Journal: bioRxiv

Article Title: Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

doi: 10.1101/2022.10.07.511362

Figure Lengend Snippet: (A) Timeline of mES cells assembly and culture into gastruloids over a 216h time period with the addition of appropriate factors for the promotion of hemato-endothelial specification. (B) Imaging of hemogenic gastruloids 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: 100μm (C) Flow cytometry analysis of hemogenic gastruloids for expression of c-Kit, CD41, and CD45 markers from 96h to 216h, assessing the emergence of c-Kit-endothelium, hemogenic progenitors, and pre-HSC/HSC at each time point. (D) Colony-forming unit (CFU) assay of disassembled gastruloids assessing the ability to form hematopoietic colonies in multipotential methylcellulose-based medium. GEMM: granulocyte-erythroid-monocyte-megakaryocyte; GM: granulocyte-monocyte; M: monocyte; E: erythroid; BFU-E: burst-forming unit erythroid. CFU frequency of 5 gastruloids, n=3, mean±SD (E) Immunostaining of whole individual gastruloids at 216h showing the localized expression of Flk-1 (green), c-Kit (yellow), and CD45 (red) and DAPI (blue) nuclear staining, in a topologically accurate configuration; scale bar: 50μm.

Article Snippet: Confocal microscopy was performed on LSM700 on a Zeiss Axiovert 200 M with Zeiss EC Plan-Neofluar 10x/0.30 M27 and Zeiss LD Plan-Neofluar 20x/0.4 M27 objective lens.

Techniques: Imaging, Marker, Flow Cytometry, Expressing, Colony-forming Unit Assay, Immunostaining, Staining

Journal: bioRxiv

Article Title: Dissecting infant leukemia developmental origins with a hemogenic gastruloid model

doi: 10.1101/2022.10.07.511362

Figure Lengend Snippet: (A) Imaging of hemogenic gastruloids with MNX1 overexpression and EV controls at 10x magnification, showing appropriate assembly and polarization of the Flk1-GFP marker. scale bar: 300μm. (B) Quantitative (q)RT-PCR confirming MNX1 overexpression in hemogenic gastruloids at endpoint (216h). Fold change in expression was calculated by normalization to HPRT1 . Statistical difference was calculated by Student’s t-test, p < 0.001 (**). Error bars indicate ± SD of 3 replicates. (C) Size of gastruloids at each timepoint, determined by the distance of the longest extremes in μm. Statistical difference was calculated by Student’s t-test, p value < 0.05 (*), 0.001 (**), 0.0001 (***), and 0.00001 (****). Error bars represent standard deviation (SD) of n=3. (D) Proportion of gastruloids exhibiting spontaneous contraction at 192h; error bars show SD of n=3. (E-G) Flow cytometry quantification of positive MNX1 and EV gastruloids cells for c-Kit (E) , CD41 (F) , and CD45 (G) at 144h and 192h. Statistical difference was calculated by Student’s t-test, p < 0.05 (*).

Article Snippet: Confocal microscopy was performed on LSM700 on a Zeiss Axiovert 200 M with Zeiss EC Plan-Neofluar 10x/0.30 M27 and Zeiss LD Plan-Neofluar 20x/0.4 M27 objective lens.

Techniques: Imaging, Over Expression, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Standard Deviation, Flow Cytometry