Development of an artificial portal vein using bioabsorbable polymers.

PURPOSE: During surgical resection of malignant tumors in the hepatobiliary pancreatic region, portal vein resection and reconstruction may be needed. However, there is no alternative to the portal vein. We therefore developed an artificial portal vein that could be used in the abdominal cavity. METHODS: In the experiments, hybrid pigs (n = 8) were included. An artificial portal vein was created using a bioabsorbable polymer sheet (BAPS). Subsequently, the portal vein's anterior wall was excised into an elliptical shape. A BAPS in the form of a patch was implanted at the same site. At 2 weeks (n = 3) and 3 months (n = 5) after the implantation, the BAPS implantation site was resected and evaluated macroscopically and histopathologically. RESULTS: Immediately after the implantation, blood leakage was not detected. Two weeks after implantation, the BAPS remained, and endothelial cells were observed. Thrombus formation was not observed. Three months after implantation, the BAPS had been completely absorbed and was indistinguishable from the surrounding portal vein. Stenosis and aneurysms were not observed. CONCLUSIONS: BAPS can replace a defective portal vein from the early stage of implantation to BAPS absorption. These results suggest that it can be an alternative material to the portal vein in surgical reconstruction.

Sequential Scalp Assessment in Hair Regeneration Therapy Using an Adipose-Derived Stem Cell-Conditioned Medium.

BACKGROUND: An adipose-derived stem cell-conditioned medium (ADSC-CM) reportedly exerts skin-rejuvenating and hair growth-promoting effects. In the therapeutic application of ADSC-CM for alopecia, changes to the interfollicular scalp remain unclear although some evidence has indicated hair growth-promoting effects. OBJECTIVE: To evaluate the effects of ADSC-CM not only on hair follicles, but also on the interfollicular scalp. METHODS: Forty patients (21 men, 19 women; age range, 23-74 years) with alopecia were treated by intradermal injection of ADSC-CM every month for 6 months. Eighty fixed sites on patients were investigated by trichograms, physiological examinations, and ultrasonographic examinations at 4 time points (before treatment and 2, 4, and 6 months after the initial treatment). RESULTS: Hair density and anagen hair rate increased significantly. As physiological parameters, transepidermal water loss value gradually increased, with significant differences at 4 and 6 months after the initial treatment, but hydration state of the stratum corneum and skin surface lipid level showed no obvious changes. As ultrasonographic parameters, dermal thickness and dermal echogenicity were increased significantly. CONCLUSION: Intradermal administration of ADSC-CM on the scalp has strong potential to provide regenerative effects for hair follicles and the interfollicular scalp. An adipose-derived stem cell-conditioned medium offers a promising prospect as an alternative treatment for alopecia.

Scar Assessment After Breast Reconstruction: Risk Factors for Hypertrophy and Hyperpigmentation in Asian Patients.

BACKGROUND: Breast scars after breast reconstruction can be hypertrophic and/or hyperpigmented, especially in Asian patients, whose skin is thicker and has increased melanin. Few studies have focused on breast scars after breast reconstruction, and the risk factors for an abnormal breast scar remain unknown. METHODS: We examined 257 Asian patients who underwent an immediate 2-stage unilateral implant-based breast reconstruction. Vascularity, hypertrophy, and hyperpigmentation of the patients' breast scars were assessed at 1 year postoperatively. Risk factors for an abnormal scar were analyzed statistically. Analyzed patient factors included age, body mass index, incision site (frontal or lateral), breast size (the weight of the resected specimen), skin necrosis at the initial operation (expander placement), and adjuvant therapy. RESULTS: At 1 year postoperatively, 161 patients (63%) showed normal vascularity, 77 patients (30%) showed mild vascularity, 18 patients (7%) showed moderate vascularity, and 1 patient (0.4%) showed severe vascularity. No patient factors were correlated with vascularity. Thirty-two patients (12%) showed hypertrophy, and the rate of hypertrophy was significantly higher in the patients with a lateral incision (n = 59) compared with those with a frontal incision (n = 198) (28.8% vs 7.6%, P < 0.01). Even in the frontal incision group, a lateral part of the frontal scar was likely to be hypertrophic. Forty-six patients (18%) showed hyperpigmentation, and the rate of hyperpigmentation was significantly higher in the patients with skin necrosis (n = 47) at the initial operation than those without skin necrosis (n = 210) (57.4% vs 9.0%, P < 0.01). Large breast was also a risk factor for hyperpigmentation due to its higher frequency of skin necrosis. CONCLUSIONS: In Asian patients who undergo breast reconstruction, the use of a lateral incision is a risk factor for hypertrophy, and skin necrosis at the initial operation is a risk factor for hyperpigmentation at the breast scar.

Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-ß1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population.

Risk Factors for Skin Flap Necrosis in Breast Cancer Patients Treated with Mastectomy Followed by Immediate Breast Reconstruction.

BACKGROUND: Skin-sparing mastectomy (SSM) and nipple-sparing mastectomy (NSM) are the standard techniques for achieving a cosmetic outcome, but necrosis of a cutaneous flap including the nipple-areolar complex (NAC) is a serious complication. To analyze the risk factors for skin flap necrosis, we retrospectively evaluated a clinical database of breast cancer patients treated with mastectomy followed by immediate breast reconstruction. METHODS: Four hundred and twelve cases were consecutively recorded between 2006 and 2016. Body weight (BW), body mass index (BMI), distance from NAC to referent tumor, distance from overlying skin to the tumor and weight of breast resection (WBR) as measured in the operating theater were included in the statistical analysis. RESULTS: NSM, SSM and total mastectomy were performed in 123 (30%), 96 (23%) and 193 cases (47%), respectively. A tissue expander was used in 379 cases (92%), a silicone implant in 8 (2%) and autologous breast reconstruction in 25 (6%). Skin flap necrosis was found in 7% of all cases and NAC necrosis in 13% of NSM cases. In a univariate analysis, BW, NSM and WBR were risk factors for skin flap necrosis, and BW, BMI and WBR were risk factors for NAC necrosis. In a multivariate analysis, NSM and WBR remained significant risk factors for skin flap necrosis, and WBR was a significant risk factor for NAC necrosis. CONCLUSIONS: WBR is an important risk factor for skin flap necrosis. Especially, NAC necrosis should be considered for patients with large-volume breasts who undergo NSM and immediate breast reconstruction.

Liposarcoma Preoperatively Diagnosed as Lipoma: 10-Year Experience at a Single Institution.

BACKGROUND: On rare occasions, a lesion preoperatively diagnosed as a lipoma is ultimately diagnosed as a liposarcoma. It is important to differentiate liposarcomas from lipomas preoperatively. OBJECTIVE: To examine characteristic features of liposarcomas preoperatively diagnosed as lipomas. METHODS: Patients (n = 637) who underwent resection of tumors preoperatively diagnosed as lipomas from January 2006 to October 2016 were retrospectively reviewed. RESULTS: Based on pathological examination, 8 of 637 lesions were diagnosed as liposarcomas postoperatively. All the liposarcomas were well-differentiated liposarcomas. The rate of male patients was higher (87.5% vs 38.9%) and the size of tumors was larger (8.75 vs 4.64 cm) in these cases than in accurately diagnosed lipoma cases. On imaging, nonfatty septa were more frequently observed (71.4% vs 20.0%) and were thicker (2.22 vs 1.33 mm) than in true lipoma cases. CONCLUSION: If the patient with a lipomatous tumor is male and the tumor is large, we should consider the possibility of it being a liposarcoma. A thick internal septum in the image findings is a good predictor of malignancy.

Comparison of Nasal Septum and Ear Cartilage as a Graft for Lower Eyelid Reconstruction.

In lower eyelid reconstruction, several types of grafts from the nasal septum, ear cartilage, buccal mucosa, and hard palate mucosa have been used for an inner layer of the lower eyelid, but there have been no studies comparing these grafts. The authors retrospectively reviewed our cases of lower eyelid reconstruction, and compared chondromucosal grafts from the nasal septum (N = 8) and ear cartilage grafts (N = 10) for an inner layer of the lower eyelid. The authors observed no significant difference in operative time, blood loss, or length of hospital stay between the "nasal septum" and "ear cartilage" groups. The final results were aesthetically and functionally satisfactory in both groups. In the nasal septum group, 1 patient suffered from perforation of the nasal septum and another patient suffered from nasal bleeding postoperatively. There were no donor site complications in the ear cartilage group. These findings indicate that both a chondromucosal graft from the nasal septum and an ear cartilage graft are good grafts for an inner layer of the lower eyelid. Regarding the donor site, however, an ear cartilage graft has the advantage of a lower complication rate.

Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing.

Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing.

Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation.

INTRODUCTION: Adipose-derived stem cells (ASCs) have shown potential for cell-based therapy in the field of plastic surgery. However, the fate of ASCs after transplantation and the mechanism(s) of their biologic capabilities remain unclear. METHODS: We isolated and cultured ASCs from transgenic mice that express both luciferase and green fluorescent protein and injected the cells into the inguinal fat pads of wild-type mice. We tested 4 experimental groups, namely, ischemic fat pads with/without ASCs and control fat pads with/without ASCs. RESULTS: Transplanted ASCs were tracked with bioluminescence imaging. The luminescence gradually decreased over 28 days, indicating cell death after transplantation. More ASCs were retained in ischemic fat pads on day 7 compared to control fat pads. On day 14, adipose tissue vascular density was higher in the ASC transplantation groups compared to those without ASCs. On day 28, there was decreased atrophy of adipose tissue in ASC-treated ischemic fat pads. Transplanted ASCs were detected as nonproliferating green fluorescent protein-positive cells, whereas native endothelial cells adjacent to the transplanted ASCs were proliferative. Protein analysis demonstrated higher expression of hepatocyte growth factor and vascular endothelial growth factor in the ASC transplantation groups, suggesting a paracrine mechanism, which was confirmed by in vitro experiments with conditioned media from ASCs. CONCLUSIONS: Transplanted ASCs are preferentially retained in ischemic adipose tissue, although most of the cells eventually undergo cell death. They exert an angiogenic effect on adipose tissue mainly through a paracrine mechanism. Increased understanding of these effects will help develop ASCs as a tool for cell-based therapy.

Efficacy of serial excisions of melanocytic nevi on the face using a carbon dioxide laser: a cosmetic point of view.

UNLABELLED: In cosmetic clinical practice, many patients express the desire for removal of melanocytic nevi, especially those on the face. The carbon dioxide (CO2) laser currently is the preferred for treatment of such lesions because of less scar formation, less bleeding, and simplicity of the procedure. However, if the diameter of the lesion is greater than 5 mm, many clinicians prefer surgical resection to laser resection because laser resection of relatively large nevi often results in the formation of a conspicuous dimple. The authors developed a serial laser excision method for melanocytic nevi larger than 5 mm, with division of the lesion into multiple segments, which allowed the ablated area to gain optimal granulation and reepithelialization, leading to a satisfactory cosmetic appearance. This novel procedure was performed for 25 patients with melanocytic nevi ranging from 5 to 10 mm in diameter. The lesions were divided into two segments in 21 patients, three segments in 3 patients, and four segments in 1 patient. The divided parts of the lesions were ablated by CO2 laser serially at intervals of 2-4 weeks. All the patients obtained optimal granulation and epithelialization in the treated lesions, and the final appearance was satisfactory. Although the therapeutic period was longer than for a surgical excision or a single laser treatment, the new treatment approach of serial excision by CO2 laser achieved favorable outcomes for the treatment of relatively large nevi 5-10 mm in size. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Therapeutic potential of fibroblast growth factor-2 for hypertrophic scars: upregulation of MMP-1 and HGF expression.

Although hypertrophic scars (HTSs) and keloids are challenging problems, their pathogenesis is not well understood, making therapy difficult. We showed that matrix metalloproteinase (MMP)-1 expression was downregulated in HTS compared with normal skin from the same patients, whereas type 1 and 3 collagen and transforming growth factor-ß (TGF-ß) were upregulated. These differences, however, were not seen in cultured fibroblasts, suggesting the involvement of microenvironmental factors in the pathogenesis of HTS. Fibroblast growth factor-2 (FGF-2) highly upregulated the expression of MMP-1 and hepatocyte growth factor (HGF) in both HTS-derived and control fibroblasts; the upregulation was reversed by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors. An animal study using human HTS tissue implanted into nude mice indicated that controlled-release FGF-2 resulted in significantly less weight and decreased hydroxyproline content in HTS. Degradation of collagen fibers in FGF-2-treated HTS was also confirmed histologically. Western blotting showed that FGF-2-treated HTS expressed significantly higher MMP-1 protein than control. Decreased MMP-1 expression may be an important transcriptional change in HTS, and its reversal as well as upregulation of HGF by FGF-2 could be a new therapeutic approach for HTS.

