Follicle Outer Root Sheath Cell Research Articles

In vitro hair growth-promoting effect of Lgr5-binding octapeptide in human primary hair cells.

Hair loss occurs due to various biological and environmental causes, which can have psychosocial consequences. The Wnt/β-catenin signaling is well-known for its role in hair growth and regeneration, as it induces the proliferation and differentiation of hair cells. When the leucine-rich G protein-coupled receptor 5 (Lgr5) interacts with the R-spondins, the frizzled receptor (FZD), a Wnt receptor, becomes stabilized, resulting in an increased β-catenin activity. We investigated whether the octapeptide that binds to Lgr5 enhances proliferation and differentiation of human primary hair cells through the activation of Wnt/β-catenin signaling. The binding affinity of the octapeptide to Lgr5 was evaluated using surface plasmon resonance (SPR). We confirmed changes in proliferation and related factors like β-catenin activation and growth factors (GFs) expression in human hair follicle dermal papilla cells (HHFDPCs). Additionally, we observed the proliferation and the expression of differentiation markers in human hair follicle outer root sheath cells (HHFORSCs), human hair follicle germinal matrix cells (HHFGMCs), and human hair follicle stem cells (HHFSCs). We used three-dimensional HHFDPC spheroid culture treated with dihydrotestosterone (DHT) to create invitro conditions that mimic androgenetic alopecia, and we studied the effects of octapeptide on Wnt expression and HHFSC differentiation. The binding of the octapeptide to Lgr5 was confirmed using SPR analysis. In HHFDPCs, treatment with octapeptide resulted in a concentration-dependent increase in proliferation. We also observed increased nuclear translocation of β-catenin and increased expression of its downstream targets. HHFDPCs treated with octapeptide exhibited increased expression of growth factors and phosphorylation of Akt and ERK. In addition, we confirmed that octapeptide increased proliferation and induced differentiation in HHFORSCs, HHFGMCs, and HHFSCs. Under the HHFDPC spheroid culture conditions, we found that octapeptide restored the inhibition of Wnt-5a and Wnt-10b expressions by DHT. In HHFSCs treated with HHFDPC spheroid culture media, we observed that octapeptide recovered the inhibition of differentiation by DHT. We found that octapeptides activated the Wnt/β-catenin signaling and induced the proliferation and differentiation of human primary hair cells by acting as an exogenous ligand for Lgr5. In addition, octapeptides recovered inhibited hair regeneration characters by DHT in androgenetic alopecia-mimic invitro model. These findings suggest that octapeptides may be a promising therapeutic option for treating hair loss.

Mechanical stimulation of human hair follicle outer root sheath cultures activates adjacent sensory neurons.

Mechanical stimuli, such as stroking or pressing on the skin, activate mechanoreceptors transmitting information to the sensory nervous system and brain. It is well accepted that deflection of the hair fiber that occurs with a light breeze or touch directly activates the sensory neurons surrounding the hair follicle, facilitating transmission of mechanical information. Here, we hypothesized that hair follicle outer root sheath cells act as transducers of mechanical stimuli to sensory neurons surrounding the hair follicle. Using electrochemical analysis on human hair follicle preparations in vitro, we were able to show that outer root sheath cells release ATP and the neurotransmitters serotonin and histamine in response to mechanical stimulation. Using calcium imaging combined with pharmacology in a coculture of outer root sheath cells with sensory neurons, we found that the release of these three molecules from hair follicle cells leads to activation of sensory neurons.

SIRT1 downregulation provokes immune-inflammatory responses in hair follicle outer root sheath cells and may contribute to development of alopecia areata

BackgroundSilent information regulator 1 (SIRT1), a type III histone deacetylase, is involved in various cutaneous and systemic autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. However, little is known about the role of SIRT1 in the development of alopecia areata (AA). ObjectivesThis study investigated whether SIRT1 regulates the hair follicle immune system and is involved in AA pathogenesis. MethodsSIRT1 expression in human scalp tissue was analyzed using immunohistochemical staining, qPCR, and western blotting. The regulatory effect of SIRT1 was evaluated after stimulation with the double-stranded RNA mimic polyinosinic:polycytidylic acid (poly I:C) in hair follicle outer root sheath (ORS) cells and C3H/HeJ mice. ResultsSIRT1 expression was significantly reduced in the AA scalp compared to the normal scalp. SIRT1 inhibition upregulated MHC class I polypeptide-related sequence A and UL16 binding protein 3 in hair follicle ORS cells. SIRT1 inhibition also promoted the production of Th1 cytokines (IFN-γ and TNF-α), IFN-inducible chemokines (CXCL9 and CXCL10), and T cell migration in ORS cells. Conversely, SIRT1 activation suppressed the autoreactive inflammatory responses. The counteractive effect of the immune response by SIRT1 was mediated through the deacetylation of NF-κB and phosphorylation of STAT3. ConclusionSIRT1 downregulation induces immune-inflammatory responses in hair follicle ORS cells and may contribute to AA development.

