental origins with a hemogenic gastruloid model
- College of Health, Medicine and Life Sciences, Centre for Genome Engineering and Maintenance, Brunel University, United Kingdom
- Department of Genetics, University of Cambridge, United Kingdom
- Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Spain
- Laboratory of Experimental Therapies in Oncology, IRCCS Istituto Giannina Gaslini, Italy
- Program in Cancer Research, Hospital del Mar Research Institute, CIBERONC, Spain
- Josep Carreras Leukemia Research Institute, Spain
- Animal Facility, IRCCS Policlinico San Martino, Italy
- Department of Pathology, University of Cambridge, United Kingdom
- Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, United Kingdom
- Centre for In Vivo Modelling, Institute of Cancer Research, United Kingdom
- Sanquin Research, Landsteiner Laboratory, Netherlands
- Amsterdam UMC Location Vrije Universiteit Amsterdam, Netherlands
Figures
Figure 1 with 1 supplement
Hemogenic gastruloids (haemGx) produced from mES cells promote hemato-endothelial specification with spatio-temporally accurate ontogeny.
(A) Timeline of mES cells assembly and culture into gastruloids over a 216 hr period with the addition of specified factors for the promotion of hemato-endothelial specification. The 24 hr pre-treatment in 2i+LIF was omitted when mES cultures showed no signs of differentiation. (B) Imaging of haemGx over time from 72 to 216 hr at 10 x magnification, showing the assembly and growth of the 3D structures and the polarization of the Flk-1-GFP marker from 96 hr; scale bar: 100 mm. (C) Flow cytometry timecourse analysis of haemGx for the presence of CD144 (VE-cadherin), C-Kit, CD41, and CD45 markers; representative plots. (D) Quantification of analysis in (C). Violin plots with median and interquartile range of up to 26 replicates; Kruskal-Wallis CD144 (p=0.0755), C-Kit (p<0.0001), CD41 (p<0.0342), CD45 (p<0.0001). Shown are significant Dunn’s multiple comparisons tests; adjusted q-value. (E) Immunostaining of whole individual haemGx at 216 hr showing the localized expression of Flk1-GFP (green), C-Kit (yellow), and CD45 (red) markers; nuclear staining is in DAPI (blue); scale bar: 100 µm.
Figure 1—figure supplement 1
Optimization and reproducibility of the hemogenic gastruloid (haemGx) protocol.
(A) Imaging of a 120 hr-haemGx with and without the addition of Activin A in conjunction with Chiron 99021 (CHIR) to achieve polarized expression of Flk-1-GFP; scale bar: 100 mm. (B) Titration of Activin A dosage (from 25 ng/ml to 150 ng/ml) required for Flk-1-GFP expression activation (green). (C) Representative flow cytometry plots of %CD45+ cells at 216 hr without and without addition of SCF, Flt3L, and TPO (SFT) from 168 hr; representative plots. (D) Quantification of %CD45+ cells by flow cytometry analysis of haemGx cultured with or without SFT, between 192–216 hr and 168–216 hr. Mean ± SD of n=7–8 independent experiments; Tukey’s multiple testing with significant q-value <0.05. (E) Quantification of %CD45+ cells at 216 hr by flow cytometry analysis of haemGx cultured in VEGF + FGF or VEGF-only during the 168–216 hr period. Mean ± SD of n=7 independent experiments; unpaired t-test; ns, not significant. (F) Flow cytometry monitoring of C-Kit, CD41, and CD45 markers in haemGx established from E14 and Flk-1-GFP mES lines; n=3–4 replicate experiments (n=1, CD45+ 120 hr). Mixed effects model with Sidak’s multiple comparison test; significant adjusted P-value <0.05. (G) Representative flow cytometry plots of CD41+ and C-Kit+ cells in E14 and Flk-1-GFP mES cell-initiated haemGx. (H) Representative flow cytometry plots of CD45+ cells in E14 and Flk-1-GFP mES cell-initiated haemGx. (I) Quantification of %CD45+ cells in Flk-1-GFP, Sox17-GFP, and TBra-GFP mES cell-initiated haemGx; n=5, mean ± SD. Welch’s t-test Sox17-GFP vs. Flk1-GFP, p=0.5254; Tbra-GFP vs. Flk1-GFP, p=0.3782.
Figure 2 with 2 supplements
Hemogenic gastruloid (HaemGx) produce hematopoietic output following the emergence of specialized endothelium.
