Intestinal fatty acid-binding protein (I-FABP), a low molecular mass (approximately 15 kDa) cytoplasmic protein, is specifically located in epithelial cells of small bowel mucosal layer. This protein is rapidly released into the circulation after injury and/or destruction of these cells due to poor mesenteric blood flow and necrosis. Therefore, it can be used as a potential diagnostic biomarker for small bowel disease. In the present study, we have succeeded in developing a sandwich enzyme-linked immunosorbent assay (ELISA) system for quantification of human I-FABP. The range of sandwich ELISA system was 0.1-50 ng/mL of I-FABP in serum, and showed excellent quantitative characteristics such as reproducibility, dilution linearity, and recovery. No cross-reactivities were detected with other types of FABPs. As measured with this ELISA system, the serum I-FABP concentration was 1.1 ± 0.9 ng/mL in 61 healthy individuals, indicating that the reference value was below 2.0 ng/mL regardless of gender and age. Furthermore, mild abdominal pain or diarrhea before blood sampling did not affect I-FABP levels. Thus, this ELISA system could be used to accurately quantify human I-FABP concentrations in serum samples. These results suggest that it could be used as a new biomarker for the diagnosis of small bowel disease.

Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. In the present study, we established efficient cryopreservation methods of the hair follicle that maintained the pluripotency of HAP stem cells. We cryopreserved the whole hair follicle from green fluorescent protein transgenic mice by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS). After 4 weeks of culture, cells from the upper part of the hair follicle grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week of culture, the growing cells formed hair spheres, each containing ∼1×10(2) HAP stem cells. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. In contrast, rapid-cooling (vitrification) cryopreservation poorly preserved the pluripotency of the hair follicle stem cells. Stem cell marker genes (nestin, Sox2, and SSEA-1) were as highly expressed in slow-rate cooled cryopreserved follicles, after thawing, as in fresh follicles. However, in the vitrification cryopreserved follicles, the expression of the stem cell marker genes was greatly reduced. Direct cryopreservation of hair spheres by either the rapid-cooling, or slow-cooling method, resulted in loss of pluripotency. These results suggest that the slow-rate cooling cryopreservation of the whole hair follicle is effective to store HAP stem cells. Stored HAP stem cells would be very useful in personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.

We demonstrate in the present study that young host mice rejuvenate aged hair follicles after transplantation. Young mice promote the hair shaft growth of transplanted old hair follicles, as well as young follicles, in contrast to old host mice, which did not support hair-shaft growth from transplanted old or young follicles. Nestin-expressing hair follicle-associated pluripotent (HAP) stem cells of transplanted old and young hair follicles remained active in young host nude mice. In contrast, the nestin-expressing HAP stem cells in young and old hair follicles transplanted to old nude mice were not as active as in young nude host mice. The present study shows that transplanted old hair follicles were rejuvenated by young host mice, suggesting that aging may be reversible.