Differentiation of human embryonic stem cells into smooth muscle cells in adherent monolayer culture

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Abstract

Smooth muscle cell (SMC) plays critical roles in many human diseases, an in vitro system that recapitulates human SMC differentiation would be invaluable for exploring molecular mechanisms leading to the human diseases. We report a directed and highly efficient SMC differentiation system by treating the monolayer-cultivated human embryonic stem cells (hESCs) with all-trans retinoid acid (atRA). When the hESCs were cultivated in differentiation medium containing 10 μM RA, more than 93% of the cells expressed SMC-marker genes along with the steadily accumulation of such SMC-specific proteins as SM α-actin and SM-MHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. This inducible and highly efficient in vitro human SMC system could be an important resource to study the mechanisms of SMC phenotype determination in human.

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Materials and methods

Cell culture. The hESC line HUES3 [12] at passage 39 were cultured on γ-irradiated mouse embryonic fibroblasts (MEFs) at 37 °C and 5% CO2 with daily change of the medium. The medium was composed of Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum, 10% knock-out serum replacement, 2 mM l-glutamine, 0.1 mM 2-β-mercaptoethanol, 1% nonessential amino acids, penicillin, and streptomycin, 10 ng/ml bFGF2 (Gibco), and 10 ng/ml human leukemia inhibitory factor (hLIF) (Chemicon).

Directed

In vitro differentiation of human ES cell into smooth muscle-like cell

Under the treatment of retinoic acid, monolayer human ES cells underwent dramatic morphological changes. Fig. 1A shows the appearance of the hES culture in the untreated (a) and treated (b–f) samples. In the absence of RA, the majority of the originally disassociated hESCs automatically re-aggregated and formed EB-like cell islands, while a small portion remained in the single-cell state. When RA was present in the medium, much fewer cells aggregated, the majority remained as single attached

Discussion

We have established an efficient in vitro SMC differentiation system using human embryonic stem cells. In the absence of feeder cells, without going through EBs and cell-sorting processes, we showed that retinoic acid treated hESCs were capable of differentiation into a morphologically homogeneous cell population stably expressing characteristics of functional SMC.

Differentiation of ESCs recapitulates the developmental processes of embryogenesis, and therefore is widely used as model system to

Acknowledgments

This work is partially supported by funds from Zhejiang Science and Technology Department (No. J 20020579-30116) and the Central Hospital of Huzhou city (No. H20010984-32536) to Ming Zhang and the Young Teachers to Liangbiao Chen from the Ministry of Education of China. We thank Hanming Chen and Yin Du for their technical assistance.

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