(A) Immunostaining of whole individual haemGx at 144 hr showing the establishment of a vascular network by co-expression of CD31 and Flk1-GFP; nuclear staining is in DAPI (blue); scale bar: 100 µm. (B) Flow cytometry analysis of haemGx cells at 144 hr and 216 rhr stained for CD41, CD45, CD31, and CD34. (C) Quantification of flow cytometry analysis in (B) of CD41+ and CD45+ fractions co-expressing CD34 and CD31. Mixed effect analysis with a Ŝidàk test for multiple comparisons. (D) Colony-forming cell (CFC) assay of 216 hr-haemGx in multipotential methylcellulose-based medium. GEM: granulocyte-erythroid-monocyte; GM: granulocyte-monocyte; M: monocyte; E: erythroid. Please note that the medium does not include TPO and does not assess the presence of megakaryocytic progenitors. CFC frequency in 3 haemGx, n=3, mean ± SD. (E) Representative photographs of colonies quantified in C; scale bar = 2 mm. (F) Real-time quantitative (qPCR) analysis of expression of hemato-endothelial genes by timepoint. Relative gene expression fold change calculated by normalization to Ppia. Bars represent mean of three replicates and show individual data points; p-values by ANOVA across datapoints; post-hoc statistical significance between specific variables by Tukey’s test and shown by brackets.
Figure 2—figure supplement 1
Multi-colour flow cytometry detection of surface markers in hemogenic gastruloid (haemGx).
(A) Representative flow cytometry plots of CD31 and CD34 staining of haemGx at 144 hr and 216 hr gated on Flk-1-GFP+. (B) Quantification of Flk-1-GFP+ haemGx populations at 144 hr and 216 hr expressing CD34 and CD31. Crossbar shows mean of four replicate experiments with individual data points shown; Welch’s t-test; p-value significant <0.05. (C–E) Unstained controls (C–D) and fluorescence minus one (FMO) controls (E) relative to flow cytometry detection of multi-colour of surface markers in Figure 2. (F) Representative flow cytometry plot of haemGx of CD45+CD41lo fractions co-expressing C-Kit and Cd144 (Ve-Cadherin).
Figure 2—figure supplement 2
Characterization of hematopoietic output from hemogenic gastruloid (haemGx).
(A) Representative image of cytospins of dissociated haemGx at 216 hr stained with Giemsa-Wright’s stain. Annotated are cells in the monocytic (dashed open arrow), granulocytic (solid open arrow), megakaryocytic (solid arrow), and erythroid (asterisk) lineages; arrowheads indicate cells with a non-specific blast-like morphology. 40 x magnification, scale bar = 100 µm. (B) Representative flow cytometry plots of CD41 and CD45 expressing populations in haemGx treated with 0.5 μM EZH2 inhibitor GSK126 or with 0.05% DMSO (control) at 144 hr and 216 hr. (C) Quantification of flow cytometry analysis in (B) showing the proportion of CD41+ and CD45+CD41lo populations in GSK126-treated haemGx. Welch’s t-test; p-value significant <0.05.
Figure 3 with 2 supplements
Time-resolved analysis of hemogenic gastruloid (haemGx) by scRNA-seq captures successive waves of hematopoietic specification.
(A) UMAP clustering of subsets of sequenced cells expressing CD41, C-Kit, and CD45 at 144 hr, 192 hr, and 216 h, annotated by timepoint (left panel), sorting condition (middle panel), and Leiden clustering (right panel). (B) UMAP panels highlighting the expression of specific hemato-endothelial markers in the transcriptional spaces defined in (A). Colour scale indicates expression levels in counts. (C) Heatmaps of differentiation of an arterial endothelial programme in haemGx from scRNA-seq data of sorted on expression of C-Kit at 144 hr and 192 hr. Colour scale indicates expression in counts. (D) Violin plots quantifying the expression of hemato-endothelial markers in C-Kit+fraction from clusters in (A) comparing 144 hr and 192 hr timepoints. Wilcoxon test; significant p-value <0.05. (E) Expression of definitive hematopoietic genes in C-Kit+, CD41+ and CD45+ cells at 144 hr and 192/216 hr timepoints; expression levels as normalized counts per million reads. Mann-Whitney or Kruskal-Wallis with Dunn’s multiple comparisons; significant p or q-value <0.05.
Figure 3—figure supplement 1
Single-cell RNA-seq (scRNA-seq) analysis of haemGx identifies time-dependent signatures of endothelial, hemogenic, and stromal cells.
