Method

• Sterile, 15 ml conical centrifuge tube

• Sterile, 5 ml transfer pipette, cut to have a wide-bore tip if necessary

• Benchtop refrigerated centrifuge

• 1000 ml micropipette

• Haemocytometer

• 35 mm tissue-culture dishes (Cat. No. 627102, Greiner)

1 Transfer EBs using a wide-bore pipette into a 15 ml conical tube and allow them to settle. Aspirate supernatant medium carefully (also using a wide-bore pipette), add 2 ml of trypsin/EDTA solution to the EBs, and incubate the tube in a water bath for 5-10 min at 37 °C.

2 Halt trypsinization by addition of 5 ml EB culture medium, and pellet cells by cent-rifugation for 5 min at 1200 rpm and 4 °C.

3 Resuspend the cells in MethoCult™ GF+ medium, count the cells and adjust the density to 1-2 x 105 cells/ml. Dispense 1ml of this mixture per 35 mm dish, and incubate.

4 CFUs can be scored by their morphology ~10d after plating, according to established criteria (see Atlas of Human Hematopoietic Colonies, Stem Cell Technologies Inc.).a

5 The differentiated cells can be assessed further (as described in refs 17-19) using FACS® analysis or immunofluorescence11

a Using this protocol and system of identification, human CFUs were generated from hES cells and identified as primitive erythroid (CFU-Ep), granulocyte-erythrocyte-macrophage--megakaryocyte (CFU-GEMM), granulocyte-macrophage (CFU-GM), macrophage (CFU-M), granulocyte (CFU-G), and erythroid burst-forming units (BFU-E) (17).

b In particular, cells cultured from day 15 EBs have been shown to express low levels of CD45 (1.4% ± 0.7%). Most of the CD45 + cells also coexpress CD34 (1.2% ± 0.5), which phenotype is similar to that of the first definitive haematopoietic cells to be detected within the wall of the dorsal aorta of human embryos (18).

providing a differentiation-inducing layer in combination with differentiationguiding medium. Depending on the desired course of differentiation, the layer may consist of either a specific ECM (such as poly-L-lysine, fibronectin, or collagen) or feeder cells (such as stromal cell lines derived from the relevant tissue). Use of a mouse bone-marrow stromal cell line to induce haematopoietic differentiation in mouse and non-human primate ES cells is described in Chapters 7 and 12, respectively.

Here we describe our method for inducing vascular differentiation of hES cells in monolayer culture using such a conditioning environment. The overall strategy (depicted in Figure 5) involves a two-step process (Protocol 7): (a) induction of differentiation into mesodermal progenitor cells, followed by (b) subculture of those mesodermal cells in growth factor-supplemented medium, to further direct differentiation into either ECs or v-SMCs.

The preliminary differentiation step (a) involves culture of single-cell suspensions in the absence of feeder cells, on collagen Type IV-coated dishes, and produces mainly two morphologically distinct cell types of mesodermal origin: (i) smaller cells with high nuclear/cytoplasmic ratios, and (ii) larger, flattened cells with an intracytoplasmic fibre arrangement (Figure 6A). Most of the type (i) cells are proliferating (Figure 6B) and express markers of endothelial progenitor cells (16); and it is these cells that give rise to vascular structures in vitro (see below). The larger cells express smooth muscle actin (SMA) as well as other SMC markers, and further studies are required to determine their exact nature and origin.

For the second differentiation step (b), mesodermal progenitors are induced further to differentiate and mature into either ECs or v-SMCs by their consecutive reculture on collagen Type IV-coated dishes, in medium supplemented with-either human vascular endothelial growth factor (hVEGF) or human platelet-derived growth factor BB (hPDGF-BB), respectively (16). Under these conditions,

Figure 6 Guided, vascular differentiation of hES cells: step (a), mesoderm differentiation in monolayer culture. Phase-contrast micrographs of hES cells cultured for 6d on type IV collagen-coated dishes for mesodermal differentiation, showing: (A) two derivative cell types comprising large, flattened cells with intracellular fibers (arrow) and smaller cells with higher nuclear/cytoplasmic ratios; and (B) the proliferative ability of the smaller cells, most of which become labelled with BrdU (dark nuclear stain), while the larger cells (arrow) fail to incorporate. Scale bars = 100mm. (Adapted from Ref. 16.)

vascular plexuses with ring- or cord-like arrangements can be observed forming from the maturing cells (16). Supplementation of medium with hVEGF results specifically in extensive uptake of Dil-acetylated low-density lipoprotein, with approximately 20% of cells producing von Willebrand factor and expressing CD31, denoting ECs (Figure 7A). Supplementation with hPDGF-BB promotes differentiation into v-SMC, with approximately 80% of cells expressing the v-SMC markers, SMA, calponin, and SM-MHC (16; Figure 7B).

