1. ¹Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, 67 Asahi-machi, Kurume, 830-0011, Japan
2. ²Department of Advanced Therapeutics and Regenerative Medicine, Kurume University, 67 Asahi-machi, Kurume, 830-0011, Japan
3. ³Department of Gene Therapy and Regenerative Medicine, School of Medicine, Graduate School of Medicine, Gifu University, 40 Tsukasa-machi, Gifu 500-8705, Japan
4. ⁴Division of Cardiology, Respiratory, and Nephrology, Graduate School of Medicine, Gifu University, 40 Tsukasa-machi, Gifu 500-8705, Japan
5. ⁵Laboratory of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
6. ⁶Division of Regeneration of Organ and Tissue Development, Regeneration, and Advanced Medical Science, Graduate School of Medicine, Gifu University, 40 Tsukasa-machi, Gifu 500-8705, Japan
7. ⁷Department of Pediatrics and Child Health, Kurume University, 67 Asahi-machi, Kurume 830-0011, Japan
8. ⁸Department of Structural Cell Biology, Neuro-musculoskeletal Disorder, Advanced Therapeutics Course, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan

Correspondence: Ken-ichiro Kosai, Department of Structural Cell Biology, Neuro-musculoskeletal Disorder, Advanced Therapeutics Course, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Fax: +81 9 265 9721. E-mail: kosai@m2.kufm.kagoshima-u.ac.jp

Received 10 January 2006; Revised 16 May 2006; Accepted 16 May 2006.

Abstract

The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters—the currently available strategy for isolating cells lacking cell-specific surface markers—is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the -myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.