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Location of stem cells of human hair follicles by clonal analysis
Abstract
We have examined the growth capacity of keratinocytes isolated from human scalp hair follicles. Like the keratinocytes of glabrous epidermis, most of the colony-forming cells are classified as holoclones or meroclones when analyzed in a clonal assay. Some of them have extensive growth potential, as they are able to undergo at least 130 doublings. Therefore, the hair follicle, like the epidermis, contains keratinocytes with the expected property of stem cells: an extensive proliferative capacity permitting the generation of a large amount of epithelium. We have also examined the distribution of clonogenic keratinocytes within the hair follicle. Several hundred colony-forming cells are concentrated at a region below the midpoint of the follicle and outside the hair bulb. This region lies deeper than the site of insertion of the arrector pili muscle, which corresponds with the position of the bulge when the latter can be identified. In contrast, few colony-forming cells are present in the hair bulb, where most of the mitotic activity is observed during the active growth phase of the follicle. Paraclones, which are present both in the midregion and in the bulb of hair follicles, are unlikely to be the transient amplifying cells expected from kinetic studies.
- ... Subsequently, dermabrasion studies corroborated the existence of stem cells in the upper hair follicle(92), and dissected follicles were able to regenerate fully differentiated interfollicular epidermis in an in vitro organotypic model (93). The hair follicles are believed to harbour the majority of the clonogenic cells, estimated at 3000-6000/mm 2 in human scalp, versus 1000-2000/mm 2 in glabrous epidermis (94). It is therefore not surprising that epidermal regeneration is proportional to the number of residual hair follicles that remain(95). ...... This was corroborated by a similar study utilising human hair that also found enrichment for colony forming cells in the presumptive bulge region and demonstrated that bulge keratinocytes had superior in vitro clonogenicity to unfractionated interfollicular keratinocytes(61). However, subsequent studies in human follicles variously reported the major clonogenic cell enrichment to be in the sub-bulge region (94,106) or upper central outer root sheath (107). Once again, all showed that the principal repository of quiescent stem cells was not an exclusive locale for colony forming cells. ...... However, it is important to note that the contributions to interfollicular epidermis have been observed only in tissue expansionary (neonatal) or regenerative phases (following wounding), after complex manipulations or from admixtures of many cells -not from foci of single cells under steady state conditions. Given the location of bulge cells within deep recesses, it is highly unlikely that these participate in routine maintenance of the interfollicular epidermis (94). This view was vindicated by exquisite long-term lineage marking studies by Ghazizadeh & Taichman that showed lineage restriction, with follicular cells contributing a mere "rim of epidermis," (Figure 1a) venturing no further than the margin of the follicle in the absence of wounding (71). ...It has been evident for some time that cell replacement in the epidermis of the skin is a highly ordered process with a central role for keratinocyte stem and progenitor cells. In recent years, many investigators have sought to distinguish keratinocyte stem cells (KSCs) from their immediate progeny using molecular markers, both in situ and ex vivo, and a number of molecular regulators that can perturb ordered cell renewal in skin epithelium have also been identified. Although we are far from having a clear understanding of the precise mechanisms that regulate ordered epidermal tissue morphogenesis and cell renewal, significant progress has been made that has begun to shed light on these processes. Unequivocal identification and isolation of viable keratinocyte stem and progenitors are now possible; this combined with the advent of molecular technologies, such as high-throughput genome-wide scanning and the ability to generate mice with designer skin, and the development of assays for these cells, albeit at an early stage, places us at an exciting time of experimental investigation and discovery, poised to capitalize on the collective efforts expended by many laboratories across the world.
- ... The success of these surgical treatments depends on efficient expansion of limbal epithelial stem cells which involved 3T3 feeder layer and fetal bovine serum (FBS) in most cases. The 3T3 feeder layer culture system was set up by Rheinwald and Green [9] and has been successfully used to expand epithelial cells from human skin, hair follicle, limbus, conjunctiva, and oral mucosa tissue [10][11][12][13][14][15][16]. However, FBS is not well-defined, and it always displays quantitative and qualitative lot-to-lot variations [17]. ...... And most of these media require high cell seeding density, which may not be practical for expansion of human corneal epithelial cells. Moreover, corneal epithelial stem cells, which are detected as holoclones in 3T3 culture 2 Journal of Ophthalmology system, could not be maintained in this serum-free culture medium for long term [12,13]. To date, there is no defined serum-free medium which could support expansion of corneal epithelial stem cells for long term. ...... During each passage, cells were trypsinized and collected when cells reached 80-90% confluence; cell numbers were calculated for population doubling assay. The result shows that epithelial cell yield from SR medium is close to that of cells cultured in FAD medium, as shown in Figure 2(a), and these data are similar to previous reports [4,[10][11][12]. In summary, the data presented here shows that SR culture medium + mitomycin C-treated 3T3 feeder layer supports human corneal epithelial cells clonal growth and proliferation. ...The limbal epithelial cells can be maintained on 3T3 feeder layer with fetal bovine serum supplemented culture medium, and these cells have been used to successfully treat limbal stem cell deficiency. However, fetal bovine serum contains unknown components and displays quantitative and qualitative lot-to-lot variations. To improve the culture condition, the defined KnockOut serum replacement was investigated to replace fetal bovine serum for culturing human limbal epithelial cell. Human primary limbal epithelial cells were cultured in KnockOut serum and fetal bovine serum supplemented medium, respectively. The cell growth rate, gene expression, and maintenance of limbal epithelial stem cells were studied and compared between these two groups. Human primary limbal epithelial cells were isolated and successfully serially cultivated in this novel KnockOut serum supplemented medium; the cell proliferation and stem cell maintenance were similar to those of cells grown in fetal bovine serum supplemented medium. These data suggests that this KnockOut serum supplemented medium is an efficient replacement to traditional fetal bovine serum supplemented medium for limbal epithelial cell culture, and this medium has great potential for long term maintenance of limbal epithelial cells, limbal epithelial stem cells transplantation, and tissue regeneration.
- ... During adult homeostasis, hair follicles undergo cycles of growth (anagen) and degeneration (catagen), followed by a resting stage (telogen) 1 . The hair follicle stem cells (HFSCs) responsible for cyclic regeneration are located in the permanent non-cyclic follicle portion called the bulge [17][18][19][20] . HFSCs were first identified based on their slowcycling properties 19,21,22 . ...... HFSCs were first identified based on their slowcycling properties 19,21,22 . They have higher clonogenicity in vitro and give rise to IFE, hair follicle and sebaceous gland lineages upon transplantation 17,18,20,23,24 . Slow-cycling HFSCs were first isolated and characterized using K5-tTA/TRE-H2BGFP and Krt15-EGFP transgenic mice 25,26 , and several studies revealed the expression of bulge specific markers, such as Cd34 (refs 18,23 ), Krt15 (refs 27,28 ), Krt19 (ref. ...... Lineage tracing targeting labelled retaining cells 26 or using Krt15-CrePR 34 , Shh-Cre 57 , K19-CreER 54 and Lgr5-CreER 54 reporter strains showed that HFSCs rapidly migrate from the bulge to the wound and contribute to epidermal repair (Fig. 2b). These data demonstrate that HFSCs are highly plastic during wound healing, similarly to their expanded fate potential upon transplantation 17,20,30,49,52 . ...Tissue repair is critical for animal survival. The skin epidermis is particularly exposed to injuries, which necessitates rapid repair. The coordinated action of distinct epidermal stem cells recruited from various skin regions together with other cell types, including fibroblasts and immune cells, is required to ensure efficient and harmonious wound healing. A complex crosstalk ensures the activation, migration and plasticity of these cells during tissue repair.