(A) Summary of plating strategy for scRNA-seq analysis of gastruloids at 120 hr, 144 hr, 168 h, 192 hr, and 216 hrwithout selection of surface markers (‘all’) or sorted as C-Kit/Sca1+ (green shading), CD41+ (blue), or CD45+ (orange) cells; P=plate. (B) ScRNA-seq quality control measures quantification of mapped reads, individual genes, and mapping of reads onto mitochondrial (mt)DNA. (C) Mapped read and gene counts for individual Smart-seq2 libraries (plate, P), biological replicate (repl) populations of single cells, and read and map distributions across unsorted and cell surface phenotype-sorted cells. (D) UMAP projection of all sequenced cells coloured by annotated clusters (left), by sorting markers C-Kit/ScaI, CD41, and CD45, or unfractionated cells (‘whole’ corresponding to ‘all’ in panel A) (center), and by haemGx culture time (right). (E) Cell type enrichment analysis of cluster classifier genes, using the PanglaoDB repository (Franzén et al., 2019); classifier genes are obtained by differential expression in comparison to all other clusters. Represented are clusters 1, 3, and 9, which are characteristic of timepoints 168–216 hr, and capture stromal cells putatively relevant for hemogenic support, including autonomic neurons (cluster 9). The statistical power of representation of individual cell types is expressed as the EnrichR combined score with a p-value threshold of <0.05.
Figure 3—figure supplement 2
Single-cell-RNAseq (scRNAseq) analysis of haemGx.
(A) Heatmap representation of the expression of endothelial and hematopoietic marker genes in individual haemGx CD41+ and CD45+ cells sorted at 192 and 216 hr. Cells and genes ordered by unsupervised hierarchical clustering. (B) Violin plots quantifying the expression of arterial markers in C-Kit + fraction from clusters in Figure 3A comparing 144 hr and 192 h timepoints. Wilcoxon test; significant p-value <0.05. (C) Summary representation of the proportion of hGx cells expressing endothelial (endo), erythroid/myeloid (Ery + My), myeloid/lymphoid (My + Ly), and HSPC gene signatures. Endo: Kdr; Ery + My: Epor ± Gata1±Klf1±Hbb ± Hba ±Hbg + Spi1±Mpo ± Anpep ±Csf1/2/3 r; My + Ly: Spi1±Csf1/2 a/3r+Ikzf1±Ighm/d±Igk; HSPC: Ptprc + Myb.
Figure 4 with 2 supplements
Late-stage hemogenic gastruloid (haemGx) contains hematopoietic output with transcriptional alignment to mouse embryonic populations and in vivo engraftment potential.
(A) Projection of clustered haemGx cells (CD41, C-Kit, and CD45 at 144 hr, 192 hr, and 216 hr; Figure 3A) onto mouse single-cell RNA-seq datasets capturing: arterial and haemogenic specification in the para-splanchnopleura (pSP) and AGM region between E8.0 and E11 (Hou et al., 2020); YS, AGM, and FL progenitors and the AGM EHT (Zhu et al., 2020); HSC emergence from the dorsal aorta HE (Thambyrajah et al., 2024). Gastruloid cell projection identified as per their cluster of origin (Figure 3A). (B) Schematic representation of the experimental workflow for execution and analysis of haemGx implantation in the adrenal gland of immunodeficient mice. (C) PCR detection of haemGx genomic (g) DNA in the bone marrow (BM) and spleen (Spl) of immunodeficient mice 4 weeks after unilateral adrenal implantation of 3 gastruloids/gland. Analysis of five replicates of 100 ng gDNA/recipient tissue; control animal was injected unilaterally with PBS in an adrenal gland in parallel with experimental implantation. Reaction positive control used 100 ng of gDNA from Rosa26-BFP::Flkj1-GFP mES cells (mES) used to generate haemGxs. NTC: no template control. (D) Flow cytometry plots of engraftment detection by BFP expression in bone marrow (BM) of recipient mice 4 weeks following implantation of 216 hr haemGx. (E) Flow cytometry plots of lineage affiliation of BFP+ engraftment in bone marrow (BM) of recipient mice 8 weeks following implantation of 216 hr haemGx.
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Figure 4—source data 1
Original files for agarose gel electrophoresis in Figure 4C.
