Endothelial Progenitor Cells Produced From Human Pluripotent Stem Cells by a Synergistic Combination of Cytokines, Small Compounds, and Serum-Free Medium

Abstract

Human pluripotent stem cells (hPSCs) are a promising source of autologous endothelial progenitor cells (EPCs) that can be used for the treatment of vascular diseases. However, this kind of treatment requires a large amount of EPCs. Therefore, a highly efficient, robust, and easily reproducible differentiation protocol is necessary. We present a novel serum-free differentiation protocol that exploits the synergy of multiple powerful differentiation effectors. Our protocol follows the proper physiological pathway by differentiating EPCs from hPSCs in three phases that mimic in vivo embryonic vascular development. Specifically, hPSCs are differentiated into (i) primitive streak, which is subsequently turned into (ii) mesoderm, which finally differentiates into (iii) EPCs. This differentiation process yields up to 15 differentiated cells per seeded hPSC in 5 days. Endothelial progenitor cells constitute up to 97% of these derived cells. The experiments were performed on the human embryonic stem cell line H9 and six human induced pluripotent stem cell lines generated in our laboratory. Therefore, robustness was verified using many hPSC lines. Two previously established protocols were also adapted and compared to our synergistic three-phase protocol. Increased efficiency and decreased variability were observed for our differentiation protocol in comparison to the other tested protocols. Furthermore, EPCs derived from hPSCs by our protocol expressed the highproliferative-potential EPC marker CD157 on their surface in addition to the standard EPC surface markers CD31, CD144, CD34, KDR, and CXCR4. Our protocol enables efficient fully defined production of autologous endothelial progenitors for research and clinical applications.

Current Concepts on Endothelial Stem Cells Definition, Location, and Markers

“…CD157 expression was significantly higher in hPSCs-derived ECFCs (46%) compared with mature human saphenous vein endothelial cells (2%); however, functional readouts of this CD157 + high proliferative potential (HPP)-ECFC population in humans is warranted. 55 Endothelial protein C receptor (EPCR) is a glycoprotein with anti-thrombotic and cytoprotective roles. EPCR expressing endothelial cells have also been suggested as vascular endothelial stem cells.…”

miR‐25‐5p regulates endothelial progenitor cell differentiation in response to shear stress through targeting ABCA1

“…Specifically, EPCs can migrate to the damaged sites where they differentiate into mature endothelial cells (ECs) to retain the endothelial integrity (Werner et al, 2002). and several EC‐specific biomarkers such as cluster of differentiation 31 (CD31) (Farkas et al, 2020), TEK tyrosine kinase (TIE2) (Khan et al, 2014) or vascular endothelial growth factor receptor 2 (VEGFR2) (Damioli et al, 2017) have been incriminated in such a process.…”

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

“…When generating ECs from hPSCs, the progenitor stage of ECs (EPCs) is normally controlled by CD34 and KDR expression, and the most common used markers to assess mature ECs in vitro are CD31 and CD144. The first stage of mesoderm commitment normally takes two/three days, and the most widely described approaches rely on the activation of the Wnt/β-catenin pathway, in some cases combined with the supplementation with BMP4 and Activin A growth factors [ 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ]. The inclusion of BMP4 activation during this first stage of differentiation has been described to enhance endothelial commitment by favoring the induction of KDR + precursor cells, which has been correlated with a higher percentage of endothelial progenitor cells at later stages of differentiation [ 47 , 48 ].…”

“…Regarding the stage of vascular commitment, activation of the VEGF pathway through an exogenous stimulus is the most widely used strategy. However, recent findings demonstrated that the addition of other cytokines or small molecules, such as (1) the TGFβ signaling inhibitor SB431542 [ 55 ], (2) the inhibitor of Notch signaling DAPT [ 47 , 54 ], and (3) the cAMP and protein kinase A pathway enhancer forskoline [ 48 , 54 ], potentiates the effect of VEGF and promotes endothelial progenitor cell (EPC) proliferation, preventing in this way the progression to a more mature phenotype at this stage. The addition of BMP4 during the stage of vascular commitment, in combination with only VEGF [ 53 ] or with VEGF/FGF2 [ 50 , 52 , 56 ], VEGF/SB [ 49 ] or VEGF/FGF2/DAPT [ 51 ], has also been used to favor the development of EPCs.…”

“…The addition of BMP4 during the stage of vascular commitment, in combination with only VEGF [ 53 ] or with VEGF/FGF2 [ 50 , 52 , 56 ], VEGF/SB [ 49 ] or VEGF/FGF2/DAPT [ 51 ], has also been used to favor the development of EPCs. The stepwise optimization of these two first stages to specify EPCs from hPSCs, in combination with a progression to more defined and serum-free protocols [ 49 , 50 , 53 , 54 ], has improved the efficiency and reproducibility of the differentiation methods. With these more recent optimized protocols to generate EPCs, it is now possible to generate more than 80% CD34 + /CD31 + EPCs within 5 days of differentiation without the need for any cell sorting strategy, contrarily to what happed in previously reported EC differentiation protocols ( Figure 4 A).…”