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

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

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

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

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

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

From hair to heart: nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells

<p>We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells located in the bulge area which are termed hair-follicle-associated pluripotent (HAP) stem cells. HAP stem cells from mouse and human could form spheres in culture, termed hair spheres, which are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. Subsequently, we demonstrated that nestin-expressing stem cells could effect nerve and spinal cord regeneration in mouse models. In the present study, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. We separated the mouse vibrissa hair follicle into 3 parts (upper, middle, and lower), and suspended each part separately in DMEM containing 10% FBS. All three parts of hair follicle differentiated to beating cardiac muscle cells as well as neurons, glial cells, keratinocytes and smooth muscle cells. The differentiation potential to cardiac muscle is greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol and inhibited by propanolol. HAP stem cells have potential for regenerative medicine for heart disease as well as nerve and spinal cord repair.</p

The growing sciatic nerve intermingled with the dorsal root ganglion in 3D Gelfoam® histoculture.

<p>(A) A sciatic nerve was placed in Gelfoam® histoculture next to a dorsal root ganglion, both from a ND-GFP mouse. At day 38, many ND-GFP-expressing fibers were seen extending from both the sciatic nerve and co-cultured dorsal root ganglion. Bar: 500 µm. (B) Immunofluorescence staining of β-III tubulin (red) demonstrated that many β-III tubulin-positive fibers extended from both the sciatic nerve and the dorsal root ganglions. The fibers consisted of ND-GFP expressing cells. Bar: 500 µm. (C) Magnified image of the area inside the box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067153#pone-0067153-g007" target="_blank">Figure 7B</a> shows that many β-III tubulin-positive fibers extended widely and radially both from the sciatic nerve and the dorsal root ganglion. β-III tubulin-positive fibers from both nerves intermingled with each other. Bar: 500 µm.</p

Early-age-dependent selective decrease of differentiation potential of hair-follicle-associated pluripotent (HAP) stem cells to beating cardiac-muscle cells

<p>We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.</p