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.

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).

Factors affecting the outcome of the suction blisters using two different harvesting techniques in vitiligo patients.

BACKGROUND: Suction blister grafting (SBG) technique has been used for long to treat various skin conditions. Different suction methods have been used such as syringes, Chinese cups, and suction device. There are some limitations of its use as time consumption, failure of induction or incomplete blister formation and pain. AIM: The aim of this work was to evaluate the outcome of using two different suction techniques, namely the syringes and the Chinese cups in induction of suction blisters. The effect of the device diameter and preheating of the donor area on the suction blister induction time (SBIT) was studied. The effect of saline injection in the blister formation and its completion was also evaluated. METHODS: The study was a left-right comparative study that included 50 patients with stable nonsegmental vitiligo. They were classified into four groups: Group 1 included 15 patients where different diameters of syringes (1.3, 1.7, and 2 cm) were compared against each other, group 2 included 15 patients where different diameters of cups (2, 3.5, and 5 cm) were compared against each other, group 3 included 20 patients subdivided into two groups; 10 patients each, where the effect of preheating the skin on blister induction was tested with use of cups (3a) and syringes (3b). Lastly, group 4 included randomly chosen 40 incomplete or multilocular blisters where the effect of saline injection on blister completion and coalescence of multilobulation was examined. SBIT was calculated in all patients. RESULTS: The use of the small diameter syringes or cups gave shorter SBIT; however, the difference, which was significant between all sizes of cups, was significant between the 1.3- and 2-cm-diameter syringes only. Preheating of the donor area shorten SBIT significantly. No complications were reported at the donor site except for transient postinflammatory hyperpigmentation in all patients. CONCLUSION: The small diameter syringes or cups and preheating of the donor area shorten the SBIT, while intra-blister saline injection increased the blister size and turns the multilocular blisters to unilocular ones.

Effect of Low-Level Environmental Lead Exposure on the Onset of Male Puberty.

The objective of this study was to evaluate male pubertal changes associated with environmental low-level lead (Pb) exposure. The study was conducted on 180 boys aged 15 years divided into 3 equal size groups: group 1 from El-Newayrat village, group 2 from Al-Shorafaa (0.5 and 10 km, respectively, from an industrialized area), and group 3 from Talla (25 km). Blood Pb levels (BLLs) were measured and pubertal changes evaluated by measurement of testicular volume (TV), and estimation of the follicle-stimulating hormone, luteinizing hormone, testosterone, estradiol, and prolactin. Blood Pb levels of children of El-Newayrat and Al-Shorafaa were significantly higher (6.38 [1.32] and 3.84 [0.79] µg/dL, respectively) than that of Talla children (1.85 [0.72]; P < 0.001), while height, weight, and TV were lower in boys in groups 1 and 2, compared to group 3. Genitalia and pubarche staging showed greatest retardation and marked bone growth delay in boys of group 1. Hormonal assays reported significant differences in boys of the industrialized areas when compared to that of Talla. Low-level Pb exposure in boys located near an industrial area was accompanied with altered male puberty indicators.

Syringes versus Chinese cups in harvesting suction-induced blister graft: a randomized split-body study.

BACKGROUND: Suction blister grafting (SBG) is a technique where the pigmented epidermis is harvested from the donor site by induction of a blister using different suction methods as syringes, Chinese cups, suction device, etc. However, pain, time consumption, incomplete blister formation, and failure of blister development are the main limitations. OBJECTIVE: To compare between cups and syringes of similar diameter in inducing suction blisters. PATIENTS AND METHODS: In 30 patients with stable nonsegmental vitiligo, 2-cm-diameter (20 ml) syringe was applied on the anterolateral aspect of one thigh and a 2-cm-diameter cup on the corresponding site of the other thigh where right and left sides were chosen randomly. Patients were observed untill complete blister development or for a maximum of 3 hours. Suction blister induction time (SBIT) and the blister diameter were recorded for each patient. Pain during the process of induction was evaluated. RESULTS: Incomplete blister development was noted in 9 out of 30 (30%) with 2 cm syringes and 6 out of 30 (20%) with the similar diameter cups with no significant difference (P = 0.49). No significant difference was found between SBIT induced by the 2 cm syringes and the similar size cups (101.17 ± 68.14 minutes, 98 ± 56.84 minutes, respectively) (P = 0.85). Meanwhile, blister diameter induced by either syringe or cup was not significantly different (P = 0.37). Anesthesia was for short duration with xylocaine, and pain was intolerable in both sides in the first seven patients. A combination of xylocaine and bupivacaine was used with prolonged loss of pain in 17 of the remaining 23 patients and tolerable pain in six patients similarly in both sides. CONCLUSION: According to present results, the differences in SIBT, diameter of blisters, and number of complete blister formation induced by either syringes or cups of similar size were not significant. Therefore, whatever the available and feasible technique for the surgeon will be the ideal choice. A combination of xylocaine and bupivacaine is recommended to overcome the accompanying pain of the procedure.