ERK1/2 Pathway Is Involved in the Enhancement of Fatty Acids from Phaeodactylum tricornutum Extract (PTE) on Hair Follicle Cell Proliferation.

Extractions from Phaeodactylum tricornutum have been widely studied and evaluated to various biological effects. The aim of this study was to investigate the promotional effect of P. tricornutum extract (PTE) on the ERK1/2 signaling pathway involved in hair follicle cell proliferation. In order to illuminate the enhancement of PTE on hair growth by promoting proliferation of hair follicle cells, the activities of human hair follicle outer root sheath cell (HFORSC), human hair follicle germinal matrix cells (HFGMC), and hair epithelial melanocytes (HEM) were observed under PET treatment. Levels of keratins, PKCζ, ERK1/2, and p38 MAPK in hair follicle cells were determined by Western blotting to illustrate the mechanisms of PTE effects on hair growth. Analyzed by GC-MS, the main polyunsaturated fatty acids which were 9.43% of total fatty acids in PTE were linolenic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid. Melanin content and tyrosinase activity in HEM were measured. The results showed that PTE exhibited remarkable enhancement on cell proliferation. Melanin production was inhibited by PTE treatment, while keratin-14, keratin-15, and keratin-17 levels on hair follicle cells were elevated at different concentrations. The promotions of ERK1/2 and p38 MAPK levels indicated that the ERK1/2 signaling pathway is involved in the proliferation of hair follicle cells. These results are the evidence that PTE potentially deserves further study as a new natural candidate for hair care applications.

Treatment of stable vitiligo using cultured and non-cultured autologous melanocytes from hair follicle outer root sheath cell suspension

Background Several surgical treatment methods have been proposed for the treatment of stable vitiligo, but they remain inadequate in terms of patient satisfaction. Objective To assess the efficacy and safety of a modified autologous cultured hair follicle outer root sheath (HF-ORS) cell suspension transplantation in the treatment of stable vitiligo lesions, and to compare it with transplantation of autologous non-cultured HF-ORS cell suspension for the same patient. Patients and methods Twenty-four patients were enrolled in this prospective controlled study. Hairs were epilated, and HF-ORS cell suspensions were prepared using two methods for the same patient. The recipient site was prepared by CO2 laser resurfacing. Assessment of cellular activity and viability included cellular melanin content determined by enzyme-linked immunosorbent assay and measurement of the fold change in premelanosome (Pmel-17) gene expression. The patients were followed-up for 6 months postprocedure. Results Nineteen patients completed the study. The results demonstrated that the cultured method showed superior repigmentation response in 15 (79%) patients, three (16%) responded equally, and one (5%) was more tolerant of the noncultured method. Conclusion Cultured autologous HF-ORS melanocytes show superior repigmentation response, increased melanin content, and upregulated gene expression of Pmel-17 than that of noncultured ones. However, the study also demonstrated that both methods are potential options for the treatment of stable vitiligo.

A Prospective Observational Comparative Study of Novel Autologous Negative Pressure Epidermal Harvesting System (ANPEHS or EHS) and Suction Blister Grafting (SBG) in Treatment of Stable Vitiligo.

