Functional Characterization of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

Abstract

Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), I_SK1-3, I_SK4 and I_K1 were similar in hiPSC-ECs and HCMECs. I_BK was larger and I_KATP was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.

Keywords: endotheline-1; exosome; human cardiac microvascular endothelial cells; human-induced pluripotent stem-derived endothelial cells; ion channel; nitric oxide.

Figure 1

Generation and identification of endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs). (A) The expression of definitive endothelial markers for CD31 (PECAM1, red), VE-cadherin (CDH5, red), and von Willebrand Factor (vWF, green) in hiPSC-ECs was confirmed using immunofluorescence staining. Cell nuclei were counterstained with DAPI (blue). (B) The mRNA level of endothelial cells markers PECAM1, CDH5 and VWF in cells from three healthy donors (D1, D2, and D3). The results are shown as mean ± SEM (n = 3). (C) The expression profile of hiPSC-EC markers PECAM1, CDH5 and VWF gene expression were examined by real-time RT-PCR. The results shown are mean ± SEM (n = 3 healthy iPSC). (D) Flow cytometry analysis of hiPSC-EC differentiation efficiency. HiPSC-ECs and Human Cardiac Microvascular Endothelial Cells (HCMEC) were stained with the endothelial markers PECAM1 (CD31), VE-cadherin (CD144). ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

Figure 2

MRNA expression level of ion channels in hiPSCs, hiPSC-ECs and HCMECs. All the data are normalized to that of hiPSC values (A–L). Transcriptional levels of small conductance calcium-activated potassium channel four isoforms, including SK1 ((A), n = 3), SK2 ((B), n = 3), SK3 ((C), n = 3), SK4 ((D), n = 3), large conductance calcium-activated potassium channel (BK, (E), n = 3), inwardly rectifying potassium channel Kir2.1 (KCNJ2, (F), n = 4), transient receptor potential vanilloid 2 (TRPV2, (G), n = 4), slowly activated delayed rectifier potassium channel (IKs, (H), n = 4), Na/Ca exchanger ((I), n = 3), hyperpolarization-activated cyclic nucleotide-gated channels (HCN2, (J), n = 3, and HCN4, (K), n = 4) as well as ATP-sensitive potassium channel (KATP, (L), n = 3) were examined by qPCR. The results are presented as mean ± SEM, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

Figure 3

Gene expression of receptor in hiPSCs, hiPSC-ECs and HCMECs. The mRNA expression level of adrenoceptors, including ADRA1A ((A), n = 6), ADRA2A ((B), n = 6), ADRB1 ((C), n = 6), ADRB2 ((D), n = 6), angiotensin II receptor, e.g., AT1 ((E), n = 6) and AT2 ((F), n = 6), muscarinic receptors, such as CHRM2 ((G), n = 6) and CHRM3 ((H), n = 3), dopamine receptor-DRD1 ((I), n = 6), DRD2 ((J), n = 6), DRD3 ((K), n = 6), DRD4 ((L), n = 6), DRD5 ((M), n = 6), and G proteins, GNA11 ((N), n = 6), Gαi2 ((O), n = 6), GNAS ((P), n = 6) and GNAQ ((Q), n = 6) were analyzed by qPCR. The p-values were determined vs. hiPSC according to the analysis of one-way ANOVA with Bonferroni post-hoc. Results are presented as mean ± SEM, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

Figure 4

Membrane potential and ion channel currents in hiPSC-ECs and HCMECs. Membrane potential and ion channel currents were recorded by the whole cell patch clamp technique. Intracellular and extracellular solutions or specific blockers were applied to confirm the identity of expected currents. (A) Mean values of resting potential (RP) in hiPSC-ECs and HCMECs, n = 10 cells/group. (B) Apamin-sensitive SK1–3 current (I_SK1–3) density from −80 mV to +50 mV in hiPSC-ECs and HCMECs. (C) The mean data of ISK1–3 at +50 mV. n = 10 cells/group. (D) Tram-34-sensitive current (I_SK4) densities from −80 mV to +50 mV in hiPSC-ECs and HCMECs. (E) I_SK4 density at +50 mV in hiPSC-ECs and HCMECs. n = 10 cells/group. (F) Iberitoxin sensitive BK currents at different potentials in hiPSC-ECs and HCMECs. (G) BK current density at +50 mV in hiPSC-ECs and HCMECs. n = 10 cells/group, * p < 0.05. (H) I_K1 current density from −120 mV to +50 mV in hiPSC-ECs and HCMECs. (I) IK1 at −120 mV in hiPSC-ECs and HCMECs. n = 10 cells/group. (J) I_KATP current density in hiPSC-ECs and HCMECs from −120 mV to +50 mV. (K) Current density of I_KATP at −120 mV in hiPSC-ECs and HCMECs. n = 10 cells/group, * p < 0.05.

Figure 5

Comparison of the functional characteristics of endothelial cells in hiPSC-ECs and HCMECs. The triplicate tube formation experiment was carried out. Representative images of tube formation are shown (A,B). The scale bar was 200 μm. (C) Tube length of formed tubes in hiPSC-ECs and HCMECs. (D) ET-1 levels in response to phenylephrine (PE) were measured using the Elisa method in hiPSC-ECs and HCMECs. n = 4, * p < 0.05; ** p < 0.005. (E) NO release was measured in hiPSC-ECs and HCMECs treated with acetylcholine (Ach). n = 4, * p < 0.05. (F,G) Fluorescence staining showing the degree of LDL uptake in hiPSC-ECs and HCMECs. Scale bar was 100 μm. (H) Relative fluorescence intensity of LDL in hiPSC-ECs and HCMECs. All data shown are mean ± SEM. n = 3, * p < 0.05; ** p < 0.005.

Figure 6

Characterization of the purified exosomes from hiPSC-EC and HCMEC conditioned media. Exosomes isolated from cell culture media of hiPSC-ECs and HCMECs. (A) Western blot showed the detection of exosome markers CD9, CD63, and CD81 in isolated exosomes. (B) Glucose-regulated protein 94 (GRP94) and Golgi marker GM130 were not found in exosomes. (C) Flow cytometry analysis using exosome marker CD9. (D) The size distribution of isolated exosomes was measured by nanoparticle tracking analysis (NTA) in hiPSC-ECs and HCMECs.