The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation.

When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and ß1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200- keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation-the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.

A novel topical combination of minoxidil and spironolactone for androgenetic alopecia: Clinical, histopathological, and physicochemical study.

Topical minoxidil 5% are effective in androgenetic alopecia (AGA). Spironolactone acts as an androgen antagonist by competitively blocking androgen receptors. Studying the effect of topical minoxidil 5% gel and spironolactone gel 1% in management of AGA. The study includes 60 patients diagnosed as AGA; (group I): treated with topical minoxidil gel 5%, (group II): with topical spironolactone gel 1% and group (III) treated with combined minoxidil 5% and spironolactone 1% gel. All patients were followed up monthly throughout the treatment period. Scalp biopsy was taken before and after 12 months. In group I, the clinical response was in 90% of patients with variable degrees in improvement, in group II, the clinical response was in 80% of patients, meanwhile, in group III the clinical response was in all patients (100%). Histopathological examination of skin biopsy after treatment revealed significant increase in anagen hair on the other hand, both telogen and vellus hair was significantly decreased meanwhile, the T/V ratio was significantly increased. The results of this work revealed that topical minoxidil gel 5% and topical spironolactone gel 1% were effective in treatment of AGA, while the combination of two agents was better in treatment.

Deriving Keratinocyte Progenitor Cells and Keratinocytes from Human-Induced Pluripotent Stem Cells.

Skin or hair loss (alopecia) may occur due to a wide variety of causes ranging from trauma to pathological processes including acquired or congenital causes. It would be ideal to replace them with immunologically compatible cells to avoid potentially exacerbating the condition. Deriving the replacement cells from human-induced pluripotent stem cells (hiPSCs) allows for sufficient scale up and using hiPSCs as the choice of human pluripotent stem cells (hPSC) will ensure immunocompatibility. Here we offer a protocol for differentiating hiPSCs into keratinocyte progenitor cells (KPC) and keratinocytes employing all-trans retinoic acid (ATRA) and L-ascorbic acid, (L-AA), bone morphogenic protein-4 (BMP4), and epidermal growth factor (EGF). We observed that the hiPSC-derived KPCs express the same panel of markers as primary hair follicle bulge stem cells (HFBSCs), including CD200, integrin α-6 (ITGA6), integrin ß-1 (ITGB1), the transcription factor P63, keratin 15 (KRT15), and keratin 19 (KRT19). If permitted to differentiate further, the hiPSC-derived KPC lose CD200 expression and rather come to express keratin 14 (KRT14) indicating emergence of more mature terminally-differentiated keratinocytes. The HFBSCs are transplantable for hair follicle (HF) restoration, and the keratinocytes may be transplantable for therapy for large burns or ulcers. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Reprogramming of normal human skin fibroblasts into normal hiPSCs using episomal DNA cocktail Basic Protocol 2: Differentiation of hiPSCs into KPCs and keratinocytes Alternate Protocol 2: EBS formation protocol using AggreWell™ plates (Antonchuk, 2013) Support Protocol 1: Passage hiPSC-KPC Support Protocol 2: Immunocytochemistry (ICC) Support Protocol 3: Immunofluorescence staining of cells for flow cytometry (FC).

Hair follicle changes following intense pulsed light axillary hair reduction: histometrical, histological and immunohistochemical evaluation.

Intense pulsed light (IPL) has been used for years in hair reduction; however, no previous studies discussed quantitative histological and immunohistochemical changes of hair follicles after IPL. Accordingly, this study aims to objectively quantify histological and immunohistochemical changes of hair follicles after IPL hair reduction. Right axillae of 21 volunteers were subjected to 6 IPL sessions using Quanta system IPL and evaluated at 1 week and 1 month after last session (i.e., 3 and 4 months from the start of treatment, respectively) in comparison to baseline and left control axillae. Using hair count, histological and immunohistochemical assessment of vertical and serial transverse sections coupled with computerized morphometric analysis, determination of hair reduction percentage, measurement of hair shaft (HS) diameter, calculation of percentage of hair follicle types and quantitative evaluation of PCNA, Ki67 and P53 markers were performed. After IPL, there was significant decrease of hair count, HS diameter, percentage of terminal anagen follicles, terminal/vellus (T/V) ratio, anagen/telogen (A/T) ratio and expression of PCNA and Ki67; however, significant increase of percentage of terminal telogen and total vellus follicles with vellus-like type and P53 expression was identified. So, reduction of hair number and thickness occurred after IPL by induction of telogenesis and miniaturization through decreased hair follicle proliferation and increase in DNA damage that could favor apoptosis.

