Hair-Shaft Growth in Gelfoam® Histoculture of Skin and Isolated Hair Follicles.

Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was histocultured for up to 40 days on Gelfoam® at the air/liquid interface. The anagen hair follicles within the histoculture scalp skin produced growing hair shafts. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. Follicle keratinocytes continued to incorporate [3H]thymidine for up to several weeks after shaft elongation had ceased. Intensive hair growth was observed in the pieces of shaved mouse skin histocultured on Gelfoam®. Isolated human and mouse hair follicles also produced growing hair shafts. By day 63 in histoculture of mouse hair follicles, the number of hair follicle-associated pluripotent (HAP) stem cells increased significantly and the follicles were intact. Gelfoam® histoculture of skin demonstrated that the hair follicle cells are the most sensitive to doxorubicin which prevented hair growth, thereby mimicking chemotherapy-induced alopecia in Gelfoam® histoculture.

Toxicology and efficacy of tumor-targeting Salmonella typhimurium A1-R compared to VNP 20009 in a syngeneic mouse tumor model in immunocompetent mice.

Salmonella typhimurium A1-R (S. typhimurium A1-R) attenuated by leu and arg auxotrophy has been shown to target multiple types of cancer in mouse models. In the present study, toxicologic and biodistribution studies of tumor-targeting S. typhimurium A1-R and S. typhimurium VNP20009 (VNP 20009) were performed in a syngeneic tumor model growing in immunocompetent BALB/c mice. Single or multiple doses of S. typhimurium A1-R of 2.5 × 105 and 5 × 105 were tolerated. A single dose of 1 × 106 resulted in mouse death. S. typhimurium A1-R (5 × 105 CFU) was eliminated from the circulation, liver and spleen approximately 3-5 days after bacterial administration via the tail vein, but remained in the tumor in high amounts. S. typhimurium A1-R was cleared from other organs much more rapidly. S. typhimurium A1-R and VNP 20009 toxicity to the spleen and liver was minimal. S. typhimurium A1-R showed higher selective targeting to the necrotic areas of the tumors than VNP20009. S. typhimurium A1-R inhibited the growth of CT26 colon carcinoma to a greater extent at the same dose of VNP20009. In conclusion, we have determined a safe dose and schedule of S. typhimurium A1-R administration in BALB/c mice, which is also efficacious against tumor growth. The results of the present report indicate similar toxicity of S. typhimurium A1-R and VNP20009, but greater antitumor efficacy of S. typhimurium A1-R in an immunocompetent animal. Since VNP2009 has already proven safe in a Phase I clinical trial, the present results indicate the high clinical potential of S. typhimurium A1-R.

Cryopreservation of Hair-Follicle Associated Pluripotent (HAP) Stem Cells Maintains Differentiation and Hair-Growth Potential.

Hair follicles contain nestin-expressing pluripotent stem cells which originate above the bulge area of the follicle, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. We have established efficient cryopreservation methods of the hair follicle that maintain the pluripotency of HAP stem cells as well as hair growth. We cryopreserved the whole hair follicle by slow-rate cooling in TC-Protector medium or in DMSO-containing medium and storage in liquid nitrogen or at -80 °C. After thawing and culture of the cryopreserved whisker follicles, growing HAP stem cells formed hair spheres. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. We have also previously demonstrated that cryopreserved mouse whisker hair follicles maintain their hair-growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. DMSO-cryopreserved hair follicles also maintained the HAP stem cells, evidenced by P75ntr expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair-shaft growth of cryopreserved hair follicles. HAP stem cells can be used for nerve and spinal-cord repair. This biobanking of hair follicles can allow each patient the potential for their own stem cell use for regenerative medicine or hair transplantation.

Protocols for Ectopic Hair Growth from Transplanted Whisker Follicles on the Spinal Cord of Mice.

Isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice, containing hair-associated pluripotent (HAP) stem cells, were histocultured in three dimensions on Gelfoam(®) for 3 weeks for subsequent transplantation to the spinal cord in order to heal an induced injury with the HAP stem cells. The hair shafts were removed from Gelfoam(®)-histocultured whisker follicles, and the remaining parts of the whisker follicles, containing GFP-nestin-expressing (HAP) 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 injuries were 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 HAP stem cells were involved in healing the spinal cord. The transplanted whisker follicles produced remarkably long hair shafts in the spinal cord over 90 days and curved and enclosed the spinal cord. This result changes our concept of hair growth, demonstrating it is not limited to the skin and that hair growth appears related to HAP stem cells as both increased in tandem on the spinal cord.

