Derivation of smooth muscle cells with neural crest origin from human induced pluripotent stem cells

Abstract

The heterogeneity of vascular smooth muscle cells (SMCs) is related to their different developmental origins such as the neural crest and mesoderm. Derivation of SMCs from different origins will provide valuable in vitro models for the investigation of vascular development and diseases. From the perspective of regenerative medicine and tissue engineering, an expandable cell source of SMCs is required for the construction of tissue-engineered blood vessels. In this study, we developed a robust protocol to derive neural crest stem cells (NCSCs) from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). NCSCs derived from ESCs and iPSCs were expandable with similar cell doubling times. NCSCs were capable of differentiating into neural and mesenchymal lineages. TGF-β1 induced the expression of SMC markers calponin-1, SM22α, and smooth muscle myosin heavy chain and resulted in the assembly of smooth muscle α-actin, calponin-1, and SM22α into stress fibers. This work provides a basis for using iPSCs to study SMC biology and deriving vascular cells for tissue engineering.

Fig. 1

Procedure to derive NCSCs from human ESCs and iPSCs. a A schematic diagram of the protocol was described. b Human ESCs and iPSCs were cultured on MEFs. c ESC and iPSC colonies were detached by collagenase and dispase while MEFs were left behind. The asterisk indicates the area occupied by the ESC/iPSC colony. d EB-like cell aggregates were cultured in suspension. e Rosette colony formed after EB-like cell aggregates attached to the CELLstart-coated surface. f Rosette colonies were mechanically harvested and cultured in suspension. The arrow indicates dead nonneural cells in suspension. g iPSC-derived NCSCs were cultured as a monolayer. Scale bar = 100 μm.

Fig. 2

Characterization of the cells in the rosette-containing colonies. The majority of cells in the colonies with rosette structures were positive for NC markers nestin (a, b), p75 (c, d), vimentin (e, f), Slug (g, h), and AP2 (i, j). Nuclei were stained using DAPI (blue in the online version). Scale bar = 100 μm.

Fig. 3

Rosette-forming capability of human ESCs and iPSCs and proliferation rate of derived NCSCs. a Percentage of rosette-containing colonies in the culture of ESCs and iPSCs. Eighty to 120 total colonies were counted from each culture. b Doubling time of NCSCs derived from ESCs and iPSCs. Values are reported as means ± standard deviation. n = 3.

Fig. 4

Characterization of NCSCs derived from iPSCs cultured as a monolayer. Cells were immunostained uniformly for NCSC markers nestin (a, b), p75 (c, d), vimentin (e, f), and HNK1 (g, h), indicating their NCSC identity. NCSC homogeneity was confirmed by flow cytometry analysis of p75. i Negative control. j p75 staining. Nuclei were stained by DAPI (blue in the online version). Scale bar = 40 μm.

Fig. 5

Characterization of NCSCs derived from iPSCs cultured as neurospheres in low-attachment cell culture plates. The cells maintained uniform expression of NCSC markers nestin/vimentin (a–c) and HNK1/p75 (d–f). Nuclei were stained by DAPI (blue in the online version). Scale bar = 100 μm.

Fig. 6

In vitro differentiation of iPSC-NCSCs into peripheral neural lineages (peripheral neurons and Schwann cells) and mesenchymal lineages (chondrocytes, osteoblasts, and adipocytes). Peripheral neurons: phase contrast image (a) and immunostaining for peripherin (b). Schwann cells: phase contrast image (c) and immunostaining for S100β (d). Chondrogenic differentiation: Alcian Blue staining for glycosaminoglycans (e) and immunofluorescent staining of collagen II (f). Osteoblastic differentiation: Alizarin Red staining for calcified matrix (g) and immunofluorescent staining of ALP (h). Adipogenic differentiation: phase contrast image (i) and Oil Red staining (j). In all immunofluorescence images, nuclei were stained by DAPI (in blue). Scale bars = 100 μm (a–d, f, h–j) and 200 μm (e, g).

Fig. 7

Differentiation of iPSC-NCSCs into smooth muscle lineage. iPSC-NCSCs were cultured in medium containing 5% FBS in the absence (a, c, e, g) or presence (b, d, f, h) of TGF-β1 (10 ng/ml) for 2 weeks and immunostained for smooth muscle markers SMA (a, b), CNN1 (c, d), SM22α (e, f), and SM-MHC (g, h). Scale bar = 100 μm.