【Summary】 In in vivo analysis, the cells were isolated from hard tissue by On-chip Sort after transplanting four kinds of stem cells from withdrawal tooth and bone marrow-derived human mesenchymal stem cell. And it became clear that the cells still keep stem cell characteristics.
【Purpose】 Because four kinds of mesenchymal stem cells coming from a withdrawal tooth of dental pulpal (DPSC), periodontal ligament (PDLSC), dental follicle (DFSC), and apical papilla (APSC) have extremely higher growth ability and pluripotent differential ability than bone marrow stem cell (BMSC), it is shown to be the stem cells which are advantageous to regenerative medicine.
１）We want to clarify whether stem cells after transplantation exist as stem cells per se.
２） In addition, if stem cells exist, we re-isolate stem cells selectively from all over the formation tissues and culture return it and clarify whether still maintain stem cell characteristics by in vitro analysis.
【Methods】 Four kinds of stem cells from a withdrawal tooth of dental pulpal (DPSC), periodontal ligament (PDLSC), dental follicle (DFSC), and apical papilla (APSC) are dissociated with the mixed solution of 3 mg/mL collagenase and 4 mg/mL despise at 37℃ for 1-hour. After that, cells were isolated with a 70-micrometer filter, and 1×104 cells were seeded to 10-cm dish. We used DMEM/F12 culture medium which contains 15% of FBS for culture. After cultivating to the passage number 3, 1×106 of various stem cells were obtained, and transplanted them to the subcutaneous of six-weeks aged immune-deficient mouse (CB17. ICR Scid-mouse), after mixing obtained cells with collagen gel (Nitta Gelatin) and 40 mg of hydroxyapatite (Zimmer). The hard tissue formed after 16 weeks of transplants was dissociated by incubation with the collagenase and dispase mixture at 37℃ for 1 hour.
Cells were isolated with the 70-micrometer cell strainer after that, and specific anti-human CD44 antibody was added.
After centrifugation, upper layer was removed and CD44 positive cells were separated using standard sheath liquid by On-chip sample buffer.
Human CD44 positive cells were sorted and plated to 35-mm dish, and culture until it reaches the number of cells for analysis.
The sorted cells by “On-chip Sort” have the differentiation capacities to bone and adipocyte, and held the features of the stem cell.
【Result】 The analysis result of the surface antigen of cells before and after sorting by On-chip Sort were shown in Fig.1 and Fig.2.
Expression pattern of cell surface antigen showed the similar result.