Protocols for Gelfoam(®) Histoculture of Hair-Shaft-Producing Mouse Whisker Follicles Containing Nestin-GFP-Expressing Hair-Follicle-Associated Pluripotent (HAP) Stem Cells for Long Time Periods.

Gelfoam(®)-histocultured whisker follicles from nestin-driven-green fluorescent protein (ND-GFP) mice 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-associated pluripotent (HAP) stem cells increased significantly and the follicles were intact. Three-dimensional Gelfoam(®) histoculture of hair follicles can provide a very long-term period for evaluating novel agents to promote hair growth.

Aging hair follicles rejuvenated by transplantation to a young subcutaneous environment.

We demonstrate in the present study that young host mice rejuvenate aged hair follicles after transplantation. Young mice promote the hair shaft growth of transplanted old hair follicles, as well as young follicles, in contrast to old host mice, which did not support hair-shaft growth from transplanted old or young follicles. Nestin-expressing hair follicle-associated pluripotent (HAP) stem cells of transplanted old and young hair follicles remained active in young host nude mice. In contrast, the nestin-expressing HAP stem cells in young and old hair follicles transplanted to old nude mice were not as active as in young nude host mice. The present study shows that transplanted old hair follicles were rejuvenated by young host mice, suggesting that aging may be reversible.

Fluorescence-guided surgery of human prostate cancer experimental bone metastasis in nude mice using anti-CEA DyLight 650 for tumor illumination.

The present report demonstrates efficacy of fluorescence-guided surgery (FGS) to resect and prevent recurrence of experimental skeletal metastasis in a nude-mouse model of human prostate cancer. Green fluorescent protein (GFP)-expressing PC-3 human prostate cancer cells were injected into the intramedullary cavity of the tibia in 25 nude mice. One week after implantation, monoclonal antibodies, specific for carcinoembryonic antigen (CEA), labeled with DyLight 650, were injected into the tail vein of 13 mice. Thirteen mice underwent FGS and 12 mice underwent bright-light surgery (BLS). Weekly GFP fluorescence imaging of the mice was performed to observe tumor recurrence. The extent of residual tumor after BLS was 13-fold greater than after FGS (p < 0.001). Time-course imaging visualized rapid growth of the residual tumor in the BLS group, whereas the FGS group showed only slight tumor growth and significantly improved disease-free survival of the treated mice. Our study demonstrated that FGS significantly reduced residual tumor as well as the recurrence of experimental prostate-cancer bone metastasis. The present results suggest that FGS will be effective for resection of skeletal metastases in selected patients with prostate cancer.

Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice.

We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.

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

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.

Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers.

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.

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

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.

Hydrogen-rich saline promotes survival of retinal ganglion cells in a rat model of optic nerve crush.

OBJECTIVE: To investigate the effect of molecular hydrogen (H2) in a rat model subjected to optic nerve crush (ONC). METHODS: We tested the hypothesis that after optic nerve crush (ONC), retinal ganglion cell (RGC) could be protected by H2. Rats in different groups received saline or hydrogen-rich saline every day for 14 days after ONC. Retinas from animals in each group underwent measurements of hematoxylin and eosin (H&E) staining, cholera toxin beta (CTB) tracing, gamma synuclein staining, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining 2 weeks post operation. Flash visual evoked potentials (FVEP) and pupillary light reflex (PLR) were then tested to evaluate the function of optic nerve. The malondialdehyde (MDA) level in retina was evaluated. RESULTS: H&E, gamma synuclein staining and CTB tracing showed that the survival rate of RGCs in hydrogen saline-treated group was significantly higher than that in saline-treated group. Apoptosis of RGCs assessed by TUNEL staining were less observed in hydrogen saline-treated group. The MDA level in retina of H2 group was much lower than that in placebo group. Furthermore, animals treated with hydrogen saline showed better function of optic nerve in assessments of FVEP and PLR. CONCLUSION: These results demonstrated that H2 protects RGCs and helps preserve the visual function after ONC and had a neuroprotective effect in a rat model subjected to ONC.