Overview of protocols

Here, we present three protocols for inducing human ES cells to differentiate into endothelial cells (Figure 1). These three methods form the basis for recent advances in endothelial differentiation of human ES cells (Wang et al., 2004; Levenberg et al.,

Protocol I (Levenberg)

Embryoid bodies

FACS Sorting A -PECAM+ JO/7

Protocol I (Levenberg)

7 days +VEGF

7 days +VEGF

EC-like precursors

7 days

+VEGF

+BPitEx

+FBS

+Heparin

Embryoid bodies

FACS Sorting A -PECAM+ JO/7

EC-like precursors

7 days

+VEGF

+BPitEx

+FBS

+Heparin

Mesodermal precursors

Figure 1 Schematic diagram of three protocols for human ES cell differentiation of endothelial cells. Outline of the key steps in each protocol presented in this chapter. Protocol I (Levenberg) and II (Wang) make use of spontaneous differentiation of embryoid bodies followed by FACS sorting of endothelial precursors. These are then cultured in endothelial differentiation conditions in the presence of VEGF. Protocol III (Gerecht-Nir) makes use of monolayer differentiation of ES cells on collagen IV, followed by size separation of cells and differentiation in the presence of VEGF.

Mesodermal precursors

EC-like cells PECAM+ Flk-1 +

VE-Cadherin+

vWF+

CD34+

Uptake of Ac-LDL Cord formation (matrigel) Anastomosis (in vivo)

EC-like cells VE-Cadherin+ vWF+ eNOS+

Uptake of Ac-LDL (CD45-)

Hematopoietic CFU-

VE-Cadherin+ Tie-2+

Uptake of Ac-LDL Cord formation (matrigel)

Figure 1 Schematic diagram of three protocols for human ES cell differentiation of endothelial cells. Outline of the key steps in each protocol presented in this chapter. Protocol I (Levenberg) and II (Wang) make use of spontaneous differentiation of embryoid bodies followed by FACS sorting of endothelial precursors. These are then cultured in endothelial differentiation conditions in the presence of VEGF. Protocol III (Gerecht-Nir) makes use of monolayer differentiation of ES cells on collagen IV, followed by size separation of cells and differentiation in the presence of VEGF.

2002; Gerecht-Nir et al., 2003) and make use of defined culture conditions, spontaneous or directed differentiation, endothelial selection and subsequent endothelial amplification. Although the protocols use slightly different techniques, the common result is the generation of highly enriched populations of human stem cell-derived endothelial cells.

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