- https://cdn.elifesciences.org/articles/102324/elife-102324-fig4-data1-v1.zip
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Figure 4—source data 2
Annotated uncropped agarose gel electrophoresis in Figure 4C.
- https://cdn.elifesciences.org/articles/102324/elife-102324-fig4-data2-v1.zip
Figure 4—figure supplement 1
Analysis of mouse embryonic scRNA-seq datasets for transcriptional similarities of haemGx outputs.
(A–C) UMAPs of mouse single-cell RNA-seq datasets annotated as in respective publications: (A) arterial and haemogenic specification in the para-splanchnopleura (pSP) and AGM region between E8.0 and E11 (Hou et al., 2020); (B) YS, AGM, and FL progenitors and the AGM EHT (Zhu et al., 2020); (C) HSC emergence from the dorsal aorta HE (Thambyrajah et al., 2024).
Figure 4—figure supplement 2
Detection of engraftment potential of late-stage hemogenic gastruloid (haemGx) in adrenal glands of Nude mice.
(A) Map of the Rosa26-BFP construct used to target Flk1-GFP mES cells employed in implant assays. Primers used for gDNA PCR analysis are indicated. (B) PCR detection limit of the BFP amplicon using the primers in (A), in a serial dilution of Rosa26-BFP::Flk1-GFP mES gDNA into human leukemia cell line K562 gDNA; total 100 ng DNA per lane. (C) Uncropped image of agarose gel electrophoresis of BFP detection shown in Figure 4C. (D) Representative flow cytometry plot of the spleen of immunodeficient mouse recipients, showing myelo-lymphoid (CD45+Ter119-) and erythroid (Ter119+CD45+/-) cell fractions at 4 weeks post-implantation. (E) PCR analysis of Rosa26-BFP::Flk1-GFP gDNA in the cell fractions in (D) of implanted animal #15 at 4 weeks post-implantation. PCR run in triplicate material from each sample; positive and NTC controls as per Figure 4C. (F–G) Representative flow cytometry interrogation of BFP detection within lineage compartments in the bone marrow (BM) of control and implanted recipient animal #2 (F) and animal #1 (G) at 8 weeks after hGx implantation. (H) Histology of implanted and control adrenal glands; paraffin sections stained with hematoxylin & eosin (H&E). Inserts in implanted animal highlight neural (orange) and hematopoietic and renal tubular epithelium (green); inserts in control animal highlight normal cortical (blue) and medullar (red) adrenal architecture, absent in implanted adrenal glands.
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Figure 4—figure supplement 2—source data 1
Original files for agarose gel electrophoresis in Figure 4—figure supplement 2B, C and E.
- https://cdn.elifesciences.org/articles/102324/elife-102324-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Annotated uncropped agarose gel electrophoresis in Figure 4—figure supplement 2B, C and E.
- https://cdn.elifesciences.org/articles/102324/elife-102324-fig4-figsupp2-data2-v1.zip
Figure 5 with 2 supplements
Hemogenic gastruloid (HaemGx) with MNX1 overexpression have increased cellularity and enhanced hemogenic endothelial potential.
(A) Cell type enrichment analysis in MNX1-r AML transcriptomes, compared to normal pediatric bone marrow (BM) or other pediatric AML from the TARGET database. GSEA used representative cell type gene sets from the 2021 DB database; bubble plot shows NES scores (colour gradient) and statistical significance (–log10(FDR), bubble size). (B) Schematic representation of generation of MNX1-overexpressing and empty vector (EV) mES cells by lentiviral transduction, with subsequent assembly into haemGxs. (C) Imaging of haemGxs with MNX1 overexpression and EV controls at 10 x magnification, showing appropriate assembly and polarization of the Flk1-GFP marker. Scale bar: 300 mm. (D) Size of haemGx at each timepoint, determined by the distance between the furthest extreme points in µm. Mean ± SD of three replicate experiments; two-tailed t-test, p<0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****). (E–G) Flow cytometry timecourse analysis of (E) C-Kit+, (F) CD41+, and (G) CD45+ cell abundance in MNX1 and EV haemGxs (120–216 hr). Mean ± standard deviation of 3–7 independent experiments; two-way ANOVA and Sidak’s multiple comparison test significant at p<0.05 for C-Kit+ cells only (construct contribution to variance p=0.0191; 144 hr comparison *p=0.0190). (H) Volcano plots of differentially expressed genes (DEGs) by bulk RNA-seq between MNX1 and EV haemGxs at 144 hr and 216 hr. Intersection of statistically significant DEGs between 144 hr and 216 hr (shown in Venn diagram on top right) identified unique DEGs for each condition (labelled in red for 144 hr and blue for 216 hr). (J) Gene Ontology (GO) term enrichment for differentially upregulated genes between MNX1 and EV haemGx at 144 h (red) and 216 h (blue), computed in EnrichR using the GO Biological Process repository. (I) Transcription factor (TF) binding site enrichment on differentially upregulated genes between MNX1 and EV haemGx at 144 h (red) and 216 hr (blue) using the ENCODE and ChEA Consensus TFs from ChIP database on EnrichR.