Fractional Microneedling: A Novel Method for Enhancement of Topical Anesthesia Before Skin Aesthetic Procedures.

BACKGROUND: Skin microneedling or fractional microneedle therapy is a recent approach used for skin rejuvenation or to enhance transdermal delivery of topical medications. OBJECTIVE: The authors evaluated the efficacy of skin microneedling, using an automated device, to enhance the numbing effect of topical anesthesia, used before minimally invasive aesthetic approaches. METHODS: Fifteen patients, looking for treatment of atrophic acne scars, were subjected to randomized split-face study comparing automated fractional skin microneedling (0.5 mm depth) followed by application of topical anesthetic cream (Lidocaine 2.5% + Prilocaine 2.5%) on one side of face, with topical anesthesia alone on the other side, followed by full face fractional microneedling treatment for postacne scars (2.5 mm depth). RESULTS: The treated sides (fractional needling + topical anesthesia) had significantly lower pain scores when compared with the nontreated sides (topical anesthesia alone). The scores of pain sensation, during the whole procedure, were statistically significantly (p < .0001) less on the treated sides (3.10 ± 1.09) of the face when compared with the nontreated sides (5.37 ± 0.99). There was also a statistically significant (p < .0001) difference in pain sensation scores between the 2 sides of the face after horizontal passes, as the mean scores of the treated and nontreated sides were 3.93 ± 0.59 and 6.20 ± 0.41, respectively. LIMITATIONS: The small number of patients, yet the results show a significant difference. CONCLUSION: Application of topical anesthesia for minimally invasive aesthetic procedures can be enhanced with fractional microneedling pretreatment.

Expression of apoptosis regulatory markers in the skin of advanced hepatitis-C virus liver patients.

BACKGROUND: Hepatitis-C virus (HCV) infection is considered a major worldwide public health problem with a global prevalence. Maintenance of skin homeostasis requires a delicate balance between proliferation, differentiation, and apoptosis. Meanwhile, it is unclear if there is an altered keratinocyte proliferation/apoptosis balance in advanced liver disease with HCV infection. AIM: This work aimed to evaluate the epidermal thickness and changes in the expression of apoptosis regulatory markers as well as apoptotic index in skin samples of advanced HCV liver patients compared to normal controls. MATERIALS AND METHODS: Twenty biopsies were taken from apparently normal skin of advanced HCV liver disease patients, as well as five healthy control subjects. These specimens were used for histometric epidermal measurement, immunohistochemical staining of apoptosis regulatory proteins (Bax, Fas, p53, Caspase-3, Bcl-2, Bcl-xL) as well as the TUNEL technique for detection of apoptotic cells. RESULTS: The mean epidermal thickness was significantly lower than the control group (P=0.000). There were significant overexpression of pro-apoptotic markers (Bax, Fas, P53, and Caspase-3) in patients (P=0.03, 0.03, 0.003, 0.003 respectively), with increased apoptotic index in HCV liver patients (P=0.002) when compared to normal controls. On the other hand, no statistically significant difference were encountered in the expression of antiapoptotic markers (Bcl-2, Bcl-xL) in HCV patients when compared to normal controls (P=0.5, 0.9, respectively). CONCLUSION: These findings suggest that an alteration in the proliferation/apoptosis balance is present in the skin of HCV liver patients.

Loose anagen hair syndrome in children of Upper Egypt.

BACKGROUND: Loose anagen hair (LAH) syndrome is a phenomenon in early childhood characterized by the presence of easily pluckable hair, where hair tufts can be pulled out easily and painlessly. AIMS: All reports in the English literature described mainly white patients with blond hair. We present the first report of LAH syndrome in dark-skinned children of Upper Egypt. PATIENTS AND METHODS: Twenty-eight children with LAH were diagnosed and examined from 1996 to 2007. The main complaints were patchy or diffuse alopecia and/or slow growth of hair. Clinical examination, hair pull test, trichogram, and scanning electron microscopy (SEM) were performed. RESULTS: Children with LAH included 21 girls (75%) and seven boys (25%). Light microscopy of hair pull tests and trichograms disclosed a striking predominance of anagen hairs (90-100%) with misshapen hair bulbs and absent inner and outer root sheaths. SEM confirmed the misshapen anagen bulbs with ruffled appearance of cuticle and the longitudinal groove parallel to the long axis of the hair shaft. Most children improved spontaneously within few years, however, hair shed continued. CONCLUSION: LAH syndrome occurs in dark-skinned children and could be under-diagnosed. The condition is of cosmetic concern and does not affect the general health.