Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin-Expressing Hair Follicle-Associated Pluripotent Stem Cells

Abstract

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.

FIG. 1.

(A) Schema of slow-rate cooling and rapid-cooling (vitrification) methods of cryopreserving hair follicles and hair spheres. (B) Schema for dividing the hair follicle into upper, middle, and lower parts.

FIG. 2.

(A) Images demonstrating the recovery of sphere formation after cryopreservation by slow-rate cooling or rapid-cooling vitrification compared to fresh follicles. (B) Comparison of the attachment rate and growing cell number from fresh and cryopreserved hair follicles using slow-rate cooling or rapid-cooling vitrification after thawing and 4 weeks of culture in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS). Results are mean±standard deviation (SD) for three independent experiments (three independent mice for each method of cryopreservation and nonfrozen control). (C) Comparison of sphere number, produced from fresh follicles and follicles cryopreserved by slow-rate cooling or rapid-cooling vitrification, per upper part of the hair follicle. Results are mean±SD for three independent experiments (three samples each).

FIG. 3.

(A) Images demonstrating the recovery of differentiation potential after cryopreservation by slow-rate cooling or rapid-cooling vitrification compared to fresh follicles. Red color is for specific antibody staining and blue color is for nuclear staining. (B) Comparison of pluripotency of fresh hair follicles and hair follicles cryopreserved by slow-rate cooling or rapid-cooling vitrification after a one-week culture of hair spheres in DMEM with 10% FBS. Results are mean±SD for three independent experiments (three independent mice for each method of cryopreservation and nonfrozen control).

FIG. 4.

(A) Comparison of the mRNA levels of stem cell marker genes (nestin, Sox2, SSEA-1) were examined using real-time polymerase chain reaction (RT-PCR) analysis. Results are mean±SD for three samples each. The mRNA levels of stem cell marker genes were maintained in the slow-rate cooling method but significantly reduced in the rapid-cooling vitrification method. (B) Western-blot analysis was performed to detect the expression of SSEA-1. The protein level of SSEA-1 was maintained in the slow-rate cooling method but reduced in the rapid-cooling vitrification method.