Hemogenic gastruloids (hGx) 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 216h period with the addition of specified factors for the promotion of hemato-endothelial specification. The 24-hour pre-treatment in 2i+LIF was omitted when mES cultures showed no signs of differentiation. (B) Imaging of hGx over time from 72h to 216h at 10x magnification, showing the assembly and growth of the 3D structures and the polarization of the Flk-1-GFP marker from 96h; scale bar: 100mm. (C) Flow cytometry timecourse analysis of hGx for the presence of CD144 (VE-cadherin), C-Kit, CD41, and CD45 markers; representative plots. (D) Quantification of analysis in (D). 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 gastruloids at 216h showing the localized expression of CD31 (yellow), C-Kit (turquoise), and CD45 (pink) markers; scale bar: 100μm.

Time-resolved analysis of scRNA-seq of hGxs captures successive waves of hematopoietic specification.

(A) UMAP projection of all sequenced cells colored 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 hGx culture time (right). (B) Heatmap representation of the expression of endothelial and hematopoietic marker genes in individual hGx C-Kit+ cells sorted at 144 and 192h. Cells and genes ordered by unsupervised hierarchical clustering; cell cluster dendrogram in Fig. S2E. (C) Heatmap representation of the expression of endothelial and hematopoietic marker genes in individual hGx CD41+ and CD45+ cells sorted at 144 and 192/216h, respectively. Cells and genes ordered by unsupervised hierarchical clustering; cell cluster dendrogram in Fig. S2F. (D) 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±Csf1/2/3r); My+Ly: Spi1±Csf1/2a/3r + Ikzf1±Ighm/d±Igk); HSPC: Ptprc + Myb). (E-G) Cell type-associations of selected surface markers and transcription factors affiliated with the HE-to-hematopoietic transition (CD44, Procr, Etv6, Gata2, Notch1, Tal1) and with hematopoietic (Gfi1b, Mllt3, Runx1) and definitive hematopoietic (Ikzf1, Hlf, Hoxa9, Myb) gene expression programs with (E) C-Kit+ (endo-haem) and CD41+/CD45+ (haem), (F) endo-haem cells obtained at 144h or 192h, and (G) CD41+ or CD45+ cells. Gene expression analysed as present (non-zero expression) or absent (zero-expression). Odds ratio with significance (-log10p<1) estimated by Fisher’s test.

HGxs specify cells with pre-HSC characteristics.

(A) Flow cytometry analysis of 216h Rosa26-BFP::Flk1-GFP hGx cells for CD41, CD45, c-Kit and CD144 (VE-Cadherin). (B) Quantification of pre-HSC-like c-Kit+CD144+ cells within the CD45+CD41lo fraction of 216h-hGx; mean of 4 independent replicate experiments. (C) Colony-forming cell (CFC) assay of 216h-hGx 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 3hGx, n=3, mean±SD. (D) Representative photographs of colonies quantified in C; scale bar=2mm. (E) Schematic representation of the experimental workflow for execution and analysis of hGx implantation in the adrenal gland of immunodeficient mice. (E) 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. (F) PCR detection of hGx genomic (g)DNA in the bone marrow (BM) and spleen (Spl) of immunodeficient mice 1 month after unilateral adrenal implantation of 3 gastruloids/gland. Analysis of 5 replicates of 100ng gDNA /recipient tissue; control animal was injected unilaterally with PBS in an adrenal gland in parallel with experimental implantation. Reaction positive control used 100ng of gDNA from Rosa26-BFP::Flkj1-GFP mES cells (mES) used to generate hGxs. NTC: no template control. See Fig. S4F-H for additional details.

HGxs with MNX1 overexpression have increased cellularity and enhanced hemogenic endothelial potential.

(A) Cell type enrichment analysis in MNX1-r AML transcriptomes, compared to normal paediatric bone marrow (BM) or other paediatric AML from the TARGET database. GSEA used representative cell type gene sets from the 2021 DB database; bubble plot shows NES scores (color 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 hGxs (haemGX). (C) Imaging of hGxs with MNX1 overexpression and EV controls at 10x magnification, showing appropriate assembly and polarization of the Flk1-GFP marker. scale bar: 300mm. (D) Size of hGx at each timepoint, determined by the distance between the furthest extreme points in µm. Mean ± SD of 3 replicate experiments; 2-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 hGxs (120-216h). Mean ± standard deviation of 3-7 independent experiments; 2-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; 144h comparison *p=0.0190). (H) Volcano plots of differentially expressed genes (DEGs) by bulk RNA-seq between MNX1 and EV hGxs at 144h and 216h. Intersection of statistically significant DEGs between 144 h and 216 h (shown in Venn diagram on top right) identified unique DEGs for each condition (labelled in red for 144 h and blue for 216 h). (J) Gene Ontology (GO) term enrichment for differentially upregulated genes between MNX1 and EV hGx at 144h (red) and 216h (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 hGx at 144h (red) and 216 hr (blue) using the ENCODE and ChEA Consensus TFs from ChIP database on EnrichR.

MNX1 selects C-Kit+ clonogenic cells and selectively transforms end-stage hGxs.

(A) Representative photographs of serial replating of colony-forming cell assays initiated from EV or MNX1 cells obtained at 144h of the hGx protocol. (B) Quantification of colony-replating efficiency of EV and MNX1 144h-hGx 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 216h of the hGx protocol. (D) Quantification of colony-replating efficiency of EV and MNX1 216h-hGx 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 144h and 216h-hGxs initiated from EV and MNX1 cells; representative plots. (F) Representative flow cytometry plots of serially re-plating MNX1 cells from 144h and 216h-gastruloids (plate 5 cells).

HGxs with MNX1 overexpression recapitulate MNX1-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) 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 Fig. S5D in MNX1-r patients (k14 LEGs) (B) using the Panglao DB 2021 database. (E) Representative Giemsa-Wright stained dissociated CFC replating MNX1 hGx cells from 144h and 216h. Solid black arrows, mast cell precursors; dashed black arrows, mast cells.