. 2021 Mar 30;65(5):473–488. doi: 10.1165/rcmb.2021-0152TR

Table 1.

Summary of Differentiation Protocols for the Generation of Various EPC Populations

Starting Cell Type	Cell Type Generated	Protocol Summary	Key Contribution	Citation
mESC	FLK1⁺/E-cadherin⁻	Generation and isolation of FLK1⁺ cells after 4 d in culture Addition of VEGF₁₆₅ to sorted cells results in sheets of CD31⁺ ECs	mESC-derived Flk1⁺ cells give rise to endothelial and mural cell types VEGF₁₆₅ promotes development of the endothelial lineage	Yamashita et al., 2000 (123)
hESC Embryoid Body	CD31⁺/KDR⁺/VE-Cadherin⁺/CD45⁻	Generation of EBs Treatment with SCF, Flt3L, IL-3, IL-6, G-CSF, and BMP4 FACS sorted CD45⁺ and CD45⁻ populations Culture in EC media	Role of cytokines and BMP4 in promoting hematopoietic differentiation Identification of CD45⁻ bipotent cells with hematopoietic and endothelial capacity	Chadwick et al., 2003 (121)
hESC Embryoid body	SCL⁺/LMO2⁺/FLT⁺/CD31⁻/CD34⁻/KDR⁻	Generation of early-stage EBs with factors such as BMP4, VEGF, SCF, and Tpo Dissociation of EBs and plating single cells in semisolid blast colony growth medium	Large-scale generation of hemangioblast cells under serum-free culture conditions	Lu et al., 2007 (122)
hESCs Embryoid body	CD144⁺/KDR⁺/CD31⁻	Generation of EBs using the established protocol by Chadwick and colleagues (2003) with varying concentrations of BMP4	BMP4 accelerates commitment of hESCs to the endothelial cell lineage while inhibiting commitment toward hematopoiesis	Goldman et al., 2009 (144)
hESC/iPSC Embryoid body + Monolayer	CD31⁺/CD146⁺/KDR⁺/CD133⁺/CD34⁺	Generation of EBs Plating of EBs on fibronectin-coated dishes in EGM-2 media supplemented with VEGF Expansion of vascular progenitor fraction in EGM-2 media	Three-dimensional and two-dimensional culture system for the simultaneous generation of hematopoietic and vascular progenitors Use of minimal factors (VEGF, BMP4, FGF2)	Park et al., 2013 (145)
iPSCs Embryoid body	CD31⁺/VE-cadherin⁺/KDR⁺/eNOS⁺	VE-cadherin⁺ cells are isolated from EBs and seeded on fibronectin-coated dishes	Characterization of humoral-, pharmacological-, and biomechanical-induced functional phenotypes of iPSC-derived ECs	Adams et al., 2013 (146)
iPSC Monolayer	CD34⁺/CD31⁺	Adherent culture system with small molecule GSK3 inhibition in the absence of exogenous growth factors	Temporal activation of WNT signaling, using small molecule CHIR, is sufficient to generate EPCs in the absence of factors VEGF and FGF2	Lian et al., 2014 (125)
hESC/iPSC Monolayer	NRP-1⁺/CD31⁺/CD144⁺/KDR⁺ (CB-ECFCs)	Adherent culture system with Activin A, BMP4, VEGF, and FGF2 in serum-free media	Two-dimensional system that does not require EB development, feeder cells, or TGF-β inhibition Protocol generates HPP-ECFCs with robust vessel-forming ability in vivo	Prasain et al., 2014 (124)
hESC Embryoid body + Monolayer	VE-cadherin⁺/CD31⁺/CD34⁺/CD14⁻/KDR^high	Three-phase protocol including both suspension and adherent culture method	Controlled modulation of BMP, Wnt, VEGF, and Notch pathways results in rapid production of EPCs by Day 6 and mature ECs by Day 14	Sahara et al., 2014 (147)
iPSC Monolayer	CD144⁺/KDR⁺/CD31⁺/CD34⁺/CD105⁺	Six-day differentiation protocol involving differentiation to mesodermal lineage with either CP21 or CHIR and/or BMP4 with subsequent exposure to VEGF-A to induce EC development	Efficient and quick production of endothelial cells with efficiencies between 61.8% and 88.8%	Patsch et al., 2015 (148)
hESC Monolayer	KDR⁺/CD34⁺/CD31⁺/VE-cadherin⁺	hESCs are cultured on stem cell niche LN matrices (LN521) This protocol involves the use of factors such as Activin A, BMP4, VEGF, and bFGF	A chemically defined, xeno-free protocol using LN coating to generate roughly 95% functional EPCs	Nguyen et al., 2016 (126)
hESC/iPSC Monolayer	CD157⁺/CD31⁺/CD144⁺/CD34⁺/KDR⁺/CXCR4⁺ (HPP-ECFCs)	Synergistic three-phase protocol using unique media compositions Generation of primitive streak → KDR⁺ mesoderm → primitive endothelium	Highly efficient, fully defined protocol for generation of EPCs with minimal variability HPP-ECFC–like cells with expression of EPC marker CD157 in addition to other standard EPC surface markers	Farkas et al., 2020 (149)

Definition of abbreviations: bFGF = basic fibroblast growth factor; BMP4 = bone morphogenetic protein 4; CB-ECFCs = cord blood endothelial colony-forming cells; EB = embryoid body; EGM-2 = endothelial growth medium-2; eNOS = endothelial nitric oxide synthase; EPCs = endothelial progenitor cells; FGF2 = fibroblast growth factor 2; hESC = human embryonic stem cell; HPP-ECFCs = high-proliferative-potential endothelial colony-forming cells; iPSC = induced pluripotent stem cell; LN = laminin; mESCs = murine embryonic stem cells; SCF = stem cell factor; TGF-β = transforming growth factor-β; VEGF = vascular endothelial growth factor.