- ... HF lineage tracing experiments using tetracyclineregulated green fluorescent protein (GFP)-tagged histone H2B (Tet-o-H2B-GFP) (Tumbar et al., 2004 ) or the expression of reporter genes under the control of HFspecific promoters such as K15 (Morris et al., 2004), Lgr5 (Jaks et al., 2008), or K19 () showed that the cells residing in the bulge and upper germ are responsible for HF regeneration during adult homeostasis. Bulge SCs isolated by microdissection (Rochat et al., 1994; Oshima et al., 2001) or by flow cytometry based on the expression of CD34 (Trempus et al., 2003; Blanpain et al., 2004) or K15-GFP (Morris et al., 2004) and cultured in vitro produced large proliferative colonies that could be propagated for a long time. Transplantation of the progeny of a single bulge SC can generate all HF lineages (Blanpain et al., 2004; Claudinot et al., 2005). ...... Pioneering studies were performed by the group of Barrandon, in which different regions of the epidermis were microdissected and transplanted into immunodeficient mice. These studies demonstrated the ability of bulge SCs to re-form all epidermal lineages (Rochat et al., 1994; Oshima et al., 2001) and suggested the existence of multipotent bulge SCs. Similarly, transplantation of fluorescenceactivated cell sorting (FACS)-isolated bulge SCs together with embryonic mesenchyme into immunodeficient mice also resulted in epidermal regeneration and differentiation into the complete repertoire of epidermal cell types (Morris et al., 2004). ...The mammalian skin is one of the best studied epithelial systems containing stem cells to date, however, the origin of most of the stem cell populations found in the adult epidermis is still largely unknown (Benitah and Frye, 2012). Far more unknown is the embryonic origin of other stem cells which populate the other layers of this tissue.
- ... [25] Özofajiyal submukozal bez kanallarının glandüler boyun bölgesinde de saç kılı folikülündekilere benzer kök hücreler bulunmaktadır. [26] Ülserasyon ya da hasar sonrasında kök hücreler lamina propriada yeni glandlar oluşturabilmekte ve sonuçta glandülar hücreler kanallardan yüzeye taşınmaktadır. Son çalışmalarda, normal skuamöz hücrelerin ve Barrett epitel hücrelerinin farklı projenitör hücrelerden kaynaklandığını gösteren bulgular elde edilmiştir. ...Barrett esophagus is described as a condition in which the normal squamous epithelium of the distal esophagus is replaced by abnormal columnar mucosa containing intestinal metaplasia. The prevalence of gastroesophageal reflux is 20% and Barrett esophagus is 0.4% in Turkey. Cronic mucosa irritation related to gastroesophageal reflux is the most important cause of the development of Barrett esophagus. In addition, obesity and some other diseases may result in Barrett esophagus development. Barrett esophagus is the only known precursor to esophageal adenocarcinoma and to date, it is one of the cancer types that has the most rapidly increasing incidence. Carcinogenic risk is 30 times higher than that in the normal population. Treatment should have the purpose of controlling symptoms of gastroesophageal reflux, preventing acid and duodenal reflux into esophagus, preventing the development of complications, such as erosion, peptic ulcus, stricture, preventing proximally extension of intestinal metaplasia, inducing regression of intestinal metaplasia to the normal mucosa, preventing the development of dysplasia. inducing regression of dysplasia to nondysplastic cells, and preventing the development of adenocarcinoma. The grade of the dysplasia determines the treatment modality. Whereas in patients with non-dysplastic or low grade dysplasia, follow-up, medical treatment, antireflux surgery or ablation therapy may be appropriate, esophagectomy should be preferred in patients with high grade dysplasia.
- ... Clonal analysis in vitro has defined three types of clonogenic cells, giving rise to the so-called holoclones, meroclones, and paraclones. Holoclone-forming cells have the highest self-renewing and proliferative capacity, and define in culture the KSCs of the epidermis or the corneal epithelium (Pellegrini et al., 1999;Rochat et al., 1994). Meroclone-and paraclone-forming cells have proportionally less proliferative capacity and terminally differentiate into keratinocytes after 5-15 cell doublings, as expected for TA progenitors (Barrandon and Green, 1987). ...Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations.
- ... Although several lines of evidence suggest that the human bulge provides a niche for SCs, it is definitely a less distinctive structure than in rodents. Also, in contrast to the bulge of murine follicles, human HFs have a different pattern of biomarkers expression [29,30]. Positive markers for bulge cells include CD200, pleckstrin homology-like domain, family A, member 1 (PHLDA1), follistatin and frizzled homolog 1 [31]. ...The skin epidermis is continuously renewed throughout life. The self-renewing capacity of human epidermis is enormous and it takes about 3 weeks for a basal cell to complete its migration, terminally differentiate and be shed from the skin surface. Epidermal stem cells have key roles in maintaining tissue homeostasis by providing a pool of new cells to replace those lost during skin turnover or after injury. In the epidermis, different populations of stem cells can be distinguished that maintain skin functions, regenerate, and repair by contributing to all skin cell types of the interfollicular epidermis, hair follicles, and sebaceous glands. However, recent data clearly show that some populations of stem cells existing in bone marrow and circulating in peripheral blood contribute to skin regeneration as well.
- ... We found that HNSCC cells cultured in monolayer had a relatively low percentage of ALDH positive cells (~2%) (Figure 1A). Landmark studies have demonstrated a correlation between stem cell properties and the morphology of colonies generated by single cells from hair follicles[18], epidermal keratinocytes[19], and HNSCC[20], among other tissues. We found that tumor cells derived from six different HNSCC cell lines growing in ultra-low adhesion conditions are able to generate three morphologically distinct sphere populations classified as holospheres, merospheres, and paraspheres (Figure 1B). ...Cancer stem cells (CSCs) are a subpopulation of tumor cells endowed with self-renewal properties and the capacity to dynamically adapt to physiological changes that occur in the tumor microenvironment. CSCs play a central role in resistance to therapy and long-term disease recurrence. Better characterization and understanding of the available in vitro tools to study the biology of CSCs will improve our knowledge of the processes underlying tumor response to therapy, and will help in the screening and development of novel strategies targeting CSCs. We investigated the behavior of different populations of head and neck CSCs grown under ultra-low adhesion conditions. We found that invasion and adhesion differ among tumorsphere subtypes (holospheres, merospheres and paraspheres), and their tumor cell progeny also harbor distinct self-renewal and clonogenic potentials. Furthermore, holospheres contained higher numbers of head and neck CSCs, as detected by the CD44 cancer stem cell marker and aldehyde dehydrogenase (ALDH) enzymatic activity. In addition, holospheres showed reduced proliferation (Ki67), hypoacetylation of histones, and increased expression of the BMI-1 epithelial stem cell marker, suggesting activation of stem cell programs. Collectively, our results suggest that holospheres enrich a specific population of CSCs with enhanced "stemness" and invasive potential.
