The Experts below are selected from a list of 327 Experts worldwide ranked by ideXlab platform
Thomas Hunziker - One of the best experts on this subject based on the ideXlab platform.
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repigmentation by Outer Root Sheath derived melanocytes proof of concept in vitiligo and leucoderma
Dermatology, 2009Co-Authors: Wolfgang Vanscheidt, Thomas HunzikerAbstract:Background: Treatment of depigmented skin is an unmet medical need. Objective: Melanocytes or stem cells thereof residing in the Outer Root Sheath (ORS) of hair follicles might be used to repigment skin. Methods: After de-epidermisation, autologous ORS cell solutions were applied to 5 patients with vitiligo and 1 with leucoderma. Results: Stable repigmentation in a variable percentage was documented in all the patients. Conclusion: Applying ORS-derived melanocytes is a promising technology to improve autologous melanocyte transplantation.
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experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle Outer Root Sheath and matrix cells
Annals of the New York Academy of Sciences, 2006Co-Authors: A Limat, Thomas Hunziker, Dirk Breitkreutz, Friedrich Noser, Hansjurgen Stark, Gabi Thikoetter, Norbert E FusenigAbstract:Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While Outer Root Sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner Root Sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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comparative analysis of surface antigens in cultured human Outer Root Sheath cells and epidermal keratinocytes persistence of low expression of class i mhc antigens in Outer Root Sheath cells in vitro
British Journal of Dermatology, 2006Co-Authors: A Limat, Thomas Hunziker, Tony Wysscoray, L R BraathenAbstract:Summary In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, Outer Root Sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular Outer Root Sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.
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use of epidermal equivalents generated from follicular Outer Root Sheath cells in vitro and for autologous grafting of chronic wounds
Cells Tissues Organs, 2002Co-Authors: Alain Limat, Thomas HunzikerAbstract:During wound healing, Outer Root Sheath (ORS) cells of hair follicles can substitute for interfollicular epidermal keratinocytes and thus act as precursor cells for interfollicular epidermal keratinocytes. Owing to improved culture techniques, ORS cells can be induced to develop highly differentiated epidermal equivalents, which are close to the normal human epidermis in terms of histological, ultrastructural, biochemical and immunohistological criteria. Such epidermal equivalents provide a versatile system for various applications in vitro, e.g. the study of epidermal homeostasis, cell interactions, pigmentation as well as toxicity testing and metabolism of xenobiotics. The easy and repeated availability of ORS cells, their successful multiplication in culture irrespective of the age of the hair follicle donor as well as the extended tissue normalization of epidermal equivalents prepared with ORS cells prompted us to test the usefulness of autologous epidermal equivalents for the treatment of recalcitrant chronic wounds. Autologous grafting of such epidermal equivalents in more than 50 recalcitrant leg ulcers of a mainly vascular origin resulted in an initial take rate of around 90%, with subsequent complete closure of the ulcers in about 45% and a significant size reduction in another 40% within 8 weeks. These positive results are probably due to the large compartment of proliferative cells as well as to the well-developed horny layer, which prevents rapid disintegration of the grafts. Practical advantages of this technology are its noninvasiveness and thus repeated availability, the fact that surgical facilities are not necessary and the short immobilization period after grafting, allowing a strategy of sequential application in an outpatient setting as an alternative to surgical autografting.
