Comparative Study of Human Pluripotent Stem Cell-Derived Endothelial Cells in Hydrogel-Based Culture Systems

Abstract

Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) are promising cell sources for drug discovery, tissue engineering, and studying or treating vascular diseases. However, hPSC-ECs derived from different culture methods display different phenotypes. Herein, we made a detailed comparative study of hPSC-ECs from three different culture systems (e.g., 2D, 3D PNIPAAm-PEG hydrogel, and 3D alginate hydrogel cultures) based on our previous reports. We expanded hPSCs and differentiated them into ECs in three culture systems. Both 3D hydrogel systems could mimic an in vivo physiologically relevant microenvironment to protect cells from shear force and prevent cell agglomeration, leading to a high culture efficiency and a high volumetric yield. We demonstrated that hPSC-ECs produced from both hydrogel systems had similar results as 2D-ECs. The transcriptome analysis showed that PEG-ECs and alginate-ECs displayed a functional phenotype due to their higher gene expressions in vasculature development, extracellular matrix, angiogenesis, and glycolysis, while 2D-ECs showed a proliferative phenotype due to their higher gene expressions in cell proliferation. Taken together, both PEG- and alginate-hydrogel systems will significantly advance the applications of hPSC-ECs in various biomedical fields.

Figure 1

Overview of three culture systems for hPSC expansion and differentiation. (A) Schematic illustration of 2D culture expansion and differentiation of hPSCs. (B) Schematic illustration of the bioprocess of the 3D PNIPAAm-PEG hydrogel for hPSC expansion and differentiation. Single hPSCs (1.0 × 10⁶ cells/mL PEG hydrogel) are mixed with a 10% PNIPAAm-PEG solution at a low temperature [e.g., 4 °(C)], which forms an elastic hydrogel at 37 °C. Single hPSCs clonally expand into uniform spheroids in the hydrogel in 5 days. Upon cooling to 4 °C, the hydrogel is liquefied, and spheroids are harvested and dissociated into single cells for the next expansion. Once the targeted cell number is reached, hPSCs are differentiated into ECs in the hydrogel. (C) Schematic illustration of the 3D alginate hydrogel for hPSC expansion and differentiation. hPSCs are processed into alginate hydrogel tubes at a low seeding density (1.0 × 10⁶ cells/mL alginate hydrogel) and expanded for 9 days to fill the tubes. The day 9 cell masses can be released via dissolving the hydrogel tubes with 0.5 mM EDTA solution, dissociated into single cells with Accutase, and processed into new hydrogel tubes for a second round of expansion. Once the targeted cell number of hPSCs is reached, hPSCs (around day 5) can be differentiated into ECs in the alginate hydrogel.

Figure 2

Comparison of hPSC expansion in three culture systems. (A) Phase image of hPSC expansion in 2D, 3D-PEG, and 3D-alginate culture systems. Scale bar, 50 μm. (B) Immunostaining of the pluripotency markers OCT3/4 and NANOG in 2D, 3D-PEG, and 3D-alginate culture systems. Scale bars, 50 μm. (C,D) Statistical analysis of OCT3/4- and NANOG-positive cells in 2D, 3D-PEG, and 3D-alginate culture systems. (E,F) Live-/dead-cell staining and statistical analysis of harvested cells from 2D, 3D-PEG, and 3D-alginate culture systems. Scale bar, 200 μm. (G,H) When seeded at 1.0 × 10⁶ cells/mL, about 7-fold expansion occurs to yield ∼7 × 10⁶ cells/mL on day 3 in the 2D culture system; about 5-, 10-, and 20-fold expansion, yielding ∼5, 10, and 20 million cells per milliliter of the hydrogel on days 3, 4, and 5, respectively, is achieved in the 3D-PEG culture system; and about ∼30-, 150-, and 480-fold expansion occur to yield ∼30×, 150×, and 480 × 10⁶ cells/mL on days 5, 7, and 9, respectively, in the 3D-alginate culture system. Data are represented as mean ± SD (n = 3).

Figure 3

Comparison of hPSC-derived ECs in three culture systems. (A) Schematic illustration of the EC differentiation protocol. (B) Phase images of hPSC-ECs on day 5 in 2D, 3D-PEG, and 3D-alginate culture systems. Scale bar, 200 μm. (C) Immunostaining analysis of EC markers PECAM1 (or CD31) and VE-cadherin (or CD144) on day 5 cells. Scale bar, 50 μm. (D,E) Flow cytometry analysis of EC markers CD31 and CD144 on day 5 cells. (F,G) When seeded at 1.0 × 10⁶ cells/mL, about 16-, 25-, and 400-fold expansion, yielding ∼1.6 × 10⁷ ECs/mL, 2.0 × 10⁷ ECs/mL PEG hydrogel, and ∼4.0 × 10⁸ ECs/mL alginate hydrogel, are produced in 2D, 3D-PEG, and 3D-alginate culture systems on day 5, respectively.

Figure 4

Functional properties of ECs derived from three culture systems. (A) Uptake by fluorescence-labeled acetylated LDL (Ac-LDL). Scale bar, 50 μm. (B–D) All ECs form a tubular network when plated on Matrigel for 24 h. The tube length (mm/field) (C) and branches (D) are calculated using the Angiogenesis Analyzer of ImageJ software. Scale bar, 50 μm. *p < 0.05, **p < 0.01. (E) TEER properties of HUVECs, 3D-alginate-ECs, 3D-PEG-ECs, and 2D-ECs (either untreated or treated with 100 ng/mL TNF-α or 100 ng/mL IL-1β or 100 ng/mL VEGF-A) are similar. ***p < 0.001. (F,G) When transplanted subcutaneously with a Matrigel matrix, all ECs form vascular structures, as indicated by H&E staining and immunostaining analysis. Scale bar, 100 and 25 μm. (H,I) qRT-PCR shows that 3D-PEG-ECs and 3D-alginate-ECs have higher expressions of the key genes related to ECs compared with 2D-ECs or HUVECs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Whole transcriptome analysis of 3D-alginate-ECs, 3D-PEG-ECs, and 2D-ECs derived from H9s. (A,B) Global heat map of expressed genes and PCA of all ECs. (C) Heat map representation of the Spearman rank correlation coefficient between methods. Correlation color key goes from white (0, no correlation) to blue (1, perfect correlation). (D) Scatterplot in the log scale of gene expression for all ECs. Three biological replicates are used for each sample.

Figure 6

Differential gene expression analysis among all ECs derived from H9s. (A,B) Top 10 upregulated GO terms in the 3D-PEG-EC and 3D-alginate-EC groups compared with 2D-ECs, respectively. (C,D) Venn diagram showing the upregulated and downregulated gene counts in 2D-EC, 3D-PEG-EC, and 3D-alginate-EC groups. (E–I) log ₂ (expression level in 3D-PEG-ECs or 3D-alginate-ECs/expression level in 2D-ECs) of extracellular matrix genes. (J–M) log ₂ (expression level in 3D-PEG-ECs or 3D-Alginate-ECs/expression level in 2D-ECs) of genes related to EC secretome (J), glycolysis (K), angiogenesis (L), and proliferation (M).