- ... Adult or, as often called, tissue stem cells have been identified throughout the years in several organs and tissues, including liver [21][22][23], lungs [24,25], bone marrow [26][27][28][29][30][31], blood vessels [32][33][34], skeletal muscle [35][36][37][38], intestine and colon [39], skin [40][41][42] and heart [43,44], just to mention a few. In tissues with slow cell turnover (like the heart, brain or liver), they are found quiescent in specific areas of each tissue designated as stem cell niche and are activated after injury. ...Liver transplantation is the standard therapy for many liver diseases. Despite being a considerable successful treatment, avoiding allograft rejection, among other complications, continues being one of the big challenges for physicians. Immunosuppression drugs significantly decrease rejection rates after liver transplantation; however, they have generally associated adverse effects which compromise liver transplantation outcome, increasing patients’ morbidity and mortality. So, a close monitoring of immunosuppression is essential to reduce drugs’ undesirable effects as long as allograft rejection is avoided. Nevertheless, monitoring of liver transplant recipients (LTRs) frequently entails the study of liver biopsies with its consequent inconvenience and risk for the patient. Identification of biomarkers that could diagnose or predict the risk of suffering allograft rejection (acute, chronic, or antibody mediated), or, on the contrary, the potential to achieve allograft tolerance, would represent a considerable progress in the managing and monitoring of LTRs. As the immune response of LTRs is responsible for the rejection or tolerance of the liver allograft, most of the potential biomarkers studied in this field are related to the immune system. For that reason, in this chapter, we attempt to review the state of the art in immunological biomarkers for the managing of patients after liver transplantation.
- ... Adult or, as often called, tissue stem cells have been identified throughout the years in several organs and tissues, including liver [21][22][23], lungs [24,25], bone marrow [26][27][28][29][30][31], blood vessels [32][33][34], skeletal muscle [35][36][37][38], intestine and colon [39], skin [40][41][42] and heart [43,44], just to mention a few. In tissues with slow cell turnover (like the heart, brain or liver), they are found quiescent in specific areas of each tissue designated as stem cell niche and are activated after injury. ...Purpose of review: Organ donation in the United States registered 9079 deceased organ donors in 2015. This high percentage of donations allowed organ transplantation in 29 851 recipients. Despite increasing numbers of transplants performed in comparison with previous years, the numbers of patients that are in need for a transplant increase every year at a higher rate. This reveals that the discrepancy between the demand and availability of organs remains fundamental problem in organ transplantation. Recent findings: Development of bioengineered organs represents a promising approach to increase the pool of organs for transplantation. The technology involves obtaining complex three-dimensional scaffolds that support cellular activity and functional remodeling though tissue recellularization protocols using progenitor cells. This innovative approach integrates cross-thematic approaches from specific areas of transplant immunology, tissue engineering and stem cell biology, to potentially manufacture an unlimited source of donor organs for transplantation. Summary: Although bioengineered organs are thought to escape immune recognition, the potential immune reactivity toward each of its components has not been studied in detail. Here, we summarize the host immune response toward different progenitor cells and discuss the potential implications of using nonself biological scaffolds to develop bioengineered organs.
- ... A far simpler explanation is that the diversity of trees arises from a single population in which the outcome of an individual cell division is unpredictable, but the distribution of PP, PD or DD outcomes are maintained. The latter interpretation is consistent with genetic lineage tracing in homeostatic mouse epidermis, where modelling argues the tissue is maintained by a single progenitor population which undergoes PP, PD and DD divisions, with the likelihood of PP and DD divisions being equal [15][16][17][18]21,22 . Strikingly, across the 70 trees, the frequency of PP divisions (38%, n= 353) was not significantly different from DD divisions (34%, n=318; Fig. 1g). ...Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.
- ... These cells are primarily found in the bulge of the outer root sheath of hair follicles [6,7]. In vitro studies showed that HFSCs have high cloning capacity with a good regenerative potential [8]. HFSCs can not only differentiate into hair follicles, but can also differentiate into nerve, melanoma, smooth muscle and epithelial cells. ...
- ... 43 Secondly, marker-dependent selection methods such as FACS or antibiotic treatment, which may leave marks in targeted cells and be a source of culture stress for epidermal stem cells, are avoided. 33 Thirdly, epidermal stem cells can be cultured for at least 180 population doublings (PDs) on feeder cells 44,45 or up to passage 20 using TGFβ/BMP inhibitors. 46 programmable nucleases are capable of targeting sites with several mismatches clearly shows that a clonal approach provides a higher degree of safety. ...Epidermolysis bullosa simplex (EBS) is a blistering skin disease due to dominant-negative mutations in either KRT5 or KRT14 resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events, and devise a strategy, which allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here, we report on TALENs, which efficiently disrupt the KRT5 locus in immortalised patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.
- ... 43 Second, markerdependent selection methods such as FACS or antibiotic treatment, which may leave marks in targeted cells and be a source of culture stress for epidermal stem cells, are avoided. 33 Third, epidermal stem cells can be cultured for at least 180 population doublings on feeder cells 44,45 or up to passage 20 using transforming growth factor b (TGF-b)/BMP inhibitors. 46 Human epidermal stem cells cultured on mouse feeder fibroblasts have been used for both cell and gene therapy, 26,28,29 and cultured autologous epidermal grafts that rely on mouse feeder layers have been approved by the Food and Drug Administration (FDA) (Epicel by Sanofi Biosurgery). ...
- ... If the stumps of HFs and SGs at the wound site are intact, skin stem cells at the HF and sweat glands also play a role in facilitating wound healing. 10,22,76 After injury, stem cells at the HF bulge and sweat duct spread out. The release of contact inhibition and the migration process are similar to basal keratinocytes: in parallel to the basal keratinocytes behind the leading edge, which are undergoing fast proliferation, these dispersed HF and sweat gland stem cells provide a source of extra proliferating cells to facilitate the covering of wound surface. ...Significance: Skin serves as a protective barrier for mammals. Epidermal stem cells are responsible for maintaining skin homeostasis. When cutaneous injuries occur, skin homeostasis and integrity are damaged, leading to dire consequences such as acute, chronic, or infected wounds. Skin wound healing is an intrinsic self-saving chain reaction, which is crucial to facilitating the replacement of damaged or lost tissue. Recent Advances: An immense amount of research has uncovered the underlying mechanisms behind the complex and highly regulated wound healing process. In this review, we will dissect the biological process of adult skin wound healing and emphasize the importance of epidermal stem cells during the wound healing. Critical Issues: We will comprehensively discuss the current clinical practices used on patients with cutaneous wounds, including both traditional skin grafting procedures and advanced grafting techniques with cultured skin stem cells. The majority of these leading techniques still retain some deficiencies during clinical use. Moreover, the regeneration of skin appendages after severe injuries remains a challenge in treatment. Future Directions: Understanding epidermal stem cells and their essential functions during skin wound healing are fundamental components behind the development of clinical treatment on patients with cutaneous wounds. It is important to improve the current standard of care and to develop novel techniques improving patient outcomes and long-term rehabilitation, which should be the goals of future endeavors in the field of skin wound healing.