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human melanocytes grown in epidermal equivalents transfer their melanin to follicular Outer Root Sheath keratinocytes
Archives of Dermatological Research, 1999Co-Authors: A Limat, Denis Salomon, Pierre Carraux, Jeanhilaire Saurat, Thomas HunzikerAbstract:Because Outer Root Sheath (ORS) cells are valuable substitutes for interfollicular epidermal keratinocytes, we wanted to determine whether epidermal equivalents generated from ORS cells and containing cultured melanocytes can serve as an in vitro model for skin pigmentation. In such epidermal equivalents prepared with ORS cells and melanocytes from donors of phototypes II, III and VI, a stratified epithelium resembling normal epidermis developed within 14 days, as documented by histological, ultrastructural (e.g. basement membrane-like structure, keratohyalin granules, keratinosomes) and immunohistochemical (e.g. keratins, integrins, gp80, involucrin, filaggrin) criteria. The melanocytes were localized in the basal layer and accounted for 10% of the total cell number. Heavily pigmented melanocytes from black donors contained regular melanosomes in all stages of maturation, whereas melanocytes derived from white donors contained predominantly melanosomes of stages I and II. Melanosome-laden dendrites were readily detected extending from the heavily pigmented melanocytes, while they were less conspicuous in melanocytes from white donors. The extent of melanosome transfer was independent of the racial origin of the ORS cells. Melanosomes could also be transferred "through racial barriers". Melanosomes, mainly of stages III and IV, were detected in the ORS cells, being distributed either as single or compound melanosomes, again irrespective of the racial origin of the ORS cells. In conclusion, pigmented epidermal equivalents generated from ORS cells offer practical advantages over other in vitro pigmentation models: (1) the ORS cells are easily and repeatedly available from any donor regardless of age; (2) primary cultures of ORS cells are free of contaminating melanocytes, a bias if using interfollicular epidermal keratinocytes; (3) a high degree of epidermal differentiation is maintained for 3 weeks in fully defined medium, enabling labelling and stimulation experiments to be performed and compounds interfering with melanin pigmentation to be tested.
A Limat - One of the best experts on this subject based on the ideXlab platform.
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experimental modulation of the differentiated phenotype of keratinocytes from epidermis and hair follicle Outer Root Sheath and matrix cells
Annals of the New York Academy of Sciences, 2006Co-Authors: A Limat, Thomas Hunziker, Dirk Breitkreutz, Friedrich Noser, Hansjurgen Stark, Gabi Thikoetter, Norbert E FusenigAbstract:Follicles of human anagen hair were separated into morphologically distinct compartments (by sequential trypsinization and microdissection) for the biochemical and immunological analysis of keratins as differentiation markers to diagnose the type of epithelial differentiation. While Outer Root Sheath contained throughout the "soft" (cyto)keratins K5, 6, 14, 16, and 17, and hair cortex contained exclusively a set of acidic and basic "hard" alpha-keratins (consistent up to the hair tip), in inner Root Sheath and hair cuticle peptides related or derived from suprabasal epidermal keratins K1 and 10 were detected. These keratin profiles served as in vivo correlates for the evaluation of type and degree of differentiation achieved by the respective isolated epithelial cells, comparing different growth or culture conditions. Cultures of ORS cells and hair matrix cells (PHS cells) as well as normal keratinocytes were initiated using postmitotic human dermal fibroblasts as efficient feeder cells. On lifted collagen gels populated with HDF ("surface" cultures), ORS and PHS cells formed stratified epithelial expressing epidermal differentiation markers such as keratins K1 and 10, involucrin, and filaggrin. Compared with NEK "surface" cultures, balance between growth and differentiation was better maintained by both follicular cell types. In contrast, epidermal tissue homeostasis was largely normalized in transplants on nude mice regardless of the epithelial cell type, apparent from orderly tissue structure, regular distribution of keratin K10, filaggrin, and involucrin, and distinct continuous deposition of basement membrane components at the epithelium-collagen interface. Embedded in Matrigel (on top of HDF collagen gels) ORS cells and NEK formed spheroids exhibiting inward-directed epidermoid differentiation, increasing with time. All epidermal maturation products found in "surface" cultures were likewise expressed, and again differentiation greatly outbalanced proliferation in spheroids of NEK but not of ORS cells. PHS cells embedded together with HDF in Matrigel produced similar spheroids as ORS cells. Size of spheroids and degree of epidermoid differentiation were dramatically reduced when HDF were replaced by follicular DP cells, demonstrating the crucial role of the mesenchymal "companion" cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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comparative analysis of surface antigens in cultured human Outer Root Sheath cells and epidermal keratinocytes persistence of low expression of class i mhc antigens in Outer Root Sheath cells in vitro
British Journal of Dermatology, 2006Co-Authors: A Limat, Thomas Hunziker, Tony Wysscoray, L R BraathenAbstract:Summary In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, Outer Root Sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular Outer Root Sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.