Background:Vitiligo surgery has evolved a long way from punch skin grafts to the epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension transplantation (EHFORSCS). In the latest development, the novel technique autologous negative pressure epidermal harvesting system (ANPEHS) seems to be a good addition to the prevailing therapy in vitiligo.Aims and Objectives:The aim of this work was to study and compare novel ANPEHS and suction blister grafting (SBG) in the treatment of stable vitiligo.Materials and Methods:This was a prospective, single-centered, observational, open-labeled comparative study of the rate and final extent of repigmentation in ANPEHS or EHS and SBG in the management of stable vitiligo. The patients in this study were drawn from the outpatient Department of Dermatology of a Tertiary Care Hospital of the Armed Forces from July 1 2015 to December 31, 2016. A total of 40 patients with at least two comparable depigmented patches of 6 months’ stability were recruited. Both procedures were performed in the same patient on the same day. Informed consents were taken from all the patients. Each patient in the study had undergone a detailed clinical, general physical, systemic, and a dermatological examination. Clinical photographs had been taken before and after grafting, monthly for the first 6 months then bimonthly for the next 6 months. Donor site was preferably inner aspect of thigh.Statistical Analysis Used:“Chi-square test” and “statistical significance” (P value) methods.Results:Of total 80 patches, excellent results were seen in 82.9% patches by using the EHS method and 80% excellent results by using the SBG method. Similarly, very good results were seen in 2.9% patches and good results in 5.7% patches by using both methods. 11.4% patches showed poor results by using the SBG method and 8.6% patches showed poor results by using the EHS method.Conclusions:The EHS method is a simple, painless, less time-consuming, expensive but effective technique to produce homogeneous repigmentation without any donor site anesthesia and complication.

Enhanced Cultivation of Hair Follicle Outer Root Sheath Cells with K + M Culture Medium

Hair follicle stem cells are vital seed cells for hair follicle and tissue engineering skin. However, there is no valid approach to obtain abundant cells within short times in vitro. The purpose of this study was to establish a new medium for culture and expansion of hair follicle outer root sheath (ORS) cells. ORS cells were harvested from human samples and cultured with a medium mixed with keratinocyte serum free medium and mouse embryonic fibroblasts suspension. The proliferation and vitality of ORS cells were evaluated. The cells exhibited typical cobblestone morphology with good adhesion and colony-forming ability. The cells were also characterized through detecting specific markers with immunofluorescent staining and the results showed that partial ORS cells could express hair follicle stem cell markers of CK15, CK19 and FST. At last, ORS cells and fibroblasts were used as seed cells and preliminarily constructed tissue engineering skin successfully. In conclusion, these findings suggested that this new medium could effectively accelerate cell proliferation and maintain the fundamental characteristics of ORS cells.

Pharmacological Activation of Thermo-Transient Receptor Potential Vanilloid 3 Channels Inhibits Hair Growth by Inducing Cell Death of Hair Follicle Outer Root Sheath.

Hair growth starts from hair follicles that reside in dermis, and abnormal hair growth is an early sign of hair follicle disease or systemic illness such as alopecia or hair loss. Therefore, identifying a target critical for dysfunctional hair follicles is fundamental to alleviating dermatologic or systemic diseases with hair abnormalities. The warm temperature-activated Ca2+-permeable transient receptor potential vanilloid 3 (TRPV3) channel protein is abundantly expressed in the skin keratinocytes, and dysfunctional TRPV3 causes human congenital Olmsted syndrome, characterized by skin diseases and alopecia, indicating an important role for TRPV3 in hair follicle development and hair growth. To validate TRPV3 as a therapeutic target, we investigated the impact of pharmacological modulation of TRPV3 on hair growth using a combination of biochemical and cell biology, immunohistochemical, whole-cell patch clamp, RNA interference, and pharmacological approaches. We found that functional TRPV3 channel proteins are highly expressed in hair follicle outer root sheath (ORS) cells as detected by Western blot analysis, immunohistochemical staining, and electrophysiological techniques. Pharmacological activation of TRPV3 by agonist natural carvacrol induces cell death of ORS cells, and topical application of carvacrol to mouse dorsal skin also inhibits hair growth. Conversely, specific inhibition of TRPV3 by inhibitor natural forsythoside B and short-hairpin RNA reverses the cell death induced by carvacrol-mediated TRPV3 activation in human ORS cells. Furthermore, forsythoside B results in a significant reversal of hair growth inhibition induced by agonist carvacrol. Altogether, our findings demonstrate that TRPV3 channel is critical for regulation of hair growth, and inhibition of TRPV3 may represent a promising therapy for hair loss or hair follicle-related skin diseases.

Noncultured Extracted Hair Follicle Outer Root Sheath Cell Suspension versus Noncultured Epidermal Cell Suspension in the Treatment of Stable Vitiligo.