- ... These stem cells were initially identified by label retaining experiments in the hair bulge, which mark the slowest proliferating cells by administering BrdU for a week in newborn mice and then assessing label retention in the skin after four weeks (Cotsarelis et al., 1990). In addition, cells dissected from segments in the bulge region from rat whisker follicles and segments extending from the bulge to the lower outer root sheath from adult human skin more efficiently formed highly proliferative colonies and can be passaged long-term in vitro (Kobayashi et al., 1993; Rochat et al., 1994). Unlike epidermal stem cells, hair bulge stem cells have been successfully enriched and isolated by three approaches. ...The skin is the largest organ of the body and is critical to survival of the organism as a barrier to the environment and for thermal regulation and hydration retention. In order to serve these critical functions, the skin is constantly undergoing renewal and possesses the capacity for repair of wounds, which are dependent on the multiple types of stem cells in the skin. Engineered skin substitutes have a critical medical application to patients with extensive burn wounds. However, current skin substitutes do not restore the normal skin anatomy, lacking the normal appendages of skin including hair follicles, sebaceous glands, and sweat glands as well as the normal mechanical properties of the skin. Advances in stem cell biology and skin morphogenesis hold promise for the ability to markedly improve the engineering of skin substitutes that would ideally be indistinguishable from normal skin.
- ... Perspective demonstrated that holoclone-forming cells have all the hallmarks of stem cells and generate meroclones and paraclones that have properties expected of transient amplifying progenitors (Dellambra et al., 2000;Pellegrini et al., 1999aPellegrini et al., , 1999bPellegrini et al., , 2001Rochat et al., 1994;Ronfard et al., 2000). ...A feature distinguishing human hematopoietic and epithelial stem cells from other equally fascinating stem cells is perhaps their easier translation into a clinical setting. We have devoted nearly our entire scientific career in trying to turn our understanding of epithelial stem cell biology into something that could help people suffering from virtually untreatable diseases of squamous epithelia. We have done that as a team, together with our numerous students, postdocs, technicians and valuable collaborators, clinicians, regulators, and, lately, industrial partners. We had rewarding successes and burning failures, but we always did our best. This award, given by friends and colleagues deserving it more than us, has been the most important recognition of our work. Below, we summarize our story.
- ... Frozen keratinocytes were thawed and cultivated at clonal density on a feeder layer of irradiated or mitomycin C-treated mouse 3T3-J2 fibroblasts. The co-culture was incubated at 37 °C with 10% CO 2 in a 3:1 mixture of Dulbecco-Vogt modification of Eagle's medium (DMEM) and Ham's F12 medium, supplemented with 10% fetal bovine serum (FBS), 1.8 × 10 −4 M adenine hemisulfate salt, 5 μg/ml insulin, 0.4 μg/ml hydrocortisone, 10 −10 M cholera toxin, and 2 × 10 −9 M triiodothyronine (T3), as described previously 1,30 . Keratinocytes between passages 2 and 7 were used for experiments, and the medium was changed every 4 days. ...Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential. Image processing and kernel density estimation were used to automatically extract the area of keratinocyte colonies from phase-contrast images of cultures containing feeder cells. The DeepFlow algorithm was then used to calculate locomotion speed of the colony area by analyzing serial images. This image-processing pipeline successfully identified keratinocyte stem cell colonies by measuring cell locomotion speed, and also assessed the effect of oligotrophic culture conditions and chemical inhibitors on keratinocyte behavior. Therefore, this study provides automated procedures for image-based quality control of stem cell cultures and high-throughput screening of small molecules targeting stem cells.
- ... The holoclone has all hallmarks of a stem cell, being the smallest colony-founding cell, uniquely endowed both with self-renewing capacity and tremendous proliferative potential (Rochat et al. 1994;Pellegrini et al. 1999a). Compelling, yet indirect, evidence of holocloneforming cells being bona fide stem cells came from the notion that clinical success of epidermal cultures for full thickness skin burns and of limbal cultures for the treatment of massive ocular burns requires a defined number of holoclone-forming cells contained in the culture (Pellegrini et al. 1999a;Ronfard et al. 2000;Rama et al. 2010). ...To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.
- ... Later, such evidence was strengthened by studies showing that limbal basal cells have a higher proliferative potential than central corneal basal cells [31,[44][45][46], and they show a differential response to chronic stimulation with phorbol esters [47]. Moreover, limbal epithelial cells have a greater ability to grow in colony forming assays [46], serving as founders of holoclones similar to those described for epidermis [48] and hair follicle [49] (see Fig. 1). Fig. 1 Example of the three types of clonal growth shown by corneal epithelial cells. ...Stem cell niche may be described as an anatomically defined and protected location that provides housing, positioning information and signaling inputs necessary to support normal stem cell activity. Based on the distribution of the differentiation-linked keratins, proliferative potential and wound healing abilities in ocular surface, it was proposed that an anatomical structure, the limbus, was the presumptive site of residence of corneal stem cells. Further analysis determined that the limbus contains a specific anatomical structure that probably provides the microenvironmental characteristics that correspond to the stem cell niche. This structure was termed as the Limbal Epithelial Crypt (LEC). Accumulated evidences show that the LEC is the site where stem cells interact directly and/or indirectly with at least six different cell types: epithelial, stromal, Langerhans cells, melanocytes, and telocytes. In addition, a rich and distinctive vasculature as well as an extensive neural network exist at limbal niches. These cell types, together with growth factors, cytokines and specific components of the Extracellular Matrix establish the conditions for the regulated growth, migration and delayed differentiation of the corneal stem cells. In spite of the wide variety of molecular markers described for limbal epithelial cells, it has been extremely difficult to isolate stem cells. This is explained by the persistence of stem cell markers in the transient amplifying cell population and in the early differentiating cells. Consequently, the use of stem cells in ophthalmic therapy shows variable outcomes and research must be increased before a formal clinical use. Nevertheless, the analysis of the different cells and factors involved in stem cell regulation, may help the development of new therapies based on the interference or stimulation of the signaling pathways and microenvironmental components that control limbal stem cells.