Figure 5—figure supplement 1
MNX1 overexpression promotes hemogenic specification in hemogenic gastruloid (haemGxFigure 5).
(A) Cell type enrichment analysis in MNX1-r AML transcriptomes, compared to normal pediatric bone marrow (BM) or other pediatric AML from the TARGET database. Gene set enrichment analysis (GSEA) used representative cell type gene sets from the 2021 DB database; bubble plot shows NES scores (colour gradient) and statistical significance (–log10(FDR), bubble size). (B) Quantitative (q) RT-PCR analysis of MNX1 overexpression in 216 hr-haemGx. Gene expression fold change calculated by normalization to HPRT1. Mean ± SD of three replicates; 2-tailed t-test, p<0.001 (**). (C) Cell counts of disassembled haemGx at 216 hr. Mean ± SD of three replicate experiments; two-tailed t-tesp, p<0.001 (**). (D) Proportion of EV and MNX1 haemGx exhibiting spontaneous unifocal contractility at 192 hr, observed in commercial N2B27 medium (see Experimental Procedures); mean ± SD of three replicate experiments; two-tailed t-test, p<0.001 (**). (E–F) Flow cytometry analysis of specification of (E) hemato-endothelial – C-Kit+ and VE-Cadherin+ – and (F) hematopoietic progenitor – CD41+ and CD45+ – cells over a 120–216 hr timecourse of EV and MNX1 haemGx cultures; representative plots.
Figure 5—figure supplement 2
Transcriptional analysis of hemogenic gastruloid (haemGx) with MNX1 overexpression.
(A) Expression of human MNX1 and murine Mnx1 genes in FPKM units from RNA-seq of MNX1 and EV haemGx. (B) UMAP of time-resolved global clustering of scRNA-seq of haemGx showing the enriched clusters in GX-MNX1 at 144 hr (orange boxes) and 216 hr (blue boxes), and MNX1-r AML (black boxes) or MLL-AML (green boxes) determined by gene set enrichment analysis (GSEA). (C) Bubble plot of GSEA NES values and statistical significance by –log10(FDR) of enrichments in specific time clusters and corresponding global clusters in GX-MNX1 at 144 hr and 216 hr compared to respective GX-EV, and MNX1-r or MLL AML samples compared to other pediatric AML.
Figure 6 with 1 supplement
MNX1 selects C-Kit+ clonogenic cells and selectively transforms end-stage haemGx.
(A) Representative photographs of serial replating of colony-forming cell assays initiated from EV or MNX1 cells obtained at 144 h of the haemGx protocol. (B) Quantification of colony-replating efficiency of EV and MNX1 144h-haemGx (GX) cells. Mean + SD of n>3 replicates; Kruskal-Wallis with Dunn’s multiple comparison testing at significant q<0.05. (C) Representative photographs of serial replating of colony-forming cell assays initiated from EV or MNX1 cells obtained at 216 h of the haemGx protocol. (D) Quantification of colony-replating efficiency of EV and MNX1 216h-haemGx cells. Mean + SD of n=5–8 replicates; Kruskal-Wallis with Dunn’s multiple comparison testing at significant q<0.05. (E) Flow cytometry analysis of hemogenic progenitor C-Kit+ cells and myeloid-affiliated CD11b+ cells at first round of plating of 144 hr and 216 hr-haemGx initiated from EV and MNX1 cells; representative plots.
Figure 6—figure supplement 1
Characterization of MNX1-overexpressing replating cells from haemGx.
(A) Representative flow cytometry plot of CD31 and C-Kit staining of CFC plating of GX-MNX1 (left) and quantification of sustained expression of CD31+CKit+ cells through replatings. (B) Representative flow cytometry plots of serially re-plating MNX1 cells from 144 h and 216h-gastruloids (plate 5 cells) stained with Ter119 and CD45.