Background:Various treatment modalities exist for vitiligo, yet none of them are curative. Vitiligo is still considered a challenging disease to manage. Surgical treatment offers an excellent option for patients with stable vitiligo, especially those who fail to respond to medical treatment. Cell suspension techniques carry the advantage of covering large surface areas.Objective:To compare noncultured extracted hair follicle outer root sheath cell suspension (NCORSHFS) and noncultured epidermal cell suspension (NCES) in producing repigmentation.Subjects and Methods:Twenty patients were randomly allocated into two groups. They were objectively evaluated for the extent of repigmentation (after 1, 2, and 3 months), complications, cosmetic outcome, and satisfaction.Results:In NCORSHFS group, 10% showed excellent pigmentation, 20% showed good pigmentation, 50% fair, and 20% poor pigmentation. In NCES group, 10% showed excellent pigmentation, 10% good pigmentation, 40% fair, and 40% poor pigmentation. This difference was not statistically significant. Excellent color match was observed in 80% of NCORSHFS and in 70% of NCES. Donor area complications were absent in NCORSHFS group, whereas in NCES, mild scarring in 20% and hyperpigmentation in 40% of patients was observed. The difference in patients’ satisfaction between the two groups was not statistically significant.Conclusion:Both NCORSHFS and NCES are effective in producing good repigmentation with perfect color match and patients’ satisfaction. NCORSHFS has no donor area complications because it is a scarless procedure.

Restoration of hair-inductive activity of cultured human follicular keratinocytes by co-culturing with dermal papilla cells

Hair follicle outer root sheath (ORS) cells can be expanded in vitro, but often lose receptivity to hair-inducing dermal signals. Recent studies have shown hair-inductive activity (trichogenicity) can be restored in rat ORS cells expanded with a fibroblast feeder by co-culturing with rat vibrissae dermal papilla (DP) cells. In this study, we investigated whether the trichogenicity of human ORS cells can be restored by co-culturing with human DP cells. ORS cells from human scalp hair follicles were cultured independently or with DP cells for 5 days and implanted into nude mice alongside freshly isolated neonatal mouse dermal cells. Although there was no hair induction when monocultured ORS cells were implanted, it was observed in co-cultured ORS cells. We also observed differential regulation of a number of genes in ORS cells co-cultured with DP cells compared to monocultured ORS cells as examined by microarray. Taken together, our data strongly suggest that human DP cells restore the trichogenicity of co-cultured ORS cells by influencing ORS gene expression through paracrine factors.

A new path in defining light parameters for hair growth: Discovery and modulation of photoreceptors in human hair follicle.

Though devices for hair growth based on low levels of light have shown encouraging results, further improvements of their efficacy is impeded by a lack of knowledge on the exact molecular targets that mediate physiological response in skin and hair follicle. The aim of this study was to investigate the expression of selected light-sensitive receptors in the human hair follicle and to study the impact of UV-free blue light on hair growth ex vivo. The expression of Opsin receptors in human skin and hair follicles has been characterized using RT-qPCR and immunofluorescence approaches. The functional significance of Opsin 3 was assessed by silencing its expression in the hair follicle cells followed by a transcriptomic profiling. Proprietary LED-based devices emitting two discrete visible wavelengths were used to access the effects of selected optical parameters on hair growth ex vivo and outer root sheath cells in vitro. The expression of OPN2 (Rhodopsin) and OPN3 (Panopsin, Encephalopsin) was detected in the distinct compartments of skin and anagen hair follicle. Treatment with 3.2 J/cm2 of blue light with 453 nm central wavelength significantly prolonged anagen phase in hair follicles ex vivo that was correlated with sustained proliferation in the light-treated samples. In contrast, hair follicle treatment with 3.2 J/cm2 of 689 nm light (red light) did not significantly affect hair growth ex vivo. Silencing of OPN3 in the hair follicle outer root sheath cells resulted in the altered expression of genes involved in the control of proliferation and apoptosis, and abrogated stimulatory effects of blue light (3.2 J/cm2 ; 453 nm) on proliferation in the outer root sheath cells. We provide the first evidence that (i) OPN2 and OPN3 are expressed in human hair follicle, and (ii) A 453 nm blue light at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. Lasers Surg. Med. 49:705-718, 2017. © 2017 Wiley Periodicals, Inc.

