Hair-follicle-associated pluripotent stem cells derived from cryopreserved intact human hair follicles sustain multilineage differentiation potential

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.

Figure 1

Protocol for hair follicle cryopreservation. A flow diagram is shown for procedures from hair follicle isolation, cryopreservation, thawing, upper-part of follicle culture, HAP-stem cell proliferation, and differentiation.

Figure 2

Images of a cultured cryopreserved human hair follicle and subsequent cell outgrowth.

Figure 3

Cell types differentiated from the cultured upper part of cryopreserved hair follicles. Nestin- and βIII-tubulin-positive neurons, GFAP-positive glial cells, K15-positive keratinocytes, smooth muscle actin (SMA)-positive smooth muscle cells, and troponin (cTnT)-positive cardiac muscle cells. Staining for nestin, βIII-tubulin, GFAP, K15, SMA, and cTnT is shown in red color in corresponding panels. DAPI is shown in blue color in each panel. Scale bar = 100 µm.

Figure 4

Stem-cell marker expression in HAP stem cell colonies produced from cultured cryopreserved human hair follicles. SSEA1-negative, SSEA3-positive, SSEA4-positive, Nanog-positive, Oct3/4-positive, and nestin-positive. Staining for SSEA1, SSEA3, SSEA4, Nanog, Oct3/4 and nestin is shown in red color in corresponding panels. DAPI is shown in blue color in each panel. Scale bar = 100 µm.

Figure 5

Cell differentiation from human HAP stem-cell colonies produced from cultured cryopreserved hair follicles. HAP stem cell colonies differentiated to nestin and βIII-tubulin-positive neurons, GFAP-positive glial cells, K15-positive keratinocytes, SMA-positive smooth muscle cells, and cTnT-positive cardiac muscle cells. Staining for nestin, βIII-tubulin, GFAP, K15, SMA, and cTnT is shown in red color in corresponding panels. DAPI is shown in blue color in each panel. Scale bar = 100 µm.

Figure 6

Gene expression in cryopreserved hair follicles compared to fresh hair follicles. The blots for nestin, Oct4 and GAPDH shown, in the lower left panel, are cropped from different gels (shown in Supplementary Information).