Figure 7 with 1 supplement
HaemGx with MNX1 overexpression recapitulate MNX1-r acute myeloid leukemia patient signatures.
(A) Quantification of human MNX1 transcripts in FPKM units from RNA-seq of GX-EV, GX-MNX1, and CFC at 144 hr and 216 hr. Bars represent mean of replicates. (B) Heatmap comparing the expression of all differentially expressed genes (DEGs) between all conditions (GX-MNX1 144 hr vs GX-EV 144 hr; GX-MNX1 216 hr vs GX-EV 216 hr; CFC MNX1 144 hr vs GX MNX1 144 hr; CFC MNX1 216 hr vs GX MNX1 216 hr) as Z score. Hierarchical clustering by Ward D method on Euclidean distances identifies 14 clusters (k). (C) Gene set enrichment analysis (GSEA) plots for gene set extracted from k14 from (B) against MNX1-r patients RNA-seq counts vs MLL, core-binding factors (CBF), or other pediatric AML. (D) Cell type analysis of the intersect of leading-edge genes LEGs (n=476) from Figure 7—figure supplement 1A.in MNX1-r patients (k14 LEGs) (B) using the Panglao DB 2021 database. (E) Representative Giemsa-Wright stained dissociated CFC replating (fourth plating) MNX1 haemGx cells from 144 hr and 216 hr. Solid black arrows, mast cell precursors; dashed black arrows, mast cells.
Figure 7—figure supplement 1
Transcriptional analyses of hemogenic gastruloid (haemGx) with MNX1 overexpression compared to patient signatures and current in vivo model.
(A) Venn diagram showing the intersection of leading-edge genes enriched in MNX1-r patient vs MLL, core-binding factors (CBF), or other pediatric AML from gene set enrichment analysis (GSEA) analysis using the K14 signature. (B) Cell type enrichment analysis in MNX1-r acute myeloid leukemia (AML) transcriptomes, compared to normal pediatric bone marrow (BM) or other pediatric AML from the TARGET database, with comparison with transcriptomes of haemGx and colony-forming assays (CFCs) from MNX1 vs EV conditions at 144 hr and 216 hr. GSEA used representative cell type gene sets from the Panglao DB 2021 database; bubble plot shows NES scores (colour gradient) and statistical significance (–log10(FDR), bubble size). (C) Representative Giemsa-Wright stained dissociated CFC replating MNX1 haemGx cells from 144 hr; purple arrows, undifferentiated blasts; solid black arrow, mast cell precursors; dashed black arrow, mast cells. (D) Transcriptional re-analysis of Waraky et al., 2024 in vivo model of MNX1-OE leukemia from transplanted fetal liver (FL) cells. Heatmap comparing the expression levels of all DEG between all conditions, i.e., FL control (FL Ctrl) vs FL with MNX1-OE (FL MNX1) in liquid culture, and bone marrow (BM) from mice transplanted with FL Ctrl (BM TX Ctrl) vs leukemic animals transplanted with FL MNX1 cells (BM TX MNX1). Hierarchical clustering by Ward D method on Euclidean distances identifies 12 clusters (k). Clusters k1, k2, k5, k6, and k9 are specifically up-regulated in BM TX MNX1 and putatively represent MNX1-OE leukemia programs. Analysis matches Figure 7B for MNX1-OE haemGx. (E) GSEA plots of significant results (by p-value <0.05) for gene set extracted from k1, k2, k5, k6, and k9, individually or combined, from (D) against MNX1-r patients RNA-seq counts vs MLL, core-binding factors (CBF), or other pediatric AML. FDR values are indicated for each enrichment point. Analysis matches Figure 7C for MNX1-OE haemGx. (F) Cell type enrichment analysis in MNX1-r AML transcriptomes, compared to normal pediatric bone marrow (BM) or other pediatric AML from the TARGET database. GSEA used representative cell type gene sets from the Panglao DB 2021 database; bubble plot shows NES scores (colour gradient) and statistical significance (–log10(FDR), bubble size). Analysis matches Figure 6E for MNX1-OE haemGx.
Author response image 1
Association of CD34 with CD41 and CD45 expression is Activin A-responsive and supports the presence of definitive haematopoiesis.
A. Flow cytometry analysis of CD34 and CD41 expression in 216h-haemogenic gastruloids; two doses of Activin A were used in the patterning pulse with CHI99021 between 48-72h. FMO controls shown. B. Flow cytometry analysis of CD34 and CD45 at 216h in the same experimental conditions.