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human melanocytes grown in epidermal equivalents transfer their melanin to follicular Outer Root Sheath keratinocytes
Archives of Dermatological Research, 1999Co-Authors: A Limat, Denis Salomon, Pierre Carraux, Jeanhilaire Saurat, Thomas HunzikerAbstract:Because Outer Root Sheath (ORS) cells are valuable substitutes for interfollicular epidermal keratinocytes, we wanted to determine whether epidermal equivalents generated from ORS cells and containing cultured melanocytes can serve as an in vitro model for skin pigmentation. In such epidermal equivalents prepared with ORS cells and melanocytes from donors of phototypes II, III and VI, a stratified epithelium resembling normal epidermis developed within 14 days, as documented by histological, ultrastructural (e.g. basement membrane-like structure, keratohyalin granules, keratinosomes) and immunohistochemical (e.g. keratins, integrins, gp80, involucrin, filaggrin) criteria. The melanocytes were localized in the basal layer and accounted for 10% of the total cell number. Heavily pigmented melanocytes from black donors contained regular melanosomes in all stages of maturation, whereas melanocytes derived from white donors contained predominantly melanosomes of stages I and II. Melanosome-laden dendrites were readily detected extending from the heavily pigmented melanocytes, while they were less conspicuous in melanocytes from white donors. The extent of melanosome transfer was independent of the racial origin of the ORS cells. Melanosomes could also be transferred "through racial barriers". Melanosomes, mainly of stages III and IV, were detected in the ORS cells, being distributed either as single or compound melanosomes, again irrespective of the racial origin of the ORS cells. In conclusion, pigmented epidermal equivalents generated from ORS cells offer practical advantages over other in vitro pigmentation models: (1) the ORS cells are easily and repeatedly available from any donor regardless of age; (2) primary cultures of ORS cells are free of contaminating melanocytes, a bias if using interfollicular epidermal keratinocytes; (3) a high degree of epidermal differentiation is maintained for 3 weeks in fully defined medium, enabling labelling and stimulation experiments to be performed and compounds interfering with melanin pigmentation to be tested.
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successful treatment of chronic leg ulcers with epidermal equivalents generated from cultured autologous Outer Root Sheath cells
Journal of Investigative Dermatology, 1996Co-Authors: A Limat, Davide Mauri, Thomas HunzikerAbstract:The Outer Root Sheath cells of hair follicles can substitute for interfollicular epidermal keratinocytes, as during healing of skin wounds when these cells migrate onto the denuded area and contribute to epidermal regeneration. Using improved culture techniques, we generated epidermal equivalents from cultured Outer Root Sheath cells of patients suffering from recalcitrant chronic leg ulcers, primarily of vascular origin. In such epidermal equivalents, tissue organization as well as immunolocalization of epidermal differentiation products (keratin 10, involucrin, filaggrin) and integrins were indistinguishable from normal epidermis. As determined by the number of bromodeoxyuridine-incorporating cells, the basal layer contained a large compartment of proliferative cells irrespective of donor age. FACS analysis of the Outer Root Sheath cells, used to prepare the epidermal equivalents, disclosed a fraction of small cells with enhanced expression of β1-integrin, a potential stem cell marker. In contrast to acute wounds, a major definitive take of grafted cultured autologous keratinocytes has not been convincingly demonstrated in chronic wounds. In a pilot study, grafting of epidermal equivalents generated in vitro from autologous Outer Root Sheath cells on 11 ulcers in five patients resulted in a definitive take rate of about 80%, with subsequent complete healing within 2 to 3 wk of five out of seven ulcers grafted with densely arranged cultures. This improvement in the treatment of chronic leg ulcers with cultured autologous keratinocytes probably depends on the large compartment of proliferative cells as well as on a well-developed horny layer which prevents disintegration of the grafts. Practical advantages of the new technique are its noninvasiveness, the lack of need for surgical facilities or anesthesia, and a short immobilization period after grafting.