Alternatively, mesodermal progenitor cells may be subjected to a three-dimensional gel assay, in step (c), with either collagen Type I or Matrigel™ (Figure 8A), to determine their capacity for in vitro vasculogenesis and the sprouting angiogenic process (Protocol 8). (These two ECMs are known to promote the formation of three-dimensional vessel-like structures from ECs in vitro (15, 16).) Transverse sections of the mesodermal cultures in Matrigel™ reveal penetration of the cells into the gel and formation of tube-like structures (Figure 8B). Ultrastructural observation by transmission electron microscopy also reveals typical, tubelike arrangements of elongated vascular ECs within the MatrigelTM, as well as intracellular Weibel-Palade bodies, lipoprotein capsules, and glycogen deposits in the developing ECs (7; Figure 8C).

Taking into consideration the limited means available for studying vascular development (comprising vasculogensis and angiogenesis) during human embry-ogensis in vivo, this sequential system for vascular-guided hES-cell differentiation offers a useful model in which to examine the mechanisms of vascular lineage

Figure 7 (see Plate 13) Guided, vascular differentiation of hES cells: step (b), maturation of mesodermal progenitor cells as analysed by immunofluorescence. (Panel A) Immunofluorescence anlayis of mesodermal cells cultured in the presence of hVEGF165 reveals expression of EC markers such as: (i) CD31 (red stain; nuclei counterstained in blue); (ii) Dil-Ac-LDL (red) and von Willebrand Factor (green). (Panel B) Similar analysis of cells cultured in the presence of hPDGF-BB reveals expression of v-SMCs markers such as: (i) SMA (red; nuclei in blue); (ii) calponin (red; nuclei in blue). Scale bars = 100mm. (Adapted from ref. 16)

Figure 7 (see Plate 13) Guided, vascular differentiation of hES cells: step (b), maturation of mesodermal progenitor cells as analysed by immunofluorescence. (Panel A) Immunofluorescence anlayis of mesodermal cells cultured in the presence of hVEGF165 reveals expression of EC markers such as: (i) CD31 (red stain; nuclei counterstained in blue); (ii) Dil-Ac-LDL (red) and von Willebrand Factor (green). (Panel B) Similar analysis of cells cultured in the presence of hPDGF-BB reveals expression of v-SMCs markers such as: (i) SMA (red; nuclei in blue); (ii) calponin (red; nuclei in blue). Scale bars = 100mm. (Adapted from ref. 16)

Figure 8 Directed, vascular differentiation of hES cells: step (c), vasculogenesls and angiogenesis in a three-dimensional gel composed of Matrigel™. (A) Inverted phase-contrast microscopy demonstrates extensive vasculogenesis with sprouting (angiogenesis) by a mesodermal aggregate on Matrigel™ in the presence of hVEGF. (B) Sections of vascular structures stained with toluidine blue reveal cord-like structures (arrows) penetrating the gel. (C) Transmission electron micrograph showing the arrangement of developing ECs (N = nucleus) within the Matrigel™ matrix (M): here the cells contain lipoprotein capsules (Li) at the lumen surfaces (Lu), and intracellular Weibel-Palade bodies (WP) and deposits of glycogen (G), the endogenous energy source for this cell type. Scale bars = 100mm.

Figure 8 Directed, vascular differentiation of hES cells: step (c), vasculogenesls and angiogenesis in a three-dimensional gel composed of Matrigel™. (A) Inverted phase-contrast microscopy demonstrates extensive vasculogenesis with sprouting (angiogenesis) by a mesodermal aggregate on Matrigel™ in the presence of hVEGF. (B) Sections of vascular structures stained with toluidine blue reveal cord-like structures (arrows) penetrating the gel. (C) Transmission electron micrograph showing the arrangement of developing ECs (N = nucleus) within the Matrigel™ matrix (M): here the cells contain lipoprotein capsules (Li) at the lumen surfaces (Lu), and intracellular Weibel-Palade bodies (WP) and deposits of glycogen (G), the endogenous energy source for this cell type. Scale bars = 100mm.

commitment and early angiogenesis. Furthermore, as considerable interest is currently centred on the inhibition of new vascular growth to treat the spread of cancer, hES cells differentiated in this manner may readily be employed for an in vitro evaluation of antiangiogenesis agents.

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