- ... Holoclone-, meroclone-and paraclone-like colonies were identified by various authors in cultures of normal epithelial cells (Larsson et al, 2014;Luo et al., 2009;Rochat et al., 1994;Shortt et al, 2007;Thangappan and Kurzrock 2009;Tudor et al., 2004) and cancer cell lines of various types (Beaver et al., 2014;Harper et al., 2007). Thangappan and Kurzrock were the first who identified holoclone-, meroclone-and paraclone-like colonies in porcine urothelial cell cultures (Thangappan and Kurzrock, 2009). ...Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclone-like colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone-like cells compared to meroclone-and paraclone-like cells (p<0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone-like cells (p<0.05). The level of Ki-67 and PanCK for meroclone-and paraclone-like cells was comparable (p>0.05). β1 and β4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 ± 0.6, 14.7 ± 0.5 and 16.1 ± 0.4, respectively. The results of actin filaments (F-actin) expression were 253 634 ± 6 920 for meroclone-like cells, 198 512 ± 7 977 for paraclone-like cells and 133 544 ± 3 169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods. ABBREVIATIONS PanCK pancytokeratin ZO-1 zonula occludens-1
- ... After the division of a keratinocyte stem cell, one daughter cell is destined to differentiate and move upwards, while the other remains in the basal cell layer as the new stem cell (Figure 3). Potentially, one epidermal keratinocyte stem cell has the capacity to give rise to enough cells to cover the complete body surface 12 . ...Slow vascularization of bioengineered organs after transplantation constitutes a major hurdle in tissue engineering and regenerative medicine. Providing these organs with functional blood and lymphatic capillaries prior to transplantation (henceforth referred to as prevascularization) may be an attractive solution for this problem, given that the bioengineered capillaries rapidly connect to the recipient’s vasculature in the wound bed. Despite considerable progress in the bioengineering of functional blood capillaries, no data were available neither on the bioengineering of functional human lymphatic capillaries, nor on the integration of a dermal lymphatic network into a skin substitute. Moreover, it remained largely unknown how pre-existing blood and lymphatic capillaries affect the reconstitution of a dermo-epidermal skin substitute (DESS) after its transplantation. The first part of my work aimed at bioengineering prevascularized DESS (PDESS) containing physiologically distinct blood and lymphatic capillaries. For this, human dermal microvascular endothelial cells (HDMEC), dermal fibroblasts and epidermal keratinocytes isolated from human skin biopsies were sequentially seeded into and onto rapidly polymerizing fibrin hydrogels. Within 3 weeks of in vitro culture, lumenized and interconnected capillary networks consisting of blood and lymphatic capillaries spontaneously assembled within PDESS. I found that during the process of prevascularization, the inoculated fibroblasts differentiated into pericytes and specifically stabilized blood capillaries, while they hardly associated with lymphatic capillaries. The lymphatic capillaries presented anchoring filaments, expressed all major lymphatic markers, and could be modulated by lymphangiogenic and anti-lymphangiogenic stimuli. In addition, the lymphatic capillaries took up fluid from the interstitial space in vitro and improved the drainage of fluids from PDESS in vivo. Analysis of PDESS after transplantation revealed a great acceleration of vascular regeneration involving two distinct mechanisms: rapid anastomosis of the bioengineered blood capillaries - followed by lymphatic anastomosis - and accelerated recipient blood and lymphatic angiogenesis. In the second part of this work, I performed a broad analysis of the effects of prevascularization on several aspects of wound healing and skin morphogenesis of DESS. By comparison of PDESS and non-prevascularized DESS (NPDESS) in vivo, I found that the rapid perfusion of the bioengineered blood capillary network increased proliferation and decreased apoptosis of transplanted cells, reduced graft shrinkage, induced the formation of rete ridges and capillary loops and promoted dermal remodelling. Furthermore, PDESS healed by the deposition of randomized collagen bundles instead of the scar-like parallel collagen bundle orientation found after transplantation of NPDESS. Collectively, my data for the first time describe the bioengineering of clinically relevant PDESS containing human blood and lymphatic capillary networks that rapidly connect with the host vasculature after transplantation, thereby markedly accelerating perfusion of the graft. In vitro and in vivo studies indicate that the rapid perfusion of PDESS unlocks latent regenerative processes that shift the healing of DESS from typical scar formation towards regeneration. Die langsame Vaskularisierung künstlicher Organe stellt ein gravierendes Problem im Bereich Tissue engineering und Regenerative Medizin dar. Das Versetzen solcher Organe mit funktionalen Blut- und lymphatischen Gefässen vor der Transplantation (Prevascularization) repräsentiert eine attraktive Lösung für dieses Problem, bedingt jedoch, dass sich die künstlichen Gefässe nach der Transplantation schnell mit den Gefässen des Empfängers verbinden (Anastomose). Trotz bemerkenswerter Fortschritte in der Herstellung künstlicher Blutgefässe gab es bisher keine Berichte über die Herstellung von funktionalen lymphatischen Gefässen oder über die Versetzung von dermo- epidermalen Hautsubstituten (DESS) mit solchen lymphatischen Gefässen. Ausserdem war nur sehr wenig über den Effekt von konstruierten Blut- und lymphatischen Gefässen auf den Heilungsprozess eines dermo-epidermalen Hautersatzes bekannt. Das Ziel des ersten Teils meiner Arbeit war die Herstellung von prevaskularisierten dermo-epidermalen Hautsubstituten (PDESS), die künstliche Blut- und lymphatische Gefässe beinhalten. Dafür wurden human dermal microvascular endothelial cells (HDMEC), dermale Fibroblasten und epidermale Keratinocyten von einer Hautbiopsie isoliert und zuerst in und dann auf ein schnell polymerisierendes Fibrin Hydrogel gesäht. Innert 3 Wochen entstand so spontan ein Netzwerk verbundener Kapillaren mit Lumen, bestehend aus Blut- und lymphatischen Gefässen. Die Fibroblasten differenzierten während der Prevaskularisierung zu Perizyten und stabilisierten spezifisch die Blutkapillaren, während sie kaum mit lymphatischen Gefässen assoziierten. Die lymphatischen Gefässe entwickelten Anchoring filaments, exprimierten alle wichtigen lymphatischen Marker und konnten mittels lymphangiogenen und anti-lymphangiogenen Stimuli moduliert werden. Ausserdem waren sie fähig zur Flüssigkeitsaufnahme in vitro und verbesserten den Flüssigkeits-Abtransport in vivo. Die Analyse von PDESS nach der Transplantation hat gezeigt, dass Prevaskularisierung die vaskuläre Regeneration mittels zwei verschiedener Mechanismen beschleunigt: schnelle Anastomose der Blutgefässe – gefolgt von der Anastomose der lymphatischen Gefässe – und Beschleunigung des Einwuchses von Blut und lymphatischen Gefässen des Empfängers in das Hautsubstitut. Im zweiten Teil meiner Arbeit führte ich eine ausgiebige Analyse der Effekte von Prevaskularisierung auf die Heilung und Hautmorphogenese von PDESS durch. Mittels eines Vergleichs von PDESS und nicht-prevaskularisierten DESS (NPDESS) in vivo habe ich herausgefunden, dass die schnelle Perfusion der künstlichen Blutgefässe die Proliferation der transplantierten Zellen unterstützt und deren Apoptose vermindert, die Schrumpfung des Hautsubstituts reduziert, die Entwicklung von Rete ridges und Capillary Loops induziert und das dermale Remodelling fördert. Ausserdem heilten PDESS mittels Deposition von zufällig orientierten Kollagenbündeln anstatt von parallelen, Narben- Kollagenbündeln wie nach der Transplantation von NPDESS. Diese Arbeit beschreibt zum ersten Mal ein klinisch relevantes dermo-epidermales Hautsubstitut, welches sowohl Blut- als auch lymphatische Gefässe beinhaltet, durch deren schnelle Anastomose die Perfusion des Transplantats merklich beschleunigt wird. In vitro und in vivo Studien deuten darauf hin, dass die schnelle Perfusion ruhende regenerative Prozesse in PDESS aktiviert, welche den Heilungsprozess von einer typischen Narbenformation in Richtung Regeneration leiten.