Adipose injury-associated factors mitigate hypoxia in ischemic tissues through activation of adipose-derived stem/progenitor/stromal cells and induction of angiogenesis.

Based on the analysis of exudates from injured adipose tissue, we prepared a mixture containing the injury-associated growth factors at the same proportion as the exudates, named adipose injury cocktail (AIC). We hypothesized that AIC induces a series of regenerating and angiogenic processes without actual wounding. The purpose of this study is to elucidate the therapeutic potentials of AIC. AIC preferentially activated adipose-derived stem/progenitor/stromal cells (ASCs) to proliferate, migrate, and form networks compared with vascular endothelial cells, whereas vascular endothelial growth factor did not induce mitogenesis or chemotaxis in human ASCs. Each component growth factor of AIC was differently responsible for the ASC activation. AIC-treated ASCs tended to differentiate into adipocytes or vessel-constituting cells rather than into other cell types. In ischemic adipose tissues of mice, induced by either a surgical intervention or diabetes, AIC administration enhanced proliferation, especially of CD31(-)/CD34(+) ASCs, and mitigated tissue hypoxia by increasing capillary density and reducing fibrogenesis. These results suggest that AIC may have therapeutic potentials for various ischemic/hypoxic conditions by inducing adipose remodeling and neovascularization through activation of ASCs and other cells. Treatment with AIC has many advantages over cell-based therapies regarding morbidity, cost, and physical risks and may be used as an alternative therapy for improving tissue oxygen.

The aspect ratio of the palpebral fissure as a new blepharoptosis parameter.

Although the margin reflex distance (MRD) is widely used to assess blepharoptosis, it has some drawbacks (e.g. inaccuracy in severe ptosis). A new parameter is desired. We digitally analyzed pre- and post-operative photographs of 95 patients with blepharoptosis. We set a rectangle with the vertical sides at the lateral and medial canthus and the horizontal sides at the highest and lowest points of the eyelids. We calculated the percentage of the vertical side (height) to the horizontal side (width) and defined this value as the vertical percentage of the palpebral fissure. The MRD and the vertical percentage values were strongly correlated (correlation coefficient 0.766). In the 77 bilateral cases, both sides showed significant improvement in vertical percentage (from 28.9 to 37.3%, right) and (28.7 to 36.1%, left). In the 18 unilateral cases, the affected side showed significant improvement in vertical percentage (from 29.6 to 38.7%), while the unaffected side showed no change. In the eight patients who underwent re-operation, the revised side's vertical percentage was not improved after the first operation (from 28.0 to 31.3%), and the revision significantly changed the vertical percentage to 39.0%. In the re-operated patients, the difference between the right and left sides decreased significantly from 6.7 to 1.9% post-revision. The aspect ratio of each palpebral fissure (the percentage of height to width) reflected the progression of blepharoptosis and the post-operative changes. The aspect ratio thus has the potential to be a new parameter for blepharoptosis.

Human CD206+ Macrophages Show Antifibrotic Effects on Human Fibroblasts through an IL-6-Dependent Mechanism In Vitro.

