Comparative study of differentiating human pluripotent stem cells into vascular smooth muscle cells in hydrogel-based culture methods

Abstract

Vascular smooth muscle cells (VSMCs), which provides structural integrity and regulates the diameter of vasculature, are of great potential for modeling vascular-associated diseases and tissue engineering. Here, we presented a detailed comparison of differentiating human pluripotent stem cells (hPSCs) into VSMCs (hPSCs-VSMCs) in four different culture methods, including 2-dimensional (2D) culture, 3-dimensional (3D) PNIPAAm-PEG hydrogel culture, 3-dimensional (3D) alginate hydrogel culture, and transferring 3-dimensional alginate hydrogel culture to 2-dimensional (2D) culture. Both hydrogel-based culture methods could mimic in vivo microenvironment to protect cells from shear force, and avoid cells agglomeration, resulting in the extremely high culture efficiency (e.g., high viability, high purity and high yield) compared with 2D culture. We demonstrated hPSC-VSMCs produced from hydrogel-based culture methods had better contractile phenotypes and the potential of vasculature formation. The transcriptome analysis showed the hPSC-VSMCs derived from hydrogel-based culture methods displayed more upregulated genes in vasculature development, angiogenesis and blood vessel development, extracellular matrix compared with 2D culture. Taken together, hPSC-VSMCs produced from hydrogel-based culture system could be applied in various biomedical fields, and further indicated the suitable development of alginate hydrogel for industrial production by taking all aspects into consideration.

Keywords: Alginate hydrogel fiber; Human pluripotent stem cells; Industrial production; PNIPAAm-PEG hydrogel; Vascular smooth muscle cells.

Fig. 1

Overview of different culture methods for hPSCs-VSMC differentiation. (i) Two-dimensional (2D) culture system. (ii) Three dimensional (3D) PNIPAAm-PEG hydrogel culture system. Single hPSCs are mixed with 10% PNIPAAm-PEG solution at 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, followed by hPSCs differentiation into VSMCs in the hydrogel. (iii) Three-dimensional (3D) alginate hydrogel culture system. hPSC are processed into the alginate hydrogel tubes at low seeding density and expanded for 9 days to fill the tubes. Once the targeted cell number of hPSCs is reached, hPSCs (around day 5) can be differentiated into VSMCs in the alginate hydrogel. (iv) Harvested VSMCs from 3D-alginate hydrogel system were transferred to 2D culture for 6 days.

Fig. 2

The comparison of hPSCs derived VSMCs in different culture systems (A) Schematic illustration of the VSMC differentiation protocol (B) Phase images of hPSC (H9) in different culture systems. Scale bar, 200 μm (C) Live/dead staining of harvested cells from 2D, 3D-PEG and 3D-Alginate culture system (n = 3) (D) Phase images of hPSC-VSMCs on day 5 in different culture systems. Scale bar, 200 μm (E) Immunostaining analysis of VSMC markers SM22A and α-SMA on day 5 cells. Scale bar, 50 μm (F) Statistical analysis of differentiation efficiency for VSMC markers SM22A and α-SMA on day 5 cells (n = 3) (G, H) When seeded at 1.0 × 10⁶ cells/mL, ∼15-, ∼24- and ∼410-fold expansion, yielding ∼1.5 × 10⁷ VSMCs/mL, 2.4 × 10⁷ VSMCs/mL PEG hydrogel and ∼4.1 × 10⁸ VSMCs/mL alginate hydrogel are produced in 2D, 3D-PEG and 3D-Alginate culture system, respectively. Data are presented as mean ± SD of three independent replicates (n = 3). ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 3

Properties of hPSC-VSMCs made in different culture systems (A) Immunostaining of fibronectin production of VSMC_2D, VSMC_3D, VSMC_F and VSMC_F-d6 after 24 h of 2.5 ng/mL TGF-β treatment (B) Quantification of produced fibronectin. Data are represented as mean ± SD (n = 3). Scale bar, 50 μm (C) Western blot analysis of produced fibronectin. GAPDH was as the control (D, E) Co-culture of VSMCs and HUVECs and statistical analysis of number of VSMCs attached to the vessels (E). Scale bar, 50 μm (F–H) Phase images (F), surface area (G) and percent change of cell surface area (n = 15) (H) of VSMC_2D, VSMC_3D, VSMC_F and VSMC_F-d6 in response to carbachol treatment. Data are represented as mean ± SD. Scale bar, 50 μm (I) The relative fluorescence unit (ΔRFU) of Fluo-4 loaded VSMCs over 10 min after adding carbachol. Data are represented as mean ± SD (n = 3) (J, K). When VSMCs and HUVECs were co-transplanted subcutaneously, VSMC_2D, VSMC_3D, VSMC_F and VSMC_F-d6 formed nice vascular structures (J) with similar number of VSMCs attached to the vessels (K). Scale bar, 50 μm. Data are presented as mean ± SD of three independent replicates (n = 3). ∗p < 0.05.

Fig. 4

Whole transcriptome analysis VSMC_F, VSMC_F-d6, VSMC_3D, VSMC_2D derived from H9s (A, B) Global heat map of expressed genes and Principal Component Analysis (PCA) of all VSMCs. Three biological replicates are used for each sample (n = 3) (C) The global gene expression correlation coefficients of all VSMC_F, VSMC_F-d6, VSMC_3D, VSMC_2D (D) Top 10 upregulated GO terms in VSMC_3D, VSMC_F and VSMC_F-d6 group compared with VSMC_2D, respectively.

Fig. 5

Differential gene expression analysis among all VSMCs derived from H9s (A, B) Venn diagram showing the up-regulated and down-regulated gene counts in VSMC_2D, VSMC_3D, VSMC_F and VSMC_F-d6 groups (C–G) Log 2 (expression level in VSMC_3D, VSMC_F and VSMC_F-d6/expression level in VSMC_2D) of extracellular matrix genes (H, I) Log2 (expression level in VSMC_3D, VSMC_F and VSMC_F-d6/expression level in VSMC_2D) of genes related to glycolysis (H), and angiogenesis (I). In Fig. 5C–I, VSMC_2D was as a control group.

Fig. 6

Differential gene expression analysis between hydrogel-based VSMCs derived from H9s (A–C) Log 2 (expression level in VSMC_3D, VSMC_F and VSMC_F-d6/expression level in VSMC_2D) of cell cycle (A), cell apoptosis(B) and cell differentiation (C). VSMC_2D was as a control group (D) qRT-PCR analyses of VSMC_2D, VSMC_3D, VSMC_F and VSMC_F-d6 for synthetic VSMC markers α-SMA, SM22A and Calponin, and contractile VSMC marker SMTN, and other genes related to VSMCs including growth factors VEGFA and VEGFC, and ECM genes FN and COL4A. Data are represented as mean ± SD of three biological replicates (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.