Development of a cell-isolation method for human prostatic smooth muscle cells based on cell type-specific activation of the SM22 gene promoter

Authors

  • Chun-Yu Wang,

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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  • Jian-Dang Shi,

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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  • Chun-He Yan,

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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  • Quan Wu,

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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  • Helmut Klocker,

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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  • Irwin Park,

    1. Northwestern University Feinberg School of Medicine, Chicago, IL, USA
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  • Chung Lee,

    1. Northwestern University Feinberg School of Medicine, Chicago, IL, USA
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  • Ju Zhang

    1. Bioactive Materials Key Laboratory of Ministry of Education, Institute for Molecular Biology, Nankai University, Tianjin, China, Department of Urology, Innsbruck Medical University, Innsbruck, Austria, and
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Ju Zhang, Institute for Molecular Biology, Nankai University, Tianjin 300071, China.
e-mail: zhangju@nankai.edu.cn

Abstract

OBJECTIVE

To separate smooth muscle cells (SMCs) from fibroblasts in cultured human prostatic stromal cells (PrSCs) by characterizing the SM22 promoter as a prostatic SMC-specific gene promoter, and to investigate its use for a promoter-based cell-sorting method, as SMCs are critical for stromal function and the pathological changes in the development of benign prostatic hyperplasia.

MATERIALS AND METHODS

Human PrSCs were cultured in SMC-selective medium or standard medium, respectively, to obtain typical cultures of SMCs and fibroblasts. SM22 promoter activity and specificity were analysed by luciferase reporter-gene assay. A dual-colour vector was constructed with the expression of the red fluorescent protein (RFP) under the control of the 1.4 kb SMC-specific SM22 promoter, and the expression of the green fluorescent protein (GFP) under cytomegalovirus promoter. Fluorescence-activated cell sorting (FACS) was used to isolate and enrich GFP+/RFP+ and GFP+/RFP– cells. Cell phenotype was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence.

RESULTS

The 1.4 kb SM22 promoter activity was much higher in PrSCs cultured in SMC-selective medium. Immunofluorescence staining and merged fluorescence microscopy ensured that SM22 promoter-driven GFP positive cells were SMCs. After transfection of the dual-colour vector into PrSCs, GFP+/RFP+ cells (SMCs) and GFP+/RFP– cells (fibroblasts) were isolated by FACS. The phenotype of FACS-enriched SMCs and fibroblasts was confirmed.

CONCLUSION

These results indicate that the 1.4 kb SM22 promoter is specific for prostatic SMCs. This dual-colour vector could be a useful tool for separating living SMCs from fibroblasts using FACS.

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