BACKGROUND: Pathologic scarring including keloid and hypertrophic scar causes aesthetic and physical problems, and there are clinical difficulties (e.g., posttreatment recurrence) in dealing with pathologic scarring. Understanding the mechanisms that underlie scar control in wound healing will help prevent and treat pathologic scarring. The authors focused on CD206+ macrophages in the wound-healing process, and hypothesized that CD206+ macrophages have antifibrotic effects on fibroblasts. METHODS: The authors established a co-culture system for CD206+ macrophages and fibroblasts (cell ratio, 1:1). The authors examined the CD206+ macrophages' antifibrotic effects on fibroblasts after a 72-hour culture, focusing on fibrosis-related genes. To identify key factor(s) in the interaction between CD206+ macrophages and fibroblasts, the authors analyzed cytokines in a conditioned medium of the co-culture system. RESULTS: Under co-culture with CD206+ macrophages, expression of the following in the fibroblasts was significantly down-regulated: type 1 (fold change, 0.38) and type 3 collagen (0.45), alpha smooth muscle actin (0.24), connective tissue growth factor (0.40), and transforming growth factor-beta (0.66); the expression of matrix metalloproteinase 1 was significantly up-regulated (1.92). Conditioned medium in the co-culture showed a high interleukin (IL)-6 concentration (419 ± 88 pg/ml). When IL-6 was added to fibroblasts, antifibrotic changes in gene expression (as observed under the co-culture) occurred in the fibroblasts. CONCLUSIONS: The authors' in vitro results revealed that CD206+ macrophages have antifibrotic effects on fibroblasts by means of a paracrine mechanism involving IL-6. Understanding these effects, especially in vivo, will help elucidate the mechanism of scar control in wound healing and contribute to the development of new scar treatments.

IFATS collection: Fibroblast growth factor-2-induced hepatocyte growth factor secretion by adipose-derived stromal cells inhibits postinjury fibrogenesis through a c-Jun N-terminal kinase-dependent mechanism.

Adipose-derived stem/stromal cells (ASCs) not only function as tissue-specific progenitor cells but also are multipotent and secrete angiogenic growth factors, such as hepatocyte growth factor (HGF), under certain circumstances. However, the biological role and regulatory mechanism of this secretion have not been well studied. We focused on the role of ASCs in the process of adipose tissue injury and repair and found that among injury-associated growth factors, fibroblast growth factor-2 (FGF-2) strongly promoted ASC proliferation and HGF secretion through a c-Jun N-terminal kinase (JNK) signaling pathway. In a mouse model of ischemia-reperfusion injury of adipose tissue, regenerative changes following necrotic and apoptotic changes were seen for 2 weeks. Acute release of FGF-2 by injured adipose tissue was followed by upregulation of HGF. During the adipose tissue remodeling process, adipose-derived 5-bromo-2-deoxyuridine-positive cells were shown to be ASCs (CD31-CD34+). Inhibition of JNK signaling inhibited the activation of ASCs and delayed the remodeling process. In addition, inhibition of FGF-2 or JNK signaling prevented postinjury upregulation of HGF and led to increased fibrogenesis in the injured adipose tissue. Increased fibrogenesis also followed the administration of a neutralizing antibody against HGF. FGF-2 released from injured tissue acts through a JNK signaling pathway to stimulate ASCs to proliferate and secrete HGF, contributing to the regeneration of adipose tissue and suppression of fibrogenesis after injury. This study revealed a functional role for ASCs in the response to injury and provides new insight into the therapeutic potential of ASCs.

Progenitor-enriched adipose tissue transplantation as rescue for breast implant complications.

Breast enhancement with artificial implants is one of the most frequently performed cosmetic surgeries but is associated with various complications, such as capsular contracture, that lead to implant removal or replacement at a relatively high rate. For replacement, we used transplantation of progenitor-supplemented adipose tissue (cell-assisted lipotransfer; CAL) in 15 patients. The stromal vascular fraction containing adipose tissue progenitor cells obtained from liposuction aspirates was used to enrich for progenitor cells in the graft. Overall, clinical results were very satisfactory, and no major abnormalities were seen on magnetic resonance imaging or mammogram after 12 months. Postoperative atrophy of injected fat was minimal and did not change substantially after 2 months. Surviving fat volume at 12 months was 155 +/- 50 mL (Right; mean +/- SD) and 143 +/- 80 mL (Left) following lipoinjection from an initial mean of 264 mL. These preliminary results suggest that CAL is a suitable methodology for the replacement of breast implants.

TGF-beta is specifically expressed in human dermal papilla cells and modulates hair folliculogenesis.