Author response image 2
Flow cytometry analysis of VE-cadherin+ cells in haemogenic gastruloids at 216h of the differentiation protocol, probing co-expression of CD45, CD41 and C-Kit.
Author response image 3
Confocal images of haematopoietic production in haemogenic gastruloids.
Wholemount, cleared haemogenic gastruloids were stained for CD45 (pseudo-coloured red) and C-Kit antigens (pseudo-coloured yellow) with indirect staining, as described in the manuscript. Flk1-GFP signal is shown in green. Nuclei are contrasted with DAPI. (A) 192h. (B) 216h.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (E. coli) | NEB 5-alpha Competent E. coli | New England Biolabs (NEB) | Cat. #C2987H | Competent cells |
| Antibody | Anti-mouse c-Kit (CD117) clone 2B8 – APC-Cy7 — Monoclonal, Rat | BioLegend | Cat. #105825 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD41 clone mwreg30 – PE-Dazzle594 — Monoclonal, Rat | BioLegend | Cat. #133935 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD45 clone 30F11 – APC — Monoclonal, Rat | BioLegend | Cat. #103111 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD144 (VE-cadherin) clone BV13 – APC — Monoclonal, Rat | BioLegend | Cat. #138011 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD43 clone S11 – PE — Monoclonal, Rat | BioLegend | Cat. #143205 | Flow cytometry (1:100) |
| Antibody | Anti-mouse Ter119 – PC7 — Monoclonal, Rat | BioLegend | Cat. #116223 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD31 – biotin — Monoclonal, Rat | BD Pharmingen | Cat. #553371 | Flow cytometry (1:100) |
| Antibody | Anti-mouse CD34 – APC — Monoclonal, Armenian Hamster | BioLegend | Cat. #128611 | Flow cytometry (1:100) |
| Antibody | CD11b anti-mouse/human clone M1/70 – biotin — Monoclonal, Rat | BioLegend | Cat. #101203 | Flow cytometry (1:100) |
| Antibody | Mouse Anti-Mouse CD45.2 clone 104 – PE — Monoclonal, Mouse | BD Biosciences | Cat. #560695 | IF (1:200) |
| Antibody | Rat Anti-Mouse CD31 clone MEC13.3 – biotin — Monoclonal, Rat | BD Biosciences | Cat. #553371 | IF (1:200) |
| Antibody | Goat Anti-Mouse c-Kit/CD117 — Polyclonal, Goat | R&D Systems | Cat. #AF1356SP | IF (1:200) |
| Peptide, recombinant protein | Murine LIF | Peprotech | Cat. #250–02 | Medium supplement |
| Peptide, recombinant protein | Stemmacs pd0325901 | Miltenyi biotec | Cat. #130-106-5411 | Medium supplement |
| Peptide, recombinant protein | Chiron (chir99021) | Biogems | Cat. #2520691 | Medium supplement |
| Peptide, recombinant protein | Activin A Plus | Qkine | Cat. #qk005 | Medium supplement |
| Peptide, recombinant protein | Murine Vegf 165 | Peprotech | Cat. #450–32 | Medium supplement |
| Peptide, recombinant protein | Murine Fgf-basic | Peprotech | Cat. #450–33 | Medium supplement |
| Peptide, recombinant protein | Murine Sonic Hedgehog (Shh) | Peprotech | Cat. #315–22 | Medium supplement |
| Peptide, recombinant protein | Murine Tpo | Peprotech | Cat. #315–14 | Medium supplement |
| Peptide, recombinant protein | Murine Flt3-ligand | Peprotech | Cat. #250–31 l | Medium supplement |
| Peptide, recombinant protein | Murine Scf | Peprotech | Cat. #250–03 | Medium supplement |
| Other | N2B27 medium (NDiff 227) | Takara bio | Cat. #y40002 | Medium |
| Other | DMEM/F-12 | Gibco | Cat. #11320033 | Medium |
| Other | Neurobasal medium | Gibco | Cat. #21103049 | Medium |
| Other | N-2 supplement | Gibco | Cat. #17502048 | Medium |
| Other | B-27 supplement | Gibco | Cat. #17504044 | Medium |
| Other | Mouse methylcellulose complete media | R&D systems | Cat. #hsc007 | Medium |
| Cell line (M. musculus) | Kdr(Flk1)-GFP mouse embryonic stem cell line | Alfonso Martinez Arias Lab; Jakobsson et al., 2010 | Maintained in ESLIF medium | |