Differentiation of nestin‑negative human hair follicle outer root sheath cells into neurons invitro.

A specialized quiescent population of hair follicle stem cells, residing in the hair follicle outer root sheath cells (ORSCs), has previously demonstrated pluripotency for differentiation into neural stem cells (NSCs). A previous study indicated that nestin-positive hair follicle ORSCs are able to differentiate into neurons. However, little has been reported on the isolation of nestin-negative human ORSCs and whether they can successfully differentiate into neurons in vitro. In the present study, nestin-positive ORSCs were significantly reduced with a prolonged incubation time in vitro. Following 9 days of primary culture, nestin-expressing ORSCs disappeared entirely, and ORSCs remained nestin-negative following 5 days of subculture. Notably, nestin was identified in ORSCs following a three-step process of neuro-induction. In addition, neruodevelopmental markers were detected in the ORSC-derived nestin-positive spherical cell mass, including the induction of the neuronal specific markers growth associated protein-43, neurotensin receptor-3 and p75 neurotrophin receptor, and also the gliocyte markers, glial fibrillary acidic protein and S100. These sphere-forming cells did not express the mature neuron-associated markers neurofilament medium, neuronal nuclei and neuron-specific enolase, which suggested that sphere-forming cells may preferentially differentiate into neural stem cell-like cells as opposed to mature neurons or neurogliocyte. In conclusion, ORSC-driven neural differentiation may be a suitable treatment strategy for neurodegenerative diseases and may possess an important value in regenerative medicine.

A Study of Noncultured Extracted Hair Follicle Outer Root Sheath Cell Suspension for Transplantation in Vitiligo.

Context:Vitiligo surgeries have come a long way from tissue grafts to cultured and non cultured cell transplantation. Extracted hair follicle outer root sheath cell transplantation (EHF ORS) suspension is more enriched with melanocyte. In a hair bulb, there is one melanocyte for every five keratinocytes which is much higher than the epidermal melanin unit.Aims:To analyse the effectiveness of cultured EHF ORS and to perform objective evaluation based on clinical improvement & photographic evidence. To observe any untoward events or side effects.Settings and Design:The study was open and uncontrolled. All the patients were screened at preliminary visit. Reviews were done every two weeks. The endpoint selected was six months post procedure.Materials and Methods:Twenty five patients of stable Vitiligo were included in the study and follicular unit were harvested by Follicular Unit Extraction method. Outer root sheath cells were extracted by trypsinization. The solution was transplanted over dermabraded recipient site. Pressure dressing was given. Patients were followed up regularly.Statistical Analysis Used:Descriptive Statistics, Chi-Square.Results:Mean ± SD repigmentation was 80.15% ± 22.9% with excellent repigmentation (90-100%) in 60% of patients.Conclusions:This method is safe, effective, and simpler than the other methods involving cell culturing and requiring a laboratory set-up but selection of patients is crucial for the success of the outcome.

Comparative Study of Efficacy of Epidermal Melanocyte Transfer Versus Hair Follicular Melanocyte Transfer in Stable Vitiligo.

Background:Vitiligo surgery has come up a long way from punch skin grafts to epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension (EHFORSCS) transplantation. The progressive development from one technique to the other is always on a quest for the best. In the latest development, EHFORSCS, which is an enriched source of follicular inactive melanocyte (melanocyte stem cells), seems to be a good addition to the prevailing cell-based therapies for vitiligo. However, it needs to be explored further in larger, clinical trials.Methodology:A total of 11 patients with sixty stable vitiligo sites attending dermatology outpatient department were included for the open-labeled, prospective, comparative study. The sites were sequentially distributed into two groups of thirty each. Sites of one group were subjected to epidermal melanocyte transfer (EMT) and the others to hair follicular melanocyte transfer (HFMT). Response to treatment was evaluated on the basis of degree of repigmentation; final evaluation of area of involvement was done after completion of 6 months.Results:At the end of 6 months, repigmentation >90% was observed in 83.33% patches of EMT group and 43.33% in HFMT group. Repigmentation >75% was observed in 90% of patches in Group A and 43.34% of patches in Group B, respectively. There was statistically significant difference in the overall pigmentation between these two groups.Conclusion:Both noncultured autologous epidermal cell suspension transfer and noncultured EHFORSCS transfer are safe and effective surgical modalities in the management of stable vitiligo though EMT has shown a better response in the present study. Outer root sheath cell suspension transfer is a novel, minimally invasive technique in its nascent stage in the surgical management of vitiligo which requires further larger clinical trials for evaluation of its efficacy.