Dermal papilla cells (DPCs) in the mammalian hair follicle have been shown to develop hair follicles through epithelial-mesenchymal interactions. A cell therapy to regenerate human hair is theoretically possible by expanding autologous human DPCs (hDPCs) and transplanting them into bald skin, though much remains to be overcome before clinical success. In this study, we compared gene signatures of hDPCs at different passages and human dermal fibroblasts, and found transforming growth factor (TGF)-beta(2) to be highly expressed in cultured hDPCs. Keratinocyte conditioned medium, which is known to help preserve the hair-inducing capacity of hDPCs, up-regulated TGF-beta(2) expression of hDPCs and also enhanced their alkaline phosphatase (ALP) activity, a known index for hair-inductive capacity. Through screening of components secreted from keratinocytes, the vitamin D(3) analogue was found to promote TGF-beta(2) expression and ALP activity of hDPCs. In animal hair folliculogenesis models using rat epidermis and expanded hDPCs, inhibition of TGF-beta(2) signalling at the ligand or receptor level significantly impaired hair folliculogenesis and maturation. These results suggest an important role for TGF-beta(2) in hair follicle morphogenesis and provide insights into the establishment of future cell therapies for hair regrowth by transplanting expanded DPCs.

Differential expression of stem-cell-associated markers in human hair follicle epithelial cells.

Several putative biomarkers have been suggested for identifying murine follicular stem cells; however, human hair follicles have a different pattern of biomarker expression, and follicular stem cell isolation methods have not been established. To isolate a stem cell population applicable to clinical settings, we conducted a comprehensive survey of the expression of stem-cell-associated (K15, CD200, CD34, and CD271) and other biomarkers (K1, K14, CD29, and CD49f) in immunohistological sections of the human epidermis and follicular outer root sheath (ORS). We also examined freshly isolated and cultured epidermal or follicular cells with single- and multicolor flow cytometry or immunocytochemistry. After sorting cells by CD200, CD34, and forward scatter (FSC) values (cell size), colony-forming assays were performed. We found that biomarkers were differentially expressed in the epidermis and ORS. Basal bulge cells were mainly K15+CD200+CD34(-)CD271(-), and suprabasal cells were K15(-)CD200+CD34(-)CD271(-). We categorized follicular cells into nine subpopulations according to biomarker expression profiles. The CD200+CD34(-) bulge cells had much higher colony-forming abilities than the CD34+ population, and were divided into two subpopulations: a CD200+CD34(-)FSC(high) (K15-rich, basal) and a CD200+CD34(-)FSC(low) (K15-poor, suprabasal) population. The former formed fewer but larger-sized colonies than the latter. Follicular epithelial cell cultivation resulted in loss of K15, CD200, CD34, and CD271 expression, but maintenance of K14, CD29, and CD49f expression. We found that the bulge contained two populations with different localizations, cell sizes, and colony-forming abilities. We showed that K15, CD200, CD34, and CD271 were useful biomarkers for characterizing freshly isolated human follicular epithelial cells in diverse stages of differentiation.

Evaluation of animal models for the hair-inducing capacity of cultured human dermal papilla cells.

BACKGROUND: The dermal papilla (DP) interacts with epithelial cells for folliculogenesis. For translational research on cell therapies for hair regrowth with cultured human DP cells (hDPCs), a model to evaluate the capacity of hDPCs to induce hair formation is inevitable. METHODS: Chamber models were constructed by transplanting 4 different combinations of mouse or human epithelial and mesenchymal cells into a silicone chamber implanted onto the back of nude mice. In parallel, 3 types of sandwich constructs were created by inserting hDPCs or human DP tissue between the epidermis and dermis of isolated rat footpad skin or human facial skin, and subcutaneously transplanting the constructs into the back of nude mice. Four to six weeks later, skin sections of each model were histologically examined. RESULTS: Folliculoneogenesis was detected in both chamber and sandwich models, although the induction rate and maturity of the hair follicles varied among cell combination subgroups in each model. The difference in hair induction rate was not statistically significant between 2 representative chamber and sandwich subgroups using cultured hDPCs. The sandwich model, however, required fewer hDPCs, did not require human keratinocytes, and exhibited a higher rate of successful sample collection. CONCLUSIONS: Although there is no significant difference in hair induction rate, the sandwich model using cultured hDPCs and the rat sole skin is more feasible than the chamber model using human cultured keratinocytes and hDPCs as a tool to evaluate the hair-inducing capacity of cultured hDPCs.