Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates

Abstract

Aims: Smooth muscle cells (SMC) play an important role in vascular homeostasis and disease. Although adult mesenchymal stem cells (MSC) have been used as a source of contractile SMC, they suffer from limited proliferation potential and culture senescence, particularly when originating from older donors. By comparison, human induced pluripotent stem cells (hiPSC) can provide an unlimited source of functional SMC for autologous cell-based therapies and for creating models of vascular disease. Our goal was to develop an efficient strategy to derive functional, contractile SMC from hiPSC.

Methods and results: We developed a robust, stage-wise, feeder-free strategy for hiPSC differentiation into functional SMC through an intermediate stage of multipotent MSC, which could be coaxed to differentiate into fat, bone, cartilage, and muscle. At this stage, the cells were highly proliferative and displayed higher clonogenic potential and reduced senescence when compared with parental hair follicle mesenchymal stem cells. In addition, when exposed to differentiation medium, the myogenic proteins such as α-smooth muscle actin, calponin, and myosin heavy chain were significantly upregulated and displayed robust fibrillar organization, suggesting the development of a contractile phenotype. Indeed, tissue constructs prepared from these cells exhibited high levels of contractility in response to receptor- and non-receptor-mediated agonists.

Conclusion: We developed an efficient stage-wise strategy that enabled hiPSC differentiation into contractile SMC through an intermediate population of clonogenic and multipotent MSC. The high yield of MSC and SMC derivation suggests that our strategy may facilitate an acquisition of the large numbers of cells required for regenerative medicine or for studying vascular disease pathophysiology.

Figure 1

The α-actin promoter activity captures iPSC as they undergo mesenchymal transition. (A and C) By day 3 of differentiation (Stage 1), cells in the periphery of F-iPSC (A) and HF-iPSC (C) colonies assumed fibroblastic morphology and started to express ZsGreen, indicating ACTA2 promoter activation. (B and D) In Stage 2, the fraction of ZsGreen expression of F-iPSC (B) and HF-iPSC increased dramatically. Bars, 50 μm. (E) The percentage of double-positive ACTA2-ZsGreen+/DsRed+ cells at the indicated stages of differentiation was measured by flow cytometry. Results are expressed as mean ± SD (n = 3).

Figure 2

A gene expression profile of hiPSC as they undergo differentiation. (A and B) RT-PCR for pluripotency-associated genes and genes associated with mesendodermal, endodermal, or ectodermal lineages in (A) F-iPSC; (B) HF-iPSC. (C and D) RT-PCR for EMT-associated genes at the indicated stages of hiPSC differentiation.

Figure 3

Stage 2 hiPSC exhibit similar immunophenotype and differentiation potential as BM-MSC. (A) Immunophenotype of Stage 2 differentiated hiPSC and BM-MSC. Flow cytometry with MSC-specific antibodies as indicated. Insets depict control samples stained with IgG alone (n = 3). (B and C) Stage 2 hiPSC were induced to differentiate into mesenchymal lineages. (B) RT-PCR for the indicated genes before and after induction of differentiation. (C) Differentiation was assessed by functional assays as indicated: Von Kossa (osteogenesis), Alcian Blue (chondrogenesis), and Oil Red O (adipogenesis) staining. Non-induced controls are shown in the corresponding insets. Bars, 50 μm.

Figure 4

Stage 2 cells express SMC-specific genes but only Stage 3 cells express SMC proteins. (A and B) RT-PCR for SMC-specific genes (ACTA2, CNN1, MYH11, SM22, and CALD1) in F-iPSC (A) or HF-iPSC (B) at different stages of differentiation as indicated. (C and D) Immunostaining for ACTA2, CNN1, and MYH11 of Stage 2 (C) or Stage 3 (D) cells.

Figure 5

Stage 3 but not Stage 2 hiPSC are highly contractile SMC. Vascular constructs were generated with Stage 3 hiPSC and vascular contractility was measured in response to receptor-mediated (U46619 and Endothelin-1) and non-receptor-mediated (118 mM KCl) vasoconstrictors. (A) Hematoxylin and eosin stain (H&E) of paraffin sections of vascular constructs with Stage 3 cells showed uniform distribution and circumferential alignment. (B) Contraction force (Pa) of Stage 2- or Stage 3-based vascular constructs in response to vasoagonists as indicated. (C) Representative graphs of isometric contraction in response to U46619. (D) Ultimate tensile stress (UTS) of vascular constructs. Results are expressed as mean ± SD (n = 3). *P < 0.05. Bar, 20 μm.

Figure 6

HF-iPSC-derived MSC were more proliferative and resistant to senescence than the parental HF-MSC. HF-iPSC-MSC (Stage 2) cells and HF-MSC (parental population) were seeded at clonal density (10 cells/cm²) and cultured for 10 days in medium containing 10% MSC-qualified FBS plus 2 ng per mL bFGF. (A) Images of representative cell culture dishes and colonies. Bar = 1000 μm. (B) The number of colonies larger or smaller than 2 mm in diameter was quantified using Image J. *P < 0.05 between the indicated samples. (C) Immunostaining of HF-iPSC-MSC and HF-MSC for Ki67 or p21 (green); nuclei were counterstained with Hoechst. Bars: 50 μm. (D) The percentage of Ki67+ and p21+ cells was determined from 10 images using Image J. **P < 0.005 between the indicated samples. (E) RT-PCR for the indicated genes. (F) Immunoblotting for p21.