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cultivation of keratinocytes from the Outer Root Sheath of human hair follicles
Methods in molecular medicine, 1996Co-Authors: A Limat, Thomas HunzikerAbstract:The Outer Root Sheath (ORS) of hair follicles is a multilayered tissue made up predominantly by undifferentiated keratinocytes (it1, it2) Although the functions of the ORS cells for hair growth are not established, it is known that the ORS cells can contribute to the regeneration of the epidermis, as during healing of superficial wounds where the ORS cells migrate out of the follicle to repopulate the denuded area (3, 4). Recent studies also suggest that stem cells for various epithelial cell populations of the skin are located in the ORS tissue (5, 6.
L R Braathen - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of surface antigens in cultured human Outer Root Sheath cells and epidermal keratinocytes persistence of low expression of class i mhc antigens in Outer Root Sheath cells in vitro
British Journal of Dermatology, 2006Co-Authors: A Limat, Thomas Hunziker, Tony Wysscoray, L R BraathenAbstract:Summary In the anagen human hair follicle, the epithelial cells from the infrainfundibular portion and the hair matrix cells express markedly lower numbers of major histocompatibility complex class I molecules than interfollicular epidermal keratinocytes. During the catagen phase of the hair cycle, class I expression on these cells increases, and activated macrophages aggregate around the follicle, which has led to the hypothesis that the cells to be resorbed are recognized by virtue of their low class I antigen expression. In the present study, we showed that, in vitro, Outer Root Sheath cells also maintain a lower constitutive expression of MHC class I molecules compared with epidermal keratinocytes. In contrast, other surface antigens such as HLA-DR, -DP and -DQ, ICAM-1, LFA-3 and CD29, which are all known to participate in leucocyte-keratinocyte interactions, were similarly expressed in both cell types. Furthermore, interferon gamma strongly upregulated MHC class I and II and ICAM-1 expression in both cell types, whereas CD29 and LFA-3 remained unaffected. Tumour necrosis factor alpha, to a lesser extent, also upregulated MHC class I and ICAM-1 expression, but not class II expression. The differences in constitutive surface antigen expression of infrainfundibular Outer Root Sheath cells compared with interfollicular epidermal keratinocytes emphasizes a distinct role of this cell type in the hair cycle, and possibly also in alopecia areata.
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formation of a regular neo epidermis by cultured human Outer Root Sheath cells grafted on nude mice
Transplantation, 1995Co-Authors: A Limat, D Breitkreutz, G Thiekoetter, C E Klein, L R Braathen, T Hunziker, N E FusenigAbstract:The Outer Root Sheath of hair follicles mainly consists of basal-like keratinocytes which can substitute for interfollicular epidermal keratinocytes, as during healing of skin wounds when Outer Root Sheath cells migrate onto the denuded area, thus contributing to epidermal regeneration. Human Outer Root Sheath cells represent a repeatedly available source of keratinocytes which can be easily and extensively expanded in culture. Close comparison of organotypic cultures of either Outer Root Sheath cells or epidermal keratinocytes grafted onto nude mice demonstrated that Outer Root Sheath cells formed a stratified epithelium resembling normal epidermis that is virtually indistinguishable from that developed by epidermal keratinocytes. Typical epidermal differentiation markers, such as the suprabasal keratins 1 and 10, involucrin, filaggrin, the basement membrane components collagen type IV and laminin, and the integrin chains alpha 2, alpha 3, alpha 6, and beta 1, were readily expressed in a mostly regular localization. These data suggest that Outer Root Sheath cells, bearing essential advantages as compared with interfollicular keratinocytes, are suitable for skin replacement.
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Outer Root Sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes
Cell and Tissue Research, 1994Co-Authors: A Limat, D Breitkreutz, C E Klein, T Hunziker, N E Fusenig, F. Noser, L R BraathenAbstract:In organotypic cultures, Outer Root Sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic “surface” epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.