- ... Hair follicles, which go through continuous cycles of regeneration (anagen), involution (cata- gen), and resting (telogen) phases are maintained by distinct multipotent stem cells located in their upper, constant region (the bulge) ( Kobayashi et al., 1993;Rochat et al., 1994). During homeostasis, these stem cells only renew the epithelial cell lineages of the hair follicle; however, if a wound needs to be healed, they can participate to the regeneration of the interfollicular epidermis ( Ito et al., 2005;Claudinot et al., 2005). ...Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demonstrate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo amplification of stem cells, the two limiting steps of cell replacement therapy.
- ... Untreated and genetically corrected RDEB-Ks (initially at 21 days post-transduction corresponding to passage 8) were analyzed for their regenerative capacity by using the colony-forming efficiency assay originally developed by Rochat and colleagues. 66 Briefly, untreated and genetically corrected RDEB-Ks were plated for three consecutive passages (at passages 9, 10, and 11) at a density of 500 or 100 cells in 10-cm Petri dishes coated with irradiated 3T3-J2 murine embryonic fibroblast (MEF) as a feeder layer. After 13 days of culture, clones were fixed, stained with 1% rhodamine, counted under a dissecting microscope, and quantified for their growth potential. ...
- ... Untreated and genetically corrected RDEB-Ks (initially at 21 days post-transduction corresponding to passage 8) were analyzed for their regenerative capacity by using the colony-forming efficiency assay originally developed by Rochat and colleagues. 66 Briefly, untreated and genetically corrected RDEB-Ks were plated for three consecutive passages (at passages 9, 10, and 11) at a density of 500 or 100 cells in 10-cm Petri dishes coated with irradiated 3T3-J2 murine embryonic fibroblast (MEF) as a feeder layer. After 13 days of culture, clones were fixed, stained with 1% rhodamine, counted under a dissecting microscope, and quantified for their growth potential. ...Recessive dystrophic epidermolysis bullosa is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by a wide variety of mutations in COL7A1-encoding type VII collagen, which is essential for dermal-epidermal adhesion. Here we demonstrate the feasibility of ex vivo COL7A1 editing in primary RDEB cells and in grafted 3D skin equivalents through CRISPR/Cas9-mediated homology-directed repair. We designed five guide RNAs to correct a RDEB causative null mutation in exon 2 (c.189delG; p.Leu64Trpfs*40). Among the site-specific guide RNAs tested, one showed significant cleavage activity in primary RDEB keratinocytes and in fibroblasts when delivered as integration-deficient lentivirus. Genetic correction was detected in transduced keratinocytes and fibroblasts by allele-specific highly sensitive TaqMan-droplet digital PCR (ddPCR), resulting in 11% and 15.7% of corrected COL7A1 mRNA expression, respectively, without antibiotic selection. Grafting of genetically corrected 3D skin equivalents onto nude mice showed up to 26% re-expression and normal localization of type VII collagen as well as anchoring fibril formation at the dermal-epidermal junction. Our study provides evidence that precise genome editing in primary RDEB cells is a relevant strategy to genetically correct COL7A1 mutations for the development of future ex vivo clinical applications.
- ... In contrast, less than 5 % clonogenic cells were found located in the matrix area of the follicle (lower part of HF, sometimes called "the bulb") (Kobayashi, Rochat et al. 1993). The same team showed that clonogenic cells of the human hair follicle are also concentrated in a region below the midpoint of the hair follicle and outside the hair bulb, in the site of insertion of arrector muscles; however it is important to notice that the morphology of the human hair follicle does not include a "bulge like" region (Rochat, Kobayashi et al. 1994). ...Squamous cell carcinoma (SCC) is the most frequent metastatic skin cancer. His etiology is linked to exposure to ultraviolet radiation (UVR). Xeroderma pigmentosum C (XP-C) is a genetic disorder characterized by a severe susceptibility to aggressive SCCs following minimal exposure to UVR. XP-C cells are deficient in nucleotide excision repair (NER) of UV-induced DNA lesions. XP-C dermal fibroblasts expresse a phenotype resembling that of stromal fibroblasts associated to cancer cells with accumulation of reactive oxygen species and over expression of matrix metalloproteinase 1 (MMP1). We explored the effects of XP-C fibroblasts on migration and invasion of SCC cells. In organotypic skin cultures, XP-C fibroblasts promote the invasion of SCC cells. Also, scratch healing of SCC cells is enhanced with culture supernatants of XP-C fibroblasts through a mitogenic effect connected to increased ratio of SCC cells in the G2-M phase of the cell cycle. We show that XP-C fibroblasts overexpress the hepatocyte growth factor/scatter factor (HGF/SF) and activate the c-Met receptor and the p38 and JNK pathways in SCC cells. Blockage of HGF inhibits c-Met, p38 and JNK activation and prevented invasiveness of SCC cells within dermal equivalents. Spheroid assays show that XP-C fibroblasts lead SCC invasions. Our data indicate for the first time that XP-C fibroblasts are responsible for the formation of a permissive microenvironment towards SCC cells proliferation and invasion. Therapies targeting XP-C fibroblasts may be considered as a way to control aggressive cancer in XP-C patients.
- Background: A previous study reported that calcineurin inhibition by cyclosporin A (CsA) showed tumor-enhancing effects through the induction of the ATF3 transcription factor and the associated suppression of p53. The development and aggressiveness of cutaneous squamous cell carcinoma (SCC) may be determined by cancer stem cell populations, which have self-renewing potential.
- Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i) compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii) the lower isthmus (comprising the bulge) harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii) unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv) the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.
- Cardiovascular disease is the leading cause of death in developed countries, but we lack the ability to regenerate cardiac tissue. Cell-based therapy holds promise to repopulate a damaged heart with functional cardiomyocytes. Developing technologies to produce cells for transplantation is key to the success of this approach. Pluripotent stem cells (PSC) are an ideal starting material for cell-based therapies because they can be expanded indefinitely in culture and their plasticity gives them the potential to regenerate any tissue or organ. The issue of teratoma formation in the host may be avoided by devising methods to differentiate PSC toward a desired lineage before transplantation. Because the heart is vital for life, and the biggest source of human morbidity and mortality, in vitro differentiation of PSC into cardiomyocytes for cell-based treatment of heart disease is an area of intense research. Exogenous expression of vital cardiogenic genes in PSC can be a powerful tool. Transduction of PSC with recombinant viral vectors can deliver genes to activate cardiac programming and drive differentiation toward a cardiac lineage. Replication defective HSV vectors efficiently transduce PSC and can be engineered to express genes that alter the cellular differentiation program. The goal of this research was to develop highly defective HSV vectors to express multiple cardiac transcription factors in embryonic stem cells to increase their cardiogenic potential. Vectors vββG4Nk, vG4Nk, and vGTM were engineered to express GATA4 and NKX2.5 or GATA4, TBX5, and MEF2c in PSC with high efficiency and low toxicity. Transduction of mESC with these vectors induced the expression of endogenous genes that are vital for cardiogenesis. Differentiation of mESC transduced with cardiogenic HSV vectors had a positive impact on terminal cardiomyocyte differentiation, producing many more embryoid bodies with beating cardiomyocytes than those transduced with control vectors. In addition, we found that delaying the drastic dilution of viral genomes that occurs over the time interval to terminal differentiation could enhance the outcome. Our results indicate that infection of mESC with cardiogenic HSV vectors has long reaching effects on mESC differentiation, supporting the suggestion that HSV vectors can be a useful tool for producing lineage related changes in differentiating PSC to generate specialized cell types.
