Extensive Hair-Shaft Elongation by Isolated Mouse Whisker Follicles in Very Long-Term Gelfoam® Histoculture

<div><p>We have previously studied mouse whisker follicles in Gelfoam® histoculture to determine the role of nestin-expressing plutipotent stem cells, located within the follicle, in the growth of the follicular sensory nerve. Long-term Gelfoam® whisker histoculture enabled hair follicle nestin-expressing stem cells to promote the extensive elongation of the whisker sensory nerve, which contained axon fibers. Transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP) were used as the source of the whiskers allowing imaging of the nestin-expressing stem cells as they formed the follicular sensory nerve. In the present report, we show that Gelfoam®-histocultured whisker follicles produced growing pigmented and unpigmented hair shafts. Hair-shaft length increased rapidly by day-4 and continued growing until at least day-12 after which the hair-shaft length was constant. By day-63 in histoculture, the number of ND-GFP hair follicle stem cells increased significantly and the follicles were intact. The present study shows that Gelfoam® histoculture can support extensive hair-shaft growth as well as hair follicle sensory-nerve growth from isolated hair follicles which were maintained over very long periods of time. Gelfoam® histoculture of hair follicles can provide a very long-term period for evaluating novel agents to promote hair growth.</p></div

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

<div><p>We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.</p></div

Hair shaft elongation of mouse whiskers in Gelfoam^® histoculture.

<p>Time-course images of hair shaft growth from individual mouse whisker follicles, isolated from nestin-driven green fluorescent protein (ND-GFP) mice, histocultured on Gelfoam®. Green fluorescence was from the ND-GFP-expressing stem cells in the whisker hair follicles which were enriched during 63 days of histoculture <i>in vitro</i>. Hair shafts lengthened rapidly in the first 4 days, extended over 9–12 days, and remained the same length until day 63.</p

Graph quantifying the time-course increase of hair follicle stem cell GFP fluorescence intensity (A) and fluorescent area (B).

<p><i>p</i><0.01 in increase of fluorescent area and fluorescence intensity at day 63 compared to day 1.</p

Ectopic hair growth in the spinal cord.

<p>Ninety days after transplantation of the 3-week Gelfoam ND-GFP-expressing whisker histocultures in the injured spinal cord, long hair shafts (arrows), were observed along and around the healed spinal cord. (<b>A</b>) Shows the elongated hair shafts that grew from whisker follicles, previously histocultured on Gelfoam into the injured spinal cord in 3 different mice at day-90 after surgery. All mice demonstrated hair shaft growth from the transplanted histoculture whisker follicles. Mouse 3 had the most remarkable hair shaft growth, which curved and enclosed the spinal cord. Arrows showed the hair growth in the spinal cord. (<b>B</b>) Panels show the hair shaft growth from the transplanted Gelfoam histoculture whisker follicles in the spine from mouse 3 at higher magnification from different views of the spinal cord (dorsal, left, and right side). The growing hair shaft reached a length of almost 14 mm and curved around the spinal cord. Arrows depict the hair shaft growing from the whisker hair follicles transplanted in the spine. Six out of 7 mice implanted with the Gelfoam whisker histoculture showed extensive ectopic hair growth on the spine.</p

Graphs quantifying the increase of shaft length over time in individual follicles during Gelfoam® histoculture.

<p>Graphs quantifying the increase of shaft length over time in individual follicles during Gelfoam® histoculture.</p

Transplantation of nestin-driven green fluorescent protein (ND-GFP)-expressing hair follicle Gelfoam histocultures to the injured spinal cord of nude mice.

<p>After Gelfoam histoculture of isolated whisker hair follicles from nestin-driven GFP (ND-GFP) mice for 3 weeks, the long hair shafts of the whisker follicle were cut off, and the follicle, along with the Gelfoam, was transplanted into the injured nude-mouse spinal cord. The transplanted mouse was sacrificed after 90 days. ND-GFP expression intensified by 90 days and expanded in the injured area of the spinal cord, which was apparently healed by the ND-GFP expressing stem cells. A total of 7 mice were studied. The figure shows typical data.</p

DAPI nuclear staining and P75^NTR immuostaining of cryopreserved or fresh follicles after 26 days in Gelfoam histoculture.

<p>(<b>A</b>) DAPI nuclear staining. Fresh hair follicles maintained intact structures of the hair shaft, inner root sheath, and outer root sheath, with a large amount of nestin-GFP HAP stem cells growing under the bulb area after 26 days of Gelfoam histoculture. In contrast, the inner structures of Gelfoam-histocultured hair follicles after DMSO- or glycerol-cryopreservation had no DAPI-stained nuclei, indicating damage of the inner structure of the follicle after cryopreservation. Most of the nestin-GFP expressing HAP stem cells grew around the outer root sheath and bulb area in cryopreserved hair follicles. (<b>B</b>) p75^NTR immunostaining of frozen sections colocalized with ND-GFP HAP stem cells. These results indicate that ND-GFP HAP stem cells in cryopreserved follicles were maintained.</p

Confocal 3D images of ND-GFP hair-follicle-associated-pluripotent (HAP) stem cells in whisker follicles grown in Gelfoam histoculture.

<p>(<b>A</b>) ND-GFP-expressing HAP stem cells in DMSO- or glycerol-cryopreserved hair follicles or fresh follicles at day 2, 10, 16 and 26 days after Gelfoam histoculture. (<b>B</b>) Quantitative analysis of ND-GFP HAP stem cells fluorescence in cultured whisker follicles. DMSO-cryopserved follicles had more extensive growth of HAP stem cells than in glycerol-cryopreserved follicles, but less than in fresh follicles. (<b>C</b>) At day 26 after culture on Gelfoam, only 2/6 hair follicles that were cryopreserved in glycerol recovered. In contrast, 6/6 hair follicles recovered after DMSO cryopreservation, similar to fresh follicles.</p

Schema of hair follicle isolation and cryopreservation, Gelfoam histocultured and transplantation.

<p>Schema of hair follicle isolation and cryopreservation, Gelfoam histocultured and transplantation.</p