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Effects of 1α,25-dihydroxy-vitamin D_3 and calcipotriol on organotypic cultures of Outer Root Sheath cells: a potential model to evaluate antipsoriatic drugs
Archives of Dermatological Research, 1993Co-Authors: A Limat, T Hunziker, L R BraathenAbstract:In the human hair follicle, Outer Root Sheath (ORS) cells constitutively express the hyperproliferation-associated keratins 6, 16 and 17 instead of keratins 1 and 10 found in interfollicular epidermis. In organotypic cultures, ORS cells form a stratified epithelium which in many respects resembles psoriatic skin: it has a hyperplastic tissue architecture and a poorly developed granular layer, and expresses hyperproliferation-associated keratins. Therefore, we studied the effects of the antipsoriatic compounds 1α,25-dihydroxy-vitamin D_3 (1α,25-(OH)_2-D_3) and its synthetic derivative calcipotriol on cultured ORS cells. In monolayer cultures, 10^−6 M 1α,25-(OH)_2-D_3 or calcipotriol completely blocked ORS cell proliferation. This inhibitory effect was substantially reduced at 10^−8 M . Incubation of organotypic ORS cultures with both vitamin D analogues resulted in a marked thinning of the living cell compartment concomitant with a thickening of the horny layer. A reduced expression of differentiation markers such as keratins 10,16 and 17, involucrin and filaggrin paralleled the thinning of the stratum Malpighi. As determined by quantification of BrdU-positive cells, ORS cell proliferation was apparently not affected by the vitamin D analogues, indicating that these compounds mainly operate by accelerating the differentiation pathway within the suprabasal living cell compartment. No alteration in the expression of the α6- and Β 1-integrin chains was found.
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soluble factors from human hair papilla cells and dermal fibroblasts dramatically increase the clonal growth of Outer Root Sheath cells
Archives of Dermatological Research, 1993Co-Authors: A Limat, Thomas Hunziker, Ernst R Waelti, Sven P Inaebnit, Ulrich N Wiesmann, L R BraathenAbstract:Depending on environmental influences, follicular Outer Root Sheath (ORS) cells in vivo can differentiate either towards interfollicular keratinocytes or, as demonstrated in the rat vibrissa, hair matrix cells. Crucial regulators of both their proliferation and differentiation are the mesenchymal cells of the respective tissues. The interactions of human ORS cells with human hair papilla cells (HPC) or human dermal fibroblasts (HDF) were studied using a two-chamber model separating the two cell types either by a microporous membrane or additionally by a medium layer. The results of 3H-thymidine incorporation studies indicated that ORS cell growth was markedly enhanced in co-culture with either HPC or HDF, the highest stimulatory effect resulting when ORS cells were in close association with the mesenchymal cells. No correlation was found between ORS cell proliferation and IL-6 production in the co-culture system, thus pointing to the secretion by HPC and HDF of growth-promoting soluble factors that are different form IL-6 as well as from EGF, bFGF and insulin present in the culture medium.
J C Simon - One of the best experts on this subject based on the ideXlab platform.
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the angiogenic potential of mesenchymal stem cells from the hair follicle Outer Root Sheath
Journal of Clinical Medicine, 2021Co-Authors: Vuk Savkovic, Alexander K Bartella, Rudiger Zimmerer, J C Simon, Danilo Obradovic, Federica Francesca Masieri, Christian Etz, Bernd LethausAbstract:Neovascularization is regarded as a pre-requisite in successful tissue grafting of both hard and soft tissues alike. This study considers mesenchymal stem cells from hair follicle Outer Root Sheath (MSCORS) as powerful tools with a neat angiogenic potential that could in the future have wide scopes of neo-angiogenesis and tissue engineering. Autologous MSCORS were obtained ex vivo by non-invasive plucking of hair and they were differentiated in vitro into both endothelial cells and vascular smooth muscle cells (SMCs), two crucial cellular components of vascular grafts. Assessment was carried out by immunostaining, confocal laser-scanning microscopy, gene expression analysis (qRT-PCR), quantitative analysis of anastomotic network parameters, and cumulative length quantification of immunostained α-smooth muscle actin-containing stress fibers (α -SMA). In comparison to adipose mesenchymal stem cells, MSCORS exhibited a significantly higher differentiation efficiency according to key quantitative criteria and their endothelial derivatives demonstrated a higher angiogenic potential. Furthermore, the cells were capable of depositing their own extracellular matrix in vitro in the form of a membrane-cell sheet, serving as a base for viable co-culture of endothelial cells and SMCs integrated with their autologous matrix. Differentiated MSCORS hereby provided a complex autologous cell-matrix construct that demonstrates vascularization capacity and can serve as a base for personalized repair grafting applications.