- Overview of modern data on morphology of the skin derivatives in the higher vertebrates is given. Analysis of convergent similarities between the hair and feathers themselves as well as between their follicles makes it possible to forward a "generative" concept of the evolutionary origin of various ecto-mesodermal derivatives, such as keratinized dermal appendages (scales, feathers, hair). This concept appeared as a result of the author's studies on the skin derivatives, as well as of the data on molecular biology and the tissue engineering showing similar mechanisms of morphogenesis of the dermal appendages. Recurrently published ideas on various heterochronies in generations of the skin derivatives both in the onto- and the phylogeneses are also taken into acount. Various dermal appendages have appeared in the evolution of the higher vertebrates as independent generations of the ecto- and mesodermal tissues. Their parallel origin was caused by similar changes in the metabolism and molecular regulation of morphogenesis.
- Purpose: To compare the regenerative potential of cultured oral mucosal epithelial cells sheets (COMECs) from Stevens-Johnson syndrome (SJS) subjects with those from non-SJS subjects. Methods: Human oral mucosal epithelial cells from SJS and non-SJS subjects were cultured, and colony-forming efficiency (CFE), proliferative and migration potential, expression of cytokines/growth factors and stem cells were compared. COMECs from SJS and non-SJS subjects were transplanted into 12 limbal stem cell-deficient rabbits, and their regenerative potential was analyzed at 1 week after transplantation. Results: CFE (p>0.05, student's t test), cell proliferation potential (p>0.05, two-way ANOVA) and expression of the cytokeratins (K3, K4, K13, K19) in the oral mucosal epithelial cells from SJS subjects were similar to those of the cells from non-SJS subjects. The initial migratory potential of SJS cells was delayed compared to that of non-SJS cells (p <0.05, RM two-way ANOVA). The SJS cells expressed lower levels of EGF and higher levels of VEGF compared to that of non-SJS cells (p<0.05, one-way ANOVA). In vivo transplanted SJS-COMECs showed similar expression of K3, K4, and K13, proliferation markers (Ki-67; p>0.05, Mann-Whitney U test), and stem cell markers (p63; p>0.05, Mann-Whitney U test) compared to non-SJS COMECs. The initial epithelial defects in vivo were larger in the eyes treated with SJS-COMECs on day 3 (p<0.01, RM two-way ANOVA), but no differences were observed by day 7 between SJS- and non-SJS-COMECs. Conclusions: These results suggest that, aside from differences in migratory potential, oral mucosal epithelial cells from SJS and non-SJS subjects are comparable in their regeneration potential in treating limbal stem cell deficiency.
- L'épidermolyse bulleuse dystrophique récessive (EBDR) est une maladie génétique cutanée due à des mutations de perte de fonction du gène COL7A1 codant pour le collagène VII. Le collagène VII forme les fibres d'ancrage, structures essentielles pour l'adhésion de l'épiderme au derme sous-jacent. Les patients EBDR développent dès la naissance, des décollements bulleux de la peau et des muqueuses responsables de plaies chroniques et de graves complications locales et systémiques. La survenue de carcinomes épidermoïdes cutanés agressifs reste la première cause de décès. Il n'existe pas de traitement à ce jour. Les cellules stromales mésenchymateuses (CSM) sont des cellules multipotentes, isolées à partir de tissus adultes (moelle osseuse, tissu adipeux) ou de tissus périnataux (cordon ombilical). Des travaux antérieurs ont montré que les injections locales et systémiques de CSM issues de la moelle osseuse (CSM-MO) allogéniques ont un potentiel pour réduire l'inflammation cutanée et améliorer la cicatrisation des plaies chez les patients EBDR. Ces améliorations cliniques sont cependant transitoires et les mécanismes d'action des CSM-MO dans l'EBDR ainsi que leur durée de vie après injection sont mal connus. Les CSM-MO pourraient agir via leurs propriétés immunomodulatrices, anti-fibrotiques, pro-angiogéniques, par un effet paracrine permettant l'expression de collagène VII endogène et/ou la sécrétion de collagène VII par les CSM-MO injectées. L'objectif de cette thèse a été d'étudier le potentiel thérapeutique des CSM dans des modèles précliniques de l'EBDR. Nous avons tout d'abord montré que les CSM-MO expriment une quantité d'ARNm de COL7A1 et de collagène VII comparable aux fibroblastes dermiques sains en culture. Nous avons ensuite évalué la capacité des CSM-MO humaines à survivre et produire du collagène VII à la jonction dermo-épidermique (JDE) à long terme après une injection locale dans des peaux équivalentes humaines EBDR greffées sur souris nude. L'injection intradermique (ID) de CSM-MO in vivo a permis de restaurer l'expression du collagène VII ainsi que la formation de fibres d'ancrage à la JDE jusqu'à 6 mois après l'injection. Les CSM-MO sont retrouvées dans la peau équivalente jusqu'à 4 mois après l'injection. Ces résultats montrent qu'une injection ID unique de CSM-MO in vivo permet de rétablir une production prolongée de collagène VII synthétisé par les cellules injectées et d'améliorer l'adhésion dermo-épidermique de la peau équivalente EBDR. Nous avons ensuite comparé l'efficacité des CSM issues de la gelée de Wharton de cordon ombilicaux (CSM-CO) humains aux CSM-MO suivant la même méthodologie que précédemment. Les CSM-CO expriment en culture une quantité d'ARNm de COL7A1 et de collagène VII supérieures aux CSM-MO et fibroblastes dermiques sains. Une injection unique ID de CSM-CO dans la peau EBDR équivalente greffée permet de rétablir une faible expression de collagène VII jusqu'à 4 mois après l'injection. Les CSM-CO sont détectées dans la peau équivalente jusqu'à 2 mois après l'injection. Ces données montrent que les CSM-CO ont une capacité moindre à restaurer l'expression du collagène VII à la JDE comparativement aux CSM-MO injectées dans le même modèle de xénogreffes EBDR. Ces résultats ouvrent la perspective d'une thérapie génique ex vivo utilisant des CSM-MO murines Col7a1-/-. Les souris Col7a1-/- reproduisent les lésions cutanées et muqueuses observées chez les patients EBDR. L'espérance de vie de ces animaux est très réduite. Les CSM-MO murines Col7a1-/- transduites en culture à l'aide d'un vecteur rétroviral SIN exprimant COL7A1 produisent en moyenne 30 fois plus de collagène VII que les CSM-MO murines WT. Des expériences in vivo sont nécessaires pour déterminer si l'injection de CSM-MO génétiquement corrigées ont le potentiel de traiter des lésions cutanées et muqueuses, pour définir la dose optimale et la durée de l'effet chez l'animal. Ceci constituerait une étape importante vers la clinique.
