We began our data analysis by verifying the expression of 'stemness' genes that are down-regulated in ESCs upon differentiation into EBs and BCs. We identified 87 genes that were down-regulated upon differentiation into EBs. Genes with the highest fold change include SOX2, LEFTY1, GAL, NODAL, OCT4, and THY1, which play a critical role in maintaining the undifferentiated status of ESCs 222324. To uncover enriched processes, data sets were analyzed by DAVID, a web-based tool that identifies over-represented biological themes in a data set based on their Gene Ontology (GO) terms. GO provides consistent descriptions of genes in terms of biological processes and molecular function. When these genes were clustered with DAVID based on their GO terms, processes involved in development, cell differentiation, and proliferation were identified. The genes identified in the development ontology were DNA methyltransferase 3B, FGF2, THY1, SFRP2, LEFTY1, GREM1, and NODAL (Additional data file 3).

We also identified 267 genes that were down-regulated upon differentiation of ESCs to BCs. These genes include GAL, TDGF, NANOG, LEFTY1, and OCT4, most of which are stemness genes 222324. When genes were clustered with DAVID using their GO terms, the processes included development, cell differentiation, and morphogenesis (Additional data file 4). These data demonstrate that OCT4, NODAL, GAL, and THY1 are initially down-regulated in stage 1 (ESCs→EBs) and are further down-regulated in stage 2 (EBs→BCs).

While the focus of this paper is to evaluate the pathways involved in hemangioblast differentiation, we begin by identifying those genes that were up-regulated in the early stage of differentiation into EBs (day 3.5) from which BCs were derived 21. We identified 128 genes that were up-regulated upon differentiation of ESCs into EBs (Additional data file 5). These genes include HAND1, WNT5, HEY1, LMO2, BMP4, TBX3, and MYL4. Clustering these genes with EASE identified processes involved in development, transcription, organ development and system development, some of which are related to hemangioblastic differentiation. These genes include SOX9, HOXB2, HOXB3, Neuregulin 1, LMO2 25 and GATA2 26. This data set also included numerous genes encoding transcription factors, such as MESP1, HAND1, TBX3, GATA2, SOX7, SOX9, HOXB2, and HOXB3.

When EBs were compared to BCs, 185 genes were identified as down-regulated upon differentiation. This data set contained processes that were similar to those that were down-regulated upon ESC differentiation into EBs, such as tissue and organ development. The most significantly down-regulated genes included NANOG, WNT5, OCT4, GAL, TDGF1, BMP4, endothelin receptor B, and VEFG (Additional data file 6). These data demonstrate that BMP4, WNT5, and HEY1 are initially up-regulated upon differentiation into EBs but then down-regulated upon further differentiation into BCs.

In contrast, 82 genes were up-regulated upon differentiation of EBs into BCs. The genes with the greatest fold change were hemoglobin genes and erythropoietic genes, such as hemoglobins γ, ζ, α, and ε, Alas2, AFP, TUBB1, GYPA, and RHAG (fold change, (FC) 31x-886x). Genes with moderate increases in expression (FC 6.2x-7.2x) were KLF1, TAL1/SCL, GATA1 and CD71. When genes were clustered with DAVID using their GO terms, processes characteristic of erythropoiesis (heme and porphyrin biosynthesis and oxygen transport) were identified (Additional data file 7).

There were 107 genes up-regulated upon differentiation of ESCs into BCs. Similar to the data set above (EBs→BCs), the genes with the greatest fold change (FC 29x-810x) were involved in hemoglobin synthesis (hemoglobins γ, ε, and α, ALA2, GYPA, and TAL1/SCL), similar to the comparison of EBs to BCs. In addition, this data set contained many key transcription factors involved in hemangioblastic differentiation, such as GATA2 26, LMO2 25 and TAL1/SCL 2728 (Additional data file 8). GATA2 and MYL4 were up-regulated upon differentiation into EBs and remained at a constant level upon differentiation into BCs. EASE analysis of up-regulated genes in BCs identified biologically relevant themes, such as oxygen and gas transport, and development (TAL1/SCL, KLF1, LMO2, GATA1; Table 2).