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.

Microneedling Therapy for Atrophic Acne Scars: An Objective Evaluation.

OBJECTIVE: Treatment of acne scarring is always a challenge. Microneedling therapy or percutaneous collagen induction is a new addition to the treatment modalities for such scars and has been reported to be simple and effective in atrophic acne scar treatment. The aim of this study is to evaluate the clinical effect and objectively quantify the histological changes of acne scarring in response to skin microneedling. DESIGN: A prospective clinical study. PARTICIPANTS: Ten patients with different types of atrophic acne scars were subjected to three months of skin microneedling treatment (six sessions at two-week intervals). MEASUREMENTS: Patients were photographed, and skin biopsies were obtained at baseline as well as one and three months from the start of treatment. Histometry for epidermal thickness and quantitative evaluation of total elastin; newly synthesized tropoelastin; collagen types I, III, and VII; and newly synthesized collagen were performed for all biopsies. RESULTS: Compared to the baseline, patients' evaluations revealed noticeable clinical improvement in atrophic post-acne scars in response to skin microneedling. There was a statistically significant increase (p<0.05) in the mean of collagen types I, III, and VII and newly synthesized collagen, while total elastin was significantly decreased (p<0.05) after the end of treatment. CONCLUSIONS: Multiple minimally invasive sessions of skin microneedling are an effective treatment for post-acne atrophic scars as it stimulates the repair processes with the advantage of being a relatively risk-free, in-office procedure with minimal patient recovery time.

Multiple microneedling sessions for minimally invasive facial rejuvenation: an objective assessment.

BACKGROUND: Microneedling or percutaneous collagen induction is a new modality used for skin rejuvenation, tightening, and scar remodeling. It offers a simple and effective treatment for photoaged skin with minimal disruption of the epidermis, thus limiting adverse effects and minimizing downtime. OBJECTIVES: To evaluate the efficacy, coupled with quantitative assessment, of the histological changes in response to multiple sessions of skin microneedling in the treatment of aging skin. PATIENTS AND METHODS: Ten patients with Fitzpatrick skin type III and IV and Glogau class II to III wrinkles were subjected to six skin microneedling sessions at 2-week intervals. Standard photographs and skin biopsy specimens were obtained at baseline and at one and three months after the start of treatment. Histometry for epidermal thickness and quantitative evaluation of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin were performed for all skin biopsies. RESULTS: Skin microneedling produced noticeable clinical improvement of photoaged skin, with corresponding histological enhancement. Compared to the baseline, collagen types I, III, and VII, as well as newly synthesized collagen, together with tropoelastin showed a statistically significant increase (P < 0.05) in response to treatment, while the mean level of total elastin was significantly decreased (P < 0.05) after treatment. CONCLUSIONS: Skin microneedling is a promising minimally invasive treatment option with the advantage of increased collagen production. However, multiple sessions are usually needed to maintain the improvement achieved.

Fractional versus ablative erbium:yttrium-aluminum-garnet laser resurfacing for facial rejuvenation: an objective evaluation.

BACKGROUND: Laser is one of the main tools for skin resurfacing. Erbium:yttrium-aluminum-garnet (Er:YAG) was the second ablative laser, after carbon dioxide, emitting wavelength of 2940 nm. Fractional laser resurfacing has been developed to overcome the drawbacks of ablative lasers. OBJECTIVE: We aimed to objectively evaluate the histopathological and immunohistochemical effects of Er:YAG 2940-nm laser for facial rejuvenation (multiple sessions of fractional vs single session of ablative Er:YAG laser). METHODS: Facial resurfacing with single-session ablative Er:YAG laser was performed on 6 volunteers. Another 6 were resurfaced using fractional Er:YAG laser (4 sessions). Histopathological (hematoxylin-eosin, orcein, Masson trichrome, and picrosirius red stains) and immunohistochemical assessment for skin biopsy specimens were done before laser resurfacing and after 1 and 6 months. Histometry for epidermal thickness and quantitative assessment for neocollagen formation; collagen I, III, and VII; elastin; and tropoelastin were done for all skin biopsy specimens. RESULTS: Both lasers resulted in increased epidermal thickness. Dermal collagen showed increased neocollagen formation with increased concentration of collagen types I, III, and VII. Dermal elastic tissue studies revealed decreased elastin whereas tropoelastin concentration increased after laser resurfacing. Neither laser showed significant difference between their effects clinically and on dermal collagen. Changes in epidermal thickness, elastin, and tropoelastin were significantly more marked after ablative laser. LIMITATIONS: The small number of patients is a limitation, yet the results show significant improvement. CONCLUSION: Multiple sessions of fractional laser have comparable effects to a single session of ablative Er:YAG laser on dermal collagen but ablative laser has more effect on elastic tissue and epidermis.