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the middle part of the plucked hair follicle Outer Root Sheath is identified as an area rich in lineage specific stem cell markers
Biomolecules, 2021Co-Authors: Federica Francesca Masieri, Marie Schneider, Alexander K Bartella, Sebastian Gaus, Sebastian Hahnel, Rudiger Zimmerer, Ulrich Sack, Danijela Maksimovicivanic, Sanja Mijatovic, J C SimonAbstract:Hair follicle Outer Root Sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.
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culturing of melanocytes from the equine hair follicle Outer Root Sheath
Processes, 2021Co-Authors: Jule Kristin Michler, Alexander K Bartella, Anna Katharina Sander, Sebastian Gaus, Sebastian Hahnel, Rudiger Zimmerer, J C Simon, Vuk Savkovic, Bernd LethausAbstract:Hair follicles harbor a heterogeneous regenerative cell pool and represent a putative low-to-non-invasively available source of stem cells. We previously reported a technology for culturing human melanocytes from the hair follicle Outer Root Sheath (ORS) for autologous pigmentation of tissue engineered skin equivalents. This study translated the ORS technology to horses. We de-veloped a culture of equine melanocytes from the ORS (eMORS) from equine forelock hair follicles cultured by means of an analogue human hair follicle-based in vitro methodology. The procedure was adjusted to equine physiology by addition of equine serum to the culture medium. The hair follicles were isolated by macerating forelock skin rests, enzymatically digested and subjected to air-medium-interface cultivation method. The procedure resulted in differentiated equine melanocytes, which exhibited typical morphology, presence of melanosomes, expression of cytoskeleton proteins vimentin, α-SMA, Sox2, S100s and tyrosinase as well as tyrosinase activity followed by production of melanin. According to all assessed parameters, eMORS could be ranked as partially melanotic melanocytes. The results of the study offer an experimental base for further insight into hair follicle biology in equine and for comparative studies of hair follicles across different species.
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sulfated hyaluronan containing artificial extracellular matrices promote proliferation of keratinocytes and melanotic phenotype of melanocytes from the Outer Root Sheath of hair follicles
Journal of Biomedical Materials Research Part A, 2019Co-Authors: Marie Schneider, J C Simon, Sandra Rother, Stephanie Moller, Matthias Schnabelrauch, Dieter Scharnweber, Vera Hintze, Vuk SavkovicAbstract:The aim of this work was to establish improved cultivation conditions for human keratinocytes (HUKORS) and melanocytes (HUMORS) from the Outer Root Sheath (ORS) of human hair follicles for purposes of generating an epidermal graft. To this end, the cells were cultivated on artificial extracellular matrix coatings composed of collagen (COLL) and hyaluronan (HA) with varying sulfation degrees. HUKORS and HUMORS were characterized based on their morphology and proliferation, marker gene expression, protein expression and melanin content in melanocytes. Depending on the sulfation degree, the matrices provided a favorable proliferation environment for HUKORS and improved the balance between proliferation and the exertion of melanotic phenotype (gene expression and melanin content) in HUMORS. Based on the increased gene expression of microphthalmia-associated transcription factor, as well as the downstream-affected melanotic genes premelanosome protein and tyrosinase in HUMORS cultivated on collagen matrices with high-sulfated HA, we assume that sulfated HA enhanced melanotic phenotype either by directly binding the CD44 receptor or by concentrating signaling mediators on site. Being a promising cultivation environment for both HUKORS and HUMORS, collagen matrices with sulfated HA have a potential of significantly improving the development of ORS-based epidermal grafts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1640-1653, 2019.