Since genes that are up-regulated in ESCs when compared to breast epithelia could represent those genes that are under-expressed in breast epithelium, we filtered out those that were also up-regulated in BCs when compared to breast epithelium. This analysis identified 2,108 genes, which comprised GO processes involved in cell cycle, DNA and RNA metabolism, and DNA replication, as expected (Additional data file 9). Genes with the highest fold change include TDGF1, GAL, LEFTY1/2, OCT4, and NANOG (FC 130.0x, 69.6x, 68.7x, 47.6x, 43.8x, and 31.5x). When this data set was clustered based on their GO terms, processes involved in development, cell differentiation and nervous system development were identified (data not shown). This data set was then analyzed with GenMapp, and then used for pathway analysis. Each genetic signature was assigned a color: ESCs, green; EBs, orange; and BCs, red. The ESC pathway confirms that most of the embryonic genes were not removed when compared to breast epithelial cells (Additional data file 1).

Since genes up-regulated in EBs when compared to breast epithelia could similarly represent those that are under-expressed in breast epithelium, we also filtered out those that were also up-regulated in ESCs when compared to breast epithelium. We identified 939 genes as up-regulated in EBs relative to breast epithelium and filtered out those that are enriched in ESCs relative to breast epithelium (Additional data file 10). When these genes were clustered with DAVID, processes involved in development, transcription, wnt and frizzle signaling, cell cycle and blood vessel morphogenesis (KDR, VEGF, Neuropilin-1 and 2, and FLT1) were identified The EBs also express genes involved in organ development, suggesting a heterogeneous mixture of cell types (GATA2/4/5/6, BMP4, NCAM1, NOG, ISL2, NKX2.5), and mesoderm genes (HAND1, T-brachyury, MESP1). Further examination of the data set identified multiple genes involved in the BMP signaling pathway in the differentiation of blood and endothelial cells. These genes include BMPR1A, BMP4, T-brachyury, KDR, GATA2, and TAL1/SCL 2628.

We identified 2,735 genes that were up-regulated in BCs relative to breast epithelium after removing genes that were enriched in ESCs when compared to breast epithelium and genes enriched in BCs when compared to breast epithelium (Additional data file 11). When genes were clustered based on their GO, we identified processes characteristic of lymphocytic cells (response to stimulus, defense response, immune response), erythrocytes (heme and porphyrin biosynthesis), coagulation, neurophysiology, development, and mesoderm and heart development (Table 3).

Genes that were up-regulated in BCs with respect to epithelia were characteristic of hematopoiesis (CD markers 5/6/9/38/41/48/55/71/74/84/244, EPOR, GATA1/2/4/5, Tcr-α, natural cytotoxicity triggering receptor-1, 2 and 3), coagulation (coagulation factor II, V, VII, XII, coagulation factor 2 receptor like 2,3/thrombin receptors, antithrombin 3, cyclooxygenase 1, plasminogen), cardiac muscle (NKX2.5, HAND1/2, GATA4, SOX6, TBX5), smooth/skeletal muscle (NOTCH1, smoothelin, acetylcholinesterase, desmin, SOX6), synaptic markers (cholinergic receptor, muscarinic 2 and 5, adrenergic α-1A-receptor, dopamine receptor D2, serotonin receptor 1B and 4, glutamate receptor Nmda 1 and 2A/B and C, gaba A receptor β1 and 2, purinergic receptor P2X 1 and 2), and hemangioblasts (GATA2 26, RUNX1 29, LMO2 25 and TAL1/SCL 2728). Some of the genes identified in the coagulation ontology are not only involved in coagulation but also angiogenesis, such as thrombin 30, plasminogen 31, and possibly coagulation factor 2 receptor like 2/3 30.

