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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).

Figure S4. Monocyte adhesion in HS27a vessels. (A) Monocytes perfused through EC, EC with HS27a-conditioned media, or HS27a co-cultured vessels. (B) Quantification of monocyte adhesion shows no changes in adhesion between EC-only and EC with HS27a-conditioned media but an increase within the HS27a co-cultured vessels. Scale bars = 100 Îźm. (PDF 858 kb)

Figure S5. Expression of VCAM-1 in monocytes co-cultured with stromal fibroblasts and conditioned media. Microarray expression analysis of (A) monocytes from two different donors alone. (B) Expression of VCAM in HS5 cells, monocytes cultured with HS5-conditioned media, and monocytes co-cultured with HS5 cells. (C) Expression of VCAM in HS27a cells, monocytes cultured with HS27a-conditioned media, and monocytes co-cultured with HS27a cells. Expression values extracted from microarray data from Iwata et al. [44] ( http://www.ncbi.nlm.nih.gov/geo/ ; accession numbers GSE9390 and GSE10595, gene ID: 203868_s_at) (PDF 152 kb)

Figure S2. Immunofluorescence staining of von Willebrand Factor in an EC-only vessel after 6 days of culture. Scale bar = 50 Îźm. (PDF 1873 kb)

Table S1. Primer information for RT-PCR analysis. (PDF 293 kb)

Figure S1. Expression of CD146 by HS27a, HS5, and MSCs is shown via RT-PCR. MSC expression of CD146 is variable compared to the HS27a and HS5 cell lines. (PDF 445 kb)

Figure S6. Monocytes, not VCAM-1, determine HSPC trafficking in HS27a vessels. (A) HSPCs were perfused through HS27a co-cultured vessels (i) alone, (ii) after monocyte perfusion, or (iii) after monocyte and VCAM-1 blocking antibody perfusion. (B) HSPCs are shown with the vessel boundary (yellow dotted line). Scale bars = 100 Îźm. Quantification of (C) HSPC adhesion and (D) migration behavior from these vessels show that monocytes change HSPC adhesion and migration but blocking VCAM-1 in the presence of monocytes does not significantly change adhesion and migration. *p < 0.05, **p < 0.01, *** p < 0.001. (PDF 889 kb)

Figure S3. RT-PCR of endothelial and stromal cells from microvessels. RT-PCR shows similar expression of CXCR4, CXCL12, E-selectin, ICAM-1, FLT-3, angiopoietin-1, IL-6, DKK3, MCP-1, HIF-1a, IL-1b, TFGb, MIP1, and GM-CSF, IL-1a (normalized to L32 ribosomal protein). KDR, P-selectin, angiopoeitin2, and FLT4 have increased expression in the endothelial-only vessels. IL-6, IL-1b, and IL-1a have increased expression in the HS5 co-cultured vessels. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. (PDF 2015 kb)

Figure S6. Monocytes, not VCAM-1, determine HSPC trafficking in HS27a vessels. (A) HSPCs were perfused through HS27a co-cultured vessels (i) alone, (ii) after monocyte perfusion, or (iii) after monocyte and VCAM-1 blocking antibody perfusion. (B) HSPCs are shown with the vessel boundary (yellow dotted line). Scale bars = 100 Îźm. Quantification of (C) HSPC adhesion and (D) migration behavior from these vessels show that monocytes change HSPC adhesion and migration but blocking VCAM-1 in the presence of monocytes does not significantly change adhesion and migration. *p < 0.05, **p < 0.01, *** p < 0.001. (PDF 889 kb)

Figure S3. RT-PCR of endothelial and stromal cells from microvessels. RT-PCR shows similar expression of CXCR4, CXCL12, E-selectin, ICAM-1, FLT-3, angiopoietin-1, IL-6, DKK3, MCP-1, HIF-1a, IL-1b, TFGb, MIP1, and GM-CSF, IL-1a (normalized to L32 ribosomal protein). KDR, P-selectin, angiopoeitin2, and FLT4 have increased expression in the endothelial-only vessels. IL-6, IL-1b, and IL-1a have increased expression in the HS5 co-cultured vessels. *p < 0.05, **p < 0.01, *** p < 0.001, ****p < 0.0001. (PDF 2015 kb)