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polycaprolactone fiber meshes provide a 3d environment suitable for cultivation and differentiation of melanocytes from the Outer Root Sheath of hair follicle
Journal of Biomedical Materials Research Part A, 2016Co-Authors: Vuk Savkovic, Marie Schneider, Franziska Flamig, Katharina Sulflow, Tina Loth, Andrea Lohrenz, Michael C Hacker, Michaela Schulzsiegmund, J C SimonAbstract:Melanocytes differentiated from the stem cells of human hair follicle Outer Root Sheath (ORS) have the potential for developing non-invasive treatments for skin disorders out of a minimal sample: of hair Root. With a robust procedure for melanocyte cultivation from the ORS of human hair follicle at hand, this study focused on the identification of a suitable biocompatible, biodegradable carrier as the next step toward their clinical implementation. Polycaprolactone (PCL) is a known biocompatible material used for a number of medical devices. In this study, we have populated electrospun PCL fiber meshes with normal human epidermal melanocytes (NHEM) as well as with hair-follicle-derived human melanocytes from the Outer Root Sheath (HUMORS) and tested their functionality in vitro. PCL fiber meshes evidently provided a niche for melanocytes and supported their melanotic properties. The cells were tested for gene expression of PAX3, PMEL, TYR and MITF, as well as for proliferation, expression of melanocyte marker proteins tyrosinase and glycoprotein 100 (gp100), L-DOPA-tautomerase enzymatic activity and melanin content. Reduced mitochondrial activity and PAX-3 gene expression indicated that the three-dimensional PCL scaffold supported differentiation rather than proliferation of melanocytes. The monitored melanotic features of both the NHEM and HUMORS cultivated on PCL scaffolds significantly exceeded those of two-dimensional adherent cultures. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 26–36, 2016.
Marie Schneider - One of the best experts on this subject based on the ideXlab platform.
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the middle part of the plucked hair follicle Outer Root Sheath is identified as an area rich in lineage specific stem cell markers
Biomolecules, 2021Co-Authors: Federica Francesca Masieri, Marie Schneider, Alexander K Bartella, Sebastian Gaus, Sebastian Hahnel, Rudiger Zimmerer, Ulrich Sack, Danijela Maksimovicivanic, Sanja Mijatovic, J C SimonAbstract:Hair follicle Outer Root Sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.
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autologous non invasively available mesenchymal stem cells from the Outer Root Sheath of hair follicle are obtainable by migration from plucked hair follicles and expandable in scalable amounts
Cells, 2020Co-Authors: Federica Francesca Masieri, Marie Schneider, Sebastian Hahnel, Danilo Obradovic, Tina Kottek, Kensuke Yamauchi, Jong Keun Seon, Sook Jung Yun, Ruben A Ferrer, Sandra FranzAbstract:Background: Regenerative therapies based on autologous mesenchymal stem cells (MSC) as well as stem cells in general are still facing an unmet need for non-invasive sampling, availability, and scalability. The only known adult source of autologous MSCs permanently available with no pain, discomfort, or infection risk is the Outer Root Sheath of the hair follicle (ORS). Methods: This study presents a non-invasively-based method for isolating and expanding MSCs from the ORS (MSCORS) by means of cell migration and expansion in air−liquid culture. Results: The method yielded 5 million cells of pure MSCORS cultured in 35 days, thereby superseding prior art methods of culturing MSCs from hair follicles. MSCORS features corresponded to the International Society for Cell Therapy characterization panel for MSCs: adherence to plastic, proliferation, colony forming, expression of MSC-markers, and adipo-, osteo-, and chondro-differentiation capacity. Additionally, MSCORS displayed facilitated random-oriented migration and high proliferation, pronounced marker expression, extended endothelial and smooth muscle differentiation capacity, as well as a paracrine immunomodulatory effect on monocytes. MSCORS matched or even exceeded control adipose-derived MSCs in most of the assessed qualities. Conclusions: MSCORS qualify for a variety of autologous regenerative treatments of chronic disorders and prophylactic cryopreservation for purposes of acute treatments in personalized medicine.
