Additional file 1 of WNT5A from the fetal liver vascular niche supports human fetal liver hematopoiesis
Creators: name: Choi, Yoon Jung
name: Heck, Adam M.
name: Hayes, Brian J.
name: Lih, Daniel
name: Rayner, Samuel G.
name: Hadland, Brandon
name: Zheng, Ying
Publisher: name: figshare
Year: 2021
Subjects: subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Table S1. Antibodies used in this study. Figure S1. A. Phenotypic hematopoietic stem and progenitor cells (HSPCs, CD45+CD34+) were found to represent 5.4 ± 0.4 % (n = 3 donors, mean ± SEM) of total liver cells. B. Representative flow cytometry profiles of (i) hESC- HPCs with isotype controls, (ii-v) fresh total cell tissue suspension from fetal human kidney(ii), heart(iii), lung(iv) and liver(v) stained with markers for HPCs. C. Multiple hematopoietic cell types were identified including (a) polychromatic normoblasts, (b) orthochromic normoblasts, (c) polychromatic erythrocytes, (d) promyelocytes, (e) myelocytes, (f) metamyelocytes (g) polymorphonuclear neutrophils, (h) eosinophils, (i) monocytes, (j) basophils, and (k) macrophages (scale bar: 10 m). D. Flow cytometry profiles of the colonies stained with erythroid and myeloid markers. Figure S2. A. Bright field image of E4ORF1 transduced liver ECs (i) and heart ECs (ii) cultured with and without serum media. Scale bar: 200 m. (iii) Ectopic expression of E4ORF1 in liver ECs (E4LECs), HUVECs (E4ECs), and cardiac ECs (E4CECs) compared with E4ECs was confirmed using a reverse-transcription quantitative PCR (RT-qPCR) ( n =3 donors, mean ± SEM) B. Comparison of mRNA expression of selected genes for primary and E4ORF1 transfected heart and liver endothelial cells for liver endo-specific genes. (n = 3, mean ± SEM, * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). Figure S3. E4LECs were transduced with lentiviral vectors containing WNT5A (WNT5A-IRES-GFP) under the control of CMV promoter (i) FACS sorting scheme for GFP positive cells. (ii) Representative images of sorted GFP+ E4LECs. Scale bar: 0.5 mm. B. Knockdown of WNT5A on E4LECs. (i) RT-qPCR confirms WNT5A knockdown on E4LECs after transduction with pGIPZ lentiviral shRNA against WNT5A (n = 3 donors, mean ±SEM, * p ≤ 0.05) (ii) Schematic diagram for preparation of KD E4LECs and co-culture with liver HPCs for 10 days. C. Overexpression of WNT5A on E4ECs. (i)RT-qPCR confirms that WNT5A is overexpressed in E4ECs transduced with lentiviral vector that encodes blasticidin resistance genes and WNT5A gene. (n = 3, mean ± SEM, * p ≤ 0.05) (ii) Schematic diagram of E4ECs co-cultured with liver HPCs for 10 days. D. RT-qPCR showed that WNT5A knockdown on E4LECs led to non-significant changes in EC gene expression including PCNA, DLL4, MMP10, NOS3 and VEGFR2 (n = 3 donors, mean ±SEM). Figure S4. The role of soluble WNT5A on ECs and HPCs. A-B. Immunostaining of E4ECs culture at 0 or 100ng/mL WNT5A (A) and E4LECs at 0 or 10 ng/mL neutralization antibody for WNT5A (B) for 24 hours. Red: VE-Cad, and Magenta: F-actin. C-D. Quantification of Ki67+ cells (C) and expression of genes (D) for both ECs before and after respective treatments. E-F. Phenotyping (E-F) of stromal free culture of CD43+CD45-CD144- cells with or without WNT5A and CFU analysis (G) after 3 days of culture shows significant increase of CD45+CD34+ cells, and appearance of CFU-GEMM with soluble WNT5A (n = 3-6, mean ± SEM, * p ≤ 0.05).