Abstract
The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco’s Modified Eagle’s Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham’s Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine.
Highlights
Hair-follicle bulge stem cells were originally shown to have the capacity to form hair-follicle cells, sebaceous-gland basal cells, and epidermis[1,2,3,4,5]
Using a slow-rate cooling method, we previously demonstrated that cryopreserved whole-mouse hair follicles were able to maintain the pluripotency of hair-follicle-associated pluripotent (HAP) stem cells[19]
The values are normalized to the expression of these genes for fresh fair follicles (Fig. 6, Supplementary Information). These results show that the mRNA levels of stem-cell marker genes such as nestin, Oct3/4, and Nanog were preserved in the HAP stem-cell colonies formed from cryopreserved human hair follicles
Summary
Hair-follicle bulge stem cells were originally shown to have the capacity to form hair-follicle cells, sebaceous-gland basal cells, and epidermis[1,2,3,4,5]. We found that nestin-expressing stem cells from both mouse and human have multilineage differentiation capacity that could produce neurons and other cell types[10,11,12,13]. We termed these nestin-expressing stem cells hair-follicle-associated pluripotent (HAP) stem cells. Using a slow-rate cooling method, we previously demonstrated that cryopreserved whole-mouse hair follicles were able to maintain the pluripotency of HAP stem cells[19]. We established effective cryopreservation procedures of the whole human hair follicle by slow-rate cooling and storage in liquid nitrogen, which preserved the multilineage-differentiation capacity of human HAP stem cells.
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