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sulfated hyaluronan containing artificial extracellular matrices promote proliferation of keratinocytes and melanotic phenotype of melanocytes from the Outer Root Sheath of hair follicles
Journal of Biomedical Materials Research Part A, 2019Co-Authors: Marie Schneider, J C Simon, Sandra Rother, Stephanie Moller, Matthias Schnabelrauch, Dieter Scharnweber, Vera Hintze, Vuk SavkovicAbstract:The aim of this work was to establish improved cultivation conditions for human keratinocytes (HUKORS) and melanocytes (HUMORS) from the Outer Root Sheath (ORS) of human hair follicles for purposes of generating an epidermal graft. To this end, the cells were cultivated on artificial extracellular matrix coatings composed of collagen (COLL) and hyaluronan (HA) with varying sulfation degrees. HUKORS and HUMORS were characterized based on their morphology and proliferation, marker gene expression, protein expression and melanin content in melanocytes. Depending on the sulfation degree, the matrices provided a favorable proliferation environment for HUKORS and improved the balance between proliferation and the exertion of melanotic phenotype (gene expression and melanin content) in HUMORS. Based on the increased gene expression of microphthalmia-associated transcription factor, as well as the downstream-affected melanotic genes premelanosome protein and tyrosinase in HUMORS cultivated on collagen matrices with high-sulfated HA, we assume that sulfated HA enhanced melanotic phenotype either by directly binding the CD44 receptor or by concentrating signaling mediators on site. Being a promising cultivation environment for both HUKORS and HUMORS, collagen matrices with sulfated HA have a potential of significantly improving the development of ORS-based epidermal grafts. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1640-1653, 2019.
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a human serum enriched medium formulation supports high viability and marker expression in primary melanocyte cultures from the Outer Root Sheath and epidermis
Experimental Dermatology, 2018Co-Authors: Marie Schneider, Andrea Lohrenz, Michael C Hacker, Michael Cross, Jan C Simon, Vuk SavkovicAbstract:Formulating clinically relevant melanocyte cultivation media that maintain the balance between proliferation and maturation to functional melanocytes is a major experimental and regulatory challenge. Within the translation of human melanocytes from the Outer Root Sheath of human hair follicle (HUMORS), we developed a melanocyte medium free of chemical mitogens, chemical melanogenesis enhancers and bovine products, enabling proliferation as well as melanotic differentiation. The formulation involved the replacement of bovine pituitary extract (BPE) and bovine serum (FBS) with human serum (HS) combined with ascorbic acid, CaCl2 , epinephrine, L-glutamine, insulin and fibroblast growth factor. The cultivation efficiency was characterized through proliferation and exertion of melanotic phenotype, gene and protein expression of melanotic markers and melanin content. Having established an application-directed BPE-free formulation, we then re-formulated a research-grade medium with BPE for purposes of even more effective in vitro cultivation, adjusted to specific requirements of HUMORS and normal human epidermal melanocytes (NHEM).
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polycaprolactone fiber meshes provide a 3d environment suitable for cultivation and differentiation of melanocytes from the Outer Root Sheath of hair follicle
Journal of Biomedical Materials Research Part A, 2016Co-Authors: Vuk Savkovic, Marie Schneider, Franziska Flamig, Katharina Sulflow, Tina Loth, Andrea Lohrenz, Michael C Hacker, Michaela Schulzsiegmund, J C SimonAbstract:Melanocytes differentiated from the stem cells of human hair follicle Outer Root Sheath (ORS) have the potential for developing non-invasive treatments for skin disorders out of a minimal sample: of hair Root. With a robust procedure for melanocyte cultivation from the ORS of human hair follicle at hand, this study focused on the identification of a suitable biocompatible, biodegradable carrier as the next step toward their clinical implementation. Polycaprolactone (PCL) is a known biocompatible material used for a number of medical devices. In this study, we have populated electrospun PCL fiber meshes with normal human epidermal melanocytes (NHEM) as well as with hair-follicle-derived human melanocytes from the Outer Root Sheath (HUMORS) and tested their functionality in vitro. PCL fiber meshes evidently provided a niche for melanocytes and supported their melanotic properties. The cells were tested for gene expression of PAX3, PMEL, TYR and MITF, as well as for proliferation, expression of melanocyte marker proteins tyrosinase and glycoprotein 100 (gp100), L-DOPA-tautomerase enzymatic activity and melanin content. Reduced mitochondrial activity and PAX-3 gene expression indicated that the three-dimensional PCL scaffold supported differentiation rather than proliferation of melanocytes. The monitored melanotic features of both the NHEM and HUMORS cultivated on PCL scaffolds significantly exceeded those of two-dimensional adherent cultures. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 26–36, 2016.