Epidermal Growth Factor Promotes Proliferation and Migration of Follicular Outer Root Sheath Cells via Wnt/β-Catenin Signaling

Background/Aims: To investigate the effect and molecular mechanism of EGF on the growth and migration of hair follicle outer root sheath (ORS) cells. Methods: Intact anagen hair follicles were isolated from mink skin and cultured with EGF in vitro to measure ORS daily growth. Meanwhile, purified primary ORS cells were treated or transfected with EGF, and their proliferation and migration were assessed by MTT assay and transwell assay, respectively. The signaling pathway downstream of EGF was characterized by using the Wnt/β-catenin signaling inhibitor, XAV-939. Results: EGF of 2-20 ng/ml, not higher or lower, promoted the growth of follicular ORS in vitro. EGF treatment or overexpression promoted the proliferation and migration of ORS cells. Moreover, EGF stimulation induced nuclear translocation of β-catenin, and upregulated the expression of Wnt10b, β-catenin, EGF receptor and SOX9. Inhibition of Wnt/β-catenin signaling by XAV-939 significantly reduced the basal and EGF-enhanced proliferation and migration of ORS cells. In addition, a number of follicle-regulatory genes, such as Survivin, Msx2 and SGK3, were upregulated by EGF in the ORS cells, which was also inhibited by XAV-939. Conclusion: EGF promotes the proliferation and migration of ORS cells and modulates the expression of several follicle-regulatory genes via Wnt/β-catenin signaling.

Comparison between autologous noncultured extracted hair follicle outer root sheath cell suspension and autologous noncultured epidermal cell suspension in the treatment of stable vitiligo: a randomized study

Vitiligo is an acquired disorder of pigmentation caused by loss of epidermal melanocytes. Autologous noncultured epidermal cell suspension (NCES) and autologous noncultured extracted hair follicle outer root sheath cell suspension (NCORSHFS) are important surgical modalities for the treatment of stable vitiligo. To compare NCES and NCORSHFS for producing repigmentation in stable vitiligo. We randomized 30 patients with 47 stable vitiligo lesions into two groups. Patients in group 1 were treated with NCES, and those in group 2 with NCORSHFS. They were evaluated 16 weeks postsurgery for the extent of repigmentation, colour match, change in Dermatology Life Quality Index (DLQI) score and patient satisfaction. The extent of repigmentation was excellent (90-100% repigmentation) in 83% of lesions in the NCES group and 65% of lesions in the NCORSHFS group (P = 0·154). Repigmentation ≥ 75% (good repigmentation) was observed in 92% of lesions in the NCES group and 78% of lesions in the NCORSHFS group (P = 0·425). There was a significant improvement in DLQI score in both the groups, but the mean decrease among groups did not differ significantly (P = 0·244). However, patients in the NCES group were significantly more satisfied than the patients in the NCORSHFS group. No significant difference was seen in colour match and pattern of repigmentation. Adverse effects were minimal. Both NCES and NCORSHFS are safe and effective techniques with comparable efficacy. To the best of our knowledge, this is the first study directly comparing two different cellular techniques.

Functions of exogenous application of connective tissue growth factor in stimulating human dermal papilla cells and human hair follicle outer root sheath cells for reconstructive tissue-engineering hair follicles

To explore the functions of connective tissue growth factor (CTGF) in the restoration of hair follicles with a mixture of human dermal papilla cells and human hair follicle outer root sheath cells in vitro in nude mice. Human hair follicle outer root sheath cells (hfORS) and human hair dermal papilla cells (hDP) were cultured in vitro and mixed in a fixed ratio (hfORS: hDP = 5:1). Flow cytometry was used to detect the content of CD200(+) cells in human hair follicle outer root sheath cells.And 8 nude mice were divided randomly into 2 groups according to a random number table and back wounds produced. Group A was transplanted with cell mixture plus 20 µg/L CTGF. Group B was transplanted with cell mixture alone. After 8 weeks of transplantation, the development of hair follicle formation was observed histologically.PCR was used to detect the expression of human specific DNA and mice DNA in transplants. The portion of CD200(+) cells in cultured hfORS was 19.65%. At 8 weeks after implantation, hair follicle formation could be observed in Group A (268 ± 96) more than Group B (62 ± 20). The difference was statistically significant (P < 0.05). And PCR showed that there was human composition in transplant. CTGF can induce the formation of hair follicle by promoting the interference between hDP and hfORS.

