Emi Inada1, Issei Saitoh2,3, Naoko Kubota1, Yoko Iwase3,4, Yuki Kiyokawa3, Hirofumi Noguchi5, Youichi Yamasaki1, Masahiro Sato6,7. 1. Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, 890-8544, Japan. 2. Department of Pediatric Dentistry, Asahi University School of Dentistry, Gifu, 501-0296, Japan. 3. Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, 951-8514, Japan. 4. Department of Dentistry for the Disabled, Asahi University School of Dentistry, Gifu, 501-0296, Japan. 5. Department of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, 903-0215, Japan. 6. Department of Genome Medicine, National Center for Child Health and Development, 2-10-1, Tokyo, 157-8535, Japan. masasato@m.kufm.kagoshima-u.ac.jp. 7. Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, 890-8544, Japan. masasato@m.kufm.kagoshima-u.ac.jp.
Abstract
BACKGROUND: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. RESULTS: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. CONCLUSION: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.
BACKGROUND: Expression of stemness factors, such as octamer-binding transcription factor 3/4 (OCT3/4), sex determining region Y-box 2 (SOX2), and alkaline phosphatase (ALP) in human deciduous tooth-derived dental pulp cells (HDDPCs) can be assessed through fixation and subsequent immuno- or cytochemical staining. Fluorescence-activated cell sorting (FACS), a powerful system to collect cells of interest, is limited by the instrument cost and difficulty in handling. Magnetic-activated cell sorting is inexpensive compared to FACS, but is confined to cells with surface expression of the target molecule. In this study, a simple and inexpensive method was developed for the molecular analysis of immuno- or cytochemically stained cells with intracellular expression of a target molecule, through isolation of a few cells under a dissecting microscope using a mouthpiece-controlled micropipette. RESULTS: Two or more colored cells (~ 10), after staining with a chromogen such a 3,3'-diaminobenzidine, were successfully segregated from unstained cells. Expression of glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene, was discernible in all samples, while the expression of stemness genes (such as OCT3/4, SOX2, and ALP) was confined to positively stained cells. CONCLUSION: These findings indicate the fidelity of these approaches in profiling cells exhibiting cytoplasmic or nuclear localization of stemness-specific gene products at a small-scale.