Diabetic Endothelial Cells Differentiated From Patient iPSCs Show Dysregulated Glycine Homeostasis and Senescence Associated Phenotypes

Abstract

Induced pluripotent stem cells derived cells (iPSCs) not only can be used for personalized cell transfer therapy, but also can be used for modeling diseases for drug screening and discovery in vitro. Although prior studies have characterized the function of rodent iPSCs derived endothelial cells (ECs) in diabetes or metabolic syndrome, feature phenotypes are largely unknown in hiPSC-ECs from patients with diabetes. Here, we used hiPSC lines from patients with type 2 diabetes mellitus (T2DM) and differentiated them into ECs (dia-hiPSC-ECs). We found that dia-hiPSC-ECs had disrupted glycine homeostasis, increased senescence, and impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. These signature phenotypes will be helpful to establish dia-hiPSC-ECs as models of endothelial dysfunction for understanding molecular mechanisms of disease and for identifying and testing new targets for the treatment of endothelial dysfunction in diabetes.

Keywords: endothelial function; endothelium; glycine; mitochondrial function; senescence.

FIGURE 1

Characterization of hiPSC-ECs. (A) ECs differentiated from 4 hiPSC lines were evaluated for the expression of CD31 and CD144, Dil-ace-LDL uptake, and tube formation on Matrigel. Quantifications of numbers of nodes (B), junctions (C), branches (D), and total branches length (E) formed by hiPSC-ECs on Matrigel (n = 6 for each cell line). Values are presented as means ± SD. One-way ANOVA.

FIGURE 2

Characterization of nitric oxide (NO) and endothelin-1 produced by hiPSC-ECs. (A) Western Blot analysis for quantification of endothelial NO synthase (eNOS) activity (n = 6 for each cell line). (B) Quantification of NO produced by hiPSC-ECs (n = 6 for each cell line). (C) Western Blot analysis for quantification of endothelin-1 protein in supernatants of hiPSC-ECs (n = 4 for each cell line). Values are presented as means ± SD. One-way ANOVA.

FIGURE 3

Disrupted glycine homeostasis in dia-hiPSC-ECs. (A) Intracellular glycine concentration in hiPSC-ECs measured by liquid chromatography–mass spectrometry (n = 3 for each cell line). (B) Representative image of Western Blot for GlyT1A, GlyT2, cSHMT, and mSHMT protein expression in hiPSC-ECs. Quantification of GlyT1A (C), GlyT2 (D), cSHMT (E), and mSHMT (F) protein expression (n = 5 for each cell line for B,C). Values are presented as means ± SD. One-way ANOVA.

FIGURE 4

Increased senescence in dia-hiPSC-ECs (A). hiPSC-EC population doubling time (n = 4 for each cell line). (B) β-gal staining (green color) in hiPSC-ECs which was count-stained with hematoxylin to show cell nuclei. (C) Quantification of β-gal density in hiPSC-ECs (n = 15 or 16 for each cell line). (D) Representative images of Western Blot for p21 and p53 protein expressions. Quantification of p21 (E) and p53 (F) protein expression (n = 6 for each cell line). Values are presented as means ± SD. One-way ANOVA.

FIGURE 5

Matrix metalloproteinase-1 (MMP-1) and adhesion molecule expression in hiPSC-ECs. (A) Matrix metalloproteinase-1 (MMP-1) content in the supernatant of hiPSC-ECs (n = 4 for each cell line). (B) Intercellular adhesion molecule-1 (ICAM-1) protein expression in hiPSC-ECs (n = 5 for each cell line). Values are presented as means ± SD. One-way ANOVA.

FIGURE 6

Impaired mitochondrial function in dia-hiPSC-ECs. (A) JC-1 staining to visualize mitochondrial membrane potential. (B) Quantification of hiPSC-EC mitochondrial membrane potential. (C) Representative images of Western Blot for mitochondrial proteins in hiPSC-ECs, including mitofusin-1 (Mfn-1), mitofusin-2 (mfn-2), and Fission-1 (Fis-1). Quantification of MFN-1 (D), MFN-2 (E), and FIS-1 (F) protein expression. (G) Quantification of ATP production in hiPSC-ECs. (H) Western Blot analysis for ATP synthase 5 (ATP5) expression. (I) Quantification of ATP5 protein expression (n = 6 for each cell line). Values are presented as means ± SD. One-way ANOVA.

FIGURE 7

Assessment of hiPSC-EC function in a mouse model of hind-limb ischemia (HLI) in vivo. (A) A schematic diagram of the HLI model and treatment. (B) Laser Doppler imaging of mouse limbs before femoral artery ligation, 3 days (i.e., at the time of treatment administration), and 17 days (i.e., at the 14 days medium or cell injection) after femoral artery ligation. (C) Recovery of right limb perfusion was expressed as a percentage of measurements in the uninjured contralateral limb (n = 6 for each cell line). (D) Fluorescence staining for human-specific CD31 (hCD31) and smooth muscle actin (SMA) expression in the injured limbs of hiPSC-EC–treated animals (Bar = 50 μm). (E) Fluorescence staining for CD31 and smooth muscle actin (SMA) in the ischemic limb (right leg) and uninjured limb (left leg) of animals treated with basal medium or hiPSC-ECs after femoral artery ligation (Bar = 100 μm). (F) Vessel density and (G) arteriole density in ischemic limbs (right limb) and uninjured contralateral limbs (left limb) (n = 6 for each cell line). Values are presented as means ± SD. One-way ANOVA.