Extracted hair follicle outer root sheath cell suspension for pigment cell restoration in vitiligo

Vitiligo surgery has come up a long way from punch skin grafts to epidermal cell suspension and latest to the extracted hair follicle outer root sheath cell suspension (EHF-ORS-CS) transplantation. The progressive development from one technique to the other is always in a quest for the best. In the latest development- EHF-ORS-CS, which is an enriched source of follicular inactive melanocyte (melanocyte stem cells), seems to be a good addition to the prevailing cell-based therapies for vitiligo; however, need to be explored further in larger, and preferably randomized blinded studies. This review discusses the principle, technical details, and stem cell composition of hair follicular outer root sheath cell suspension.

Enhanced Proliferation and Functions ofIn VitroExpanded Human Hair Follicle Outer Root Sheath Cells by Low Oxygen Tension Culture

Human hair follicle outer root sheath (hORS) cells are known to contain hair follicle stem cells and play an important role in healing large size wounds, and thus can serve as the cell source for skin engineering. This study investigated the effect of low oxygen tension culture on hORS cell proliferation potential and functional maintenance during in vitro expansion. Spared postsurgery scalp tissues were donated by 15 patients aged 20-45 (13 men and 2 women) and were randomly divided into three groups, and isolated hORS cells were combined into three pooled cell samples. They were cultured either in 4% O(2) or 21% O(2) and were analyzed for cell proliferation, colony forming efficiency (CFE), and their ability in forming engineered skin in vitro. The results showed that freshly isolated hORS cells expressed CD200 (22.88±8.76), cytokeratin 15 (CK15) (62.57±4.72), CD29 (22.53±2.49/strong and 29.80±4.09/dim), and CD49f (28.07±15.76/strong and 49.73±5.65/dim). When exposed in 4% O(2), hORS cells proliferated significantly faster than the cells in 21% O(2) for the first three passages (p<0.05), could better maintain cobblestone morphology, respectively, generate 3.63-folds more and 23.26-folds more cell yields after one and three passages. Additionally, enhanced CFE with significantly higher total and holoclone colony numbers were found in the 4% O(2) group than in the 21% O(2) group (p<0.05) for the first three passages along with better maintained CK15 expression. Furthermore, hORS cells expanded in 4% O(2) could form better epidermal structure of in vitro engineered skin comparing to the skin engineered by the control cells. The low oxygen culture method of hORS cells is simple, low cost, less labor intensive, and less biosafety concern, which may potentially be applied in skin engineering and clinical application.

Noncultured extracted hair follicle outer root sheath cell suspension for transplantation in vitiligo

Current noncultured cell-based transplantation therapies for vitiligo largely involve shave skin biopsy for preparation of noncultured melanocyte suspension. As the overall proportion of melanocytes is low in the epidermis, these techniques require basal cell layer enrichment, which adds additional steps. We tried follicular unit extraction (FUE) to harvest hair follicles as a source of melanocytes. To evaluate the efficacy of a novel surgical method for vitiligo: noncultured extracted hair follicular outer root sheath (ORS) cell suspension transplantation. Fourteen patients with vitiligo, stable for at least 3 months, were included in this prospective study. Fifteen to 25 hair follicles were extracted from occipital scalp using the FUE method. Hair follicles were incubated with trypsin-ethylenediamine tetraacetic acid solution at 37°C for 90 min to separate ORS cells. The cell suspension was filtered through a 70-μm cell strainer, then centrifuged for 5 min at 1000 r.p.m. to obtain a cell pellet. The pellet was resuspended and applied to the dermabraded recipient area and dressed. The mean ± SD repigmentation was 65·7 ± 36·7%. Overall, nine of 14 patients achieved > 75% repigmentation. Mean percentage repigmentation was significantly higher in patients with ≥ 1 year stability than those with < 1 year stability (P = 0·02). Extracted hair follicular ORS cell suspension can be a useful simplified transplantation method for vitiligo. The transplantation procedure should be reserved for patients with vitiligo stable for at least 1 year. A larger study is needed for further evaluation.