This was also recapitulated by DAVID analysis, which identified the following pathways as statistically over-represented: porphyrin metabolism, acute myocardial infarction, hematopoietic cell lineage pathways and calcium signaling pathways. GenMapp was then used for pathway analysis. Each genetic signature was assigned a color: ESC, green; EBs, orange; and BCs, red. GenMapp analysis of pathways involved in whole blood, bone marrow, coagulation and complement, heme and porphyrin synthesis are indicative of hematopoietic cell types (Figure 2). Genes identified in GenMapp's heme biosynthesis pathway are indicative of erythoblasts (Figure 3). We also identified myogenic (cardiac and smooth) pathways, which correlates with the GO analysis.

We identified 2,101 genes that were up-regulated in BCs relative to leukocytes (after removing genes that were enriched in both hESCs and BCs when compared to leukocytes (Additional data file 12). When these genes were clustered based on their GO, we identified processes involved in development, nervous system development, blood vessel development and angiogenesis, and erythrocytes (Table 4). The presence of development ontology not only indicates a 'progenitor' status of BCs, but contains genes involved in hemangioblast development, such as LMO2, TAL1/SCL, and RUNX1. This comparison identified genes that are characteristic of endothelia (PECAM1, VE-Cadherin, CD34, vWF, EPOR 32, endothelin 1 33, and thrombin receptor). There were also genes that indicate the presence of erythrocytes (GATA1, spectrin and ankyrin), blood vessel development (neuropilin-1 and 2, stabilin 1 and 2, EGFR, FGF1 and 6, NOTCH4), and neurons/neuronal junctions (glutamate receptor 1/6, serotonin receptor 1e/6, nestin, neurogenic differentiation 4, neuroligin 2, myelin basic protein, peripheral myelin protein 22).

Of particular note is the absence of leukocytic processes, such as response to stimulus and defense response identified in the level two analyses. Thus, this comparison allowed for the masking of the 'lymphocytic' signature and, thus, the identification of other endothelial and blood vessel development genes (vWF, bradykinin receptor b1, and thrombin receptor). When this data set was analyzed using GenMapp, more endothelial genes were mapped to the coagulation cascade pathway (vWF, bradykinin receptor b1, thrombin receptor-pathway; data not shown).

We identified 904 genes that were up-regulated in BCs relative to prostate-derived endothelium after filtering out those genes that are enriched in ESCs relative to endothelium (Additional data file 13). Comparing BCs to endothelial cells identified fewer genes when compared to other comparisons of BCs in level II and III analyses, and, thus, identified fewer GO terms. However, it did identify more erythrocytic processes (nine in total; Table 5) than the other comparisons. Another predominant theme in this data set was development (Table 5). By comparing BCs to a more mature yet similar cell type (adult endothelium), we were able to mask the endothelial signature, thus identifying predominantly development genes (indicating stem/progenitor type signature), such as caudal type homeobox transcription factor 2 (CDX2), delta-like homolog (DLK1), lamin A/C, secreted frizzled-related protein 5 (SFRP5), patched (PTCH), dishevelled 2 (DVL2), and even-skipped homeobox homolog 1 (EVX1).

The BCs were then compared to prostate-derived stromal fibromuscular (CD49a immunoselected) tissue. This comparison identified the most number of genes (3,277 genes; Additional data file 14), and had the most diverse GO terms (lymphocytic, developmental, erythrocytic, coagulation, synapses and neurogenesis, and heart development; Table 6). This data set contained lymphocytic processes according to their GO (response to stimulus, defense response) and numerous lymphocytic markers (CD 6/38/41/43/48/55/61/71/84/244, immunoglobulin genes heavy constant γ1, constant κ, constant λ1, and CD 158A/B/D/F/H (killer cell immunoglobulin-like receptor)). This comparison identified genes involved in hemangioblast differentiation (TAL1/SCL, LMO2, RUNX1), endothelial genes (neuropilin 1 and 2), and coagulation genes (fibrinogen α and β chain, coagulation factor 5, plasminogen, but not KDR, FLT1, CD4, PECAM, VE-Cadherin, vWF). Although EASE analysis for both epithelial and stromal comparisons identified similar heart GO terms, the stromal comparison identified different heart development genes, such as MEF2C, aortic preferentially expressed (APEG1), POU6F1, TBX1, and ryanodine receptor 2 (cardiac).