| Cell line (M. musculus) | Sox17-GFP mouse embryonic stem cell line | Alfonso Martinez Arias Lab; Niakan et al., 2010 | Maintained in ESLIF medium | |
| Cell line (M. musculus) | T/Bra::GFP mouse embryonic stem cell line | Alfonso Martinez Arias Lab; Fehling et al., 2003 | Maintained in ESLIF medium | |
| Cell line (M. musculus) | E14Tg2a mouse embryonic stem cell line | MMRRC, University of California Davis, US; Hooper et al., 1987 | Maintained in ESLIF medium | |
| Cell line (M. musculus) | Kdr(Flk1)-GFP::Rosa26-BFP mouse embryonic stem cell line | This paper | N/A | Materials and Methods (Cell culture), Figure 4—figure supplement 2A |
| Cell line (H. sapiens) | HEK293T | ATCC | CRL-3216 | Maintained in DMEM-F12, 10% FBS, 1% P/S |
| Sequence-based reagent | MNX1 forward | Gulino et al., 2021 | qPCR primers | 5-GTTCAAGCTCAACAAGTACC-3 |
| Sequence-based reagent | MNX1 reverse | Gulino et al., 2021 | qPCR primers | GGTTCTGGAACCAAATCTTC-3 |
| Sequence-based reagent | Ppia forward | Moris et al., 2018 | qPCR primers | TTACCCATCAAACCATTCCTTCTG-3 |
| Sequence-based reagent | Ppia reverse | Moris et al., 2018 | qPCR primers | AACCCAAAGAACTTCAGTGAGAGC-3 |
| Sequence-based reagent | BFP forward | This paper | PCR primers | 5-GCACCGTGGACAACCATCACTT-3 |
| Sequence-based reagent | BFP reverse | This paper | PCR primers | 5-CAGTTTGCTAGGGAGGTCGC-3 |
| Recombinant DNA reagent | Pwpt-lssmorange-PQR | Ghevaert Lab, WT-MRC Cambridge Stem Cell Institute, UK Dalby et al., 2018 | Vector | |
| Recombinant DNA reagent | Pwpt-LssmOrange-PQR-MNX1 | Cloned by Biomatik Corporation (Kitchener, Canada) | Vector | |
| Recombinant DNA reagent | Pcdna3-Rosa26-Rosa26-BFP | This paper | Vector | Construct described in Figure 4—figure supplement 2A; Materials and methods (Lentiviral vector packaging and transduction) |
| Other | CELLSTAR cell-repellent cell culture plate, 96 well, U-bottom | Greiner (Bio-One) | Cat. #650970 |
Additional files
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Supplementary file 1
Cluster classifier gene lists for each cluster by differential gene expression analysis of scRNA-seq of hemogenic gastruloid (haemGx) (all conditions), obtained by Wilcoxon rank test of each cluster against all other clusters.
- https://cdn.elifesciences.org/articles/102324/elife-102324-supp1-v1.xlsx
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Supplementary file 2
Cell type enrichment analysis of cluster classifier genes, showing the inferred identity of clusters of scRNA-seq of hemogenic gastruloid (haemGx) (all conditions).
Classifier genes (in Supplementary file 1) were obtained by differential expression in comparison to all other clusters and subjected to enrichment analysis using the PanglaoDB repository (Franzén et al., 2019).
- https://cdn.elifesciences.org/articles/102324/elife-102324-supp2-v1.xlsx
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Supplementary file 3
Summary of projections of scRNA-seq of hemogenic gastruloid (haemGx) cells onto available datasets by Hou et al., 2020, Zhu et al., 2020, and Thambyrajah et al., 2024, showing the identity of each haemGx cell projecting to annotated clusters of the respective studies.
- https://cdn.elifesciences.org/articles/102324/elife-102324-supp3-v1.xlsx
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Supplementary file 4
List of differentially expressed genes between hemogenic gastruloid (haemGx)-MNX1 and haemGx-MNX1 at 144 hr and 216 hr compared to empty vector (EV) control.
- https://cdn.elifesciences.org/articles/102324/elife-102324-supp4-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/102324/elife-102324-mdarchecklist1-v1.pdf
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Dissecting infant leukemia developmental origins with a hemogenic gastruloid model
eLife 14:RP102324.
https://doi.org/10.7554/eLife.102324.3