Efficacy of mesotherapy in facial rejuvenation: a histological and immunohistochemical evaluation.

BACKGROUND: Mesotherapy, commonly known as "biorejuvenation" or "biorevitalization", is a technique used to rejuvenate the skin by means of a transdermal injection of a multivitamin solution and natural plant extracts that are thought to improve the signs of skin aging. OBJECTIVES: This prospective study aimed to evaluate the clinical effect of mesotherapy applied to periorbital wrinkles and to quantitatively evaluate histological changes in the skin occurring in response to the same treatment. METHODS: Six volunteers with Fitzpatrick skin types III or IV and Glogau class I-III wrinkles were subjected to a three-month course of mesotherapy injections in the periocular area (six sessions administered at two-week intervals). Standard photographs and skin biopsies were obtained from the treatment area at baseline, at the end of treatment, and at three months post-treatment. Quantitative evaluation of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin was performed using a computerized morphometric analysis. RESULTS: The clinical evaluation of volunteers at baseline, end of treatment, and three months post-treatment revealed no significant differences. Histological and immunostaining analysis of collagen types I, III, and VII, newly synthesized collagen, total elastin, and tropoelastin showed no statistically significant changes (P > 0.05) after mesotherapy injection. CONCLUSIONS: The present study indicates that mesotherapy for skin rejuvenation does not result in statistically significant histological changes or clinical improvement.

The expression and distribution of deoxyribonucleic acid repair and apoptosis markers in testicular germ cells of infertile varicocele patients resembles that of old fertile men.

OBJECTIVE: To explore the expression and distribution of DNA damage repair and apoptosis marker proteins in human testicular germ cells of infertile varicocele patients; and to compare the expression and distribution with that of young and old fertile men. DESIGN: Retrospective case-control study. SETTING: Academic institutions. PATIENT(S): Testicular specimens were obtained from 8 infertile varicocele patients aged 20-30 years and from 16 fertile volunteers aged 20-82 years. INTERVENTION(S): Testicular germ cell DNA repair markers were assessed using immunohistochemical staining for the cell proliferation marker (proliferating cell nuclear antigen), DNA repair markers [poly(ADP-ribose) polymerase-1 (PARP-1), poly(ADP-ribose), X-ray repair cross-complementing 1, and apurinic/apyrimidinic endonuclease 1], and apoptosis markers (caspase 9, active caspase 3, and cleaved PARP-1). MAIN OUTCOME MEASURE(S): The prevalence and cellular localization of the above markers in testicular tissues of varicocele patients and fertile men of varying ages. RESULT(S): Statistically significant differences in DNA damage repair-associated proteins and apoptosis markers were observed in infertile men with varicocele compared with fertile young men. Old fertile men showed similar expression of the same markers when compared with infertile varicocele patients. CONCLUSION(S): The study demonstrates that there is an increase in human testicular germ cell DNA repair and apoptosis in infertile varicocele patients and that their profile resembles that of premature aging.

Deoxyribonucleic acid repair and apoptosis in testicular germ cells of aging fertile men: the role of the poly(adenosine diphosphate-ribosyl)ation pathway.

OBJECTIVE: To explore the relationship between men's age and DNA damage repair proteins related to apoptosis in human testicular germ cells. DESIGN: Retrospective case-control study. SETTING: Academic institutions. PATIENT(S): Testicular specimens were obtained from 22 fertile volunteers aged 20-82 years. INTERVENTION(S): Deoxyribonucleic acid repair markers were assessed using immunohistochemical staining for the cell proliferation marker [proliferating cell nuclear antigen (PCNA)]; DNA repair markers [poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), poly(adenosine diphosphate-ribose) (PAR), X-ray repair cross-complementing1(XRCC1), and apurinic/apyrimidinic endonuclease 1 (APE1)]; and apoptosis-associated markers (caspase 9, active caspase 3, and cleaved PARP-1). MAIN OUTCOME MEASURE(S): The prevalence and cellular localization of the above markers in testicular tissues of young, middle aged, and old men. RESULT(S): Statistically significant differences in DNA damage repair-associated proteins (PARP-1, PAR, XRCC1, and APE1), and apoptosis markers (caspase 9, active caspase 3, and cleaved PARP-1) were observed in testicular samples from older men. These differences were most marked in spermatocytes. CONCLUSION(S): The study demonstrates that there is an age-related increase in human testicular germ cell DNA break repair and apoptosis with age.