- Cancers arising in the oesophageal epithelium are among the most common fatal tumors in the world. Despite this, comparatively little is known about the cell biology and organization of this tissue. Recently, in vitro and in vivo techniques developed over the past 30 years for the study of the epidermis have been applied to the study of the oesophageal epithelium. This approach, combined with data from previous histochemical studies, has lead to the identification and isolation of putative oesophageal epithelial stem cells. Oesophageal epithelial stem cells demonstrate several unusual properties, and their identification may facilitate studies on oesophageal carcinogenesis.
- Epithelial stem cells of the ocular surface are essential for the maintenance of corneal transparency and therefore for vision. Human corneal/limbal epithelial stem cells (LESCs) are believed to reside in the limbus, the interface between the peripheral cornea and neighboring conjunctiva. A specific anatomical microenvironment called the niche regulates the proliferative and differentiation potential of LESCs and their daughter cells. This review covers multiple structural and functional aspects of the human limbal epithelial stem cell niche, including: anatomical features of the niche, composition of the local extracellular matrix, soluble factors and signaling pathways, cell-to-cell interactions with surrounding stromal niche cells and melanocytes.
- Since the hair follicle undergoes cycles of degeneration and regeneration throughout life, it has been assumed that hair follicle growth is controlled by stem cells. Currently, the best characterized hair follicle stem cell population is the bulge stem cell which resides in a discrete microenvironment called the bulge. Bulge stem cells have the capacity to differentiate into all epithelial components of the hair follicle, and also participate in the repair of the skin after injury. The ability to purify and culture human hair follicle bulge stem cells offers fascinating therapeutic potential for alopecia as well as for epithelial tissue engineering.
- Repair of large skin defects caused by burns, trauma, or tumor operations is a clinical challenge. Hair follicle stem cells (HFSCs) are involved in epithelialization of wounds, formation of new hair follicles and promote vascularization in the newly formed skin, and human acellular amniotic membrane (hAAM) is a promising scaffold for skin substitute. Here, we investigated the ability of rat HFSCs (rHFSCs) combined with an hAAM to repair full thickness skin defects in nude mice. The effect of the rHFSC‐hAAM composite on the repair of skin defects in nude mice was assessed by hematoxylin and eosin staining, immunohistochemistry, and EdU‐labeled cell tracking. Isolated and cultured rHFSCs had strong cloning and proliferation potentials. Immunofluorescence staining and flow cytometry assays showed that rHFSCs expressed high levels of integrin α6, CK15, p63, and Sox9. Cells cultured in hAAM showed flaky and cluster‐like morphology and were able to adhere and grow effectively. After transplantation, the rHFSC‐hAAM composite promoted wound healing in nude mice. Moreover, cells in the rHFSC‐hAAM composite were directly involved in hair follicle formation and angiogenesis of tissue around the hair follicle. These results provide an experimental and theoretical basis for the clinical application of HFSCs in repair of human skin defects and a new approach for skin tissue engineering.
- The epidermis basal layer is composed of two keratinocyte populations: Keratinocyte Stem cells (KSC) and Transitory Amplifying (TA) cells that arise from KSC division. Unfortunately, no specific marker exists to differ between KSC and TA cells. Here, we aimed at comparing two different methods that pretended to isolate these two populations: (i) the rapid adhesion method on coated substrate and (ii) the flow cytometry method, which is based on the difference in cell surface expressions of the α6 integrin and transferrin receptor (CD71). Then, we compared different parameters that are known to discriminate KSC and TA populations. Interestingly, we showed that both methods allow enrichment in stem cells. However, cell sorting by flow cytometry (α6high/CD71low) phenotype leads to a better enrichment of KSC since the colony forming efficiency is five times increased versus total cell suspension, whereas it is only 1.4 times for the adhesion method. Moreover, α6high/CD71low cells give rise to a thicker pluristratified epithelium with lower seeding density and display a low Ki67 positive cells number, showing that they have reached the balance between proliferation and differentiation. We clearly demonstrated that cells isolated by a rapid adherent method are not the same population as KSC isolated by flow cytometry following α6high/CD71low phenotype.
- Background: Crocodile oil and its products are used as ointments for burns and scalds in traditional medicines. A new ointment formulation - crocodile oil burn ointment (COBO) was developed to provide more efficient wound healing activity. The purpose of the study was to evaluate the burn healing efficacy of this new formulation by employing deep second-degree burns in a Wistar rat model. The analgesic and anti-inflammatory activities of COBO were also studied to provide some evidences for its further use. Materials and methods: The wound healing potential of this formulation was evaluated by employing a deep second-degree burn rat model and the efficiency was comparatively assessed against a reference ointment - (1% wt/wt) silver sulfadiazine (SSD). After 28 days, the animals were euthanized and the wounds were removed for transversal and longitudinal histological studies. Acetic acid-induced writhing in mice was used to evaluate the analgesic activity and its anti-inflammatory activity was observed in xylene -induced edema in mice. Results: COBO enhanced the burn wound healing (20.5±1.3 d) as indicated by significant decrease in wound closure time compared with the burn control (25.0±2.16 d) (P<0.01). Hair follicles played an importance role in the physiological functions of the skin, and their growth in the wound could be revealed for the skin regeneration situation. Histological results showed that the hair follicles were well-distributed in the post-burn skin of COBO treatment group, and the amounts of total, active, primary and secondary hair follicles in post-burn 28-day skin of COBO treatment groups were more than those in burn control and SSD groups. On the other hand, the analgesic and anti-inflammatory activity of COBO were much better than those of control group, while they were very close to those of moist exposed burn ointment (MEBO). Conclusions: COBO accelerated wound closure, reduced inflammation, and had analgesic effects compared with SSD in deep second degree rat burn model. These findings suggest that COBO would be a potential therapy for treating human burns. Abbreviations: COBO, crocodile oil burn ointment; SSD, silver sulfadiazine; MEBO, moist exposed burn ointment; TCM, traditional Chinese medicine; CHM, Chinese herbal medicine; GC-MS, gas chromatography-mass spectrometry.
- Hair follicle stem cells (HFSCs) are noted for their relative quiescence and therefore can be distinguished from other cells by their differential history of cell division. Replicating cells can be labeled by pulsing the animals repeatedly with 5-bromo-2′-deoxyuridine (BrdU) or tritiated thymidine ([³H]TdR), thymidine analogs that get incorporated into DNA during DNA synthesis. Because dividing cells dilute the label after each cell division, frequently dividing cells will lose the label over time while slow cycling cells will retain the label and thus are termed label retaining cells (LRCs). [³H]TdR can be visualized by autoradiography and BrdU can be detected by immunofluorescence with anti-BrdU antibodies. Alternatively, a well-established tet-regulatable transgenic mouse model can be used to express histone H2B-GFP in epithelial proliferative cells and their dilution and retention of the GFP signal can be followed. In this chapter, we detail the steps to perform BrdU pulse-chase and H2B-GFP pulse-chase experiments to identify quiescent cells in the hair follicle.
- Background Recent studies have shown that human beta-defensin-1 (hBD-1) and (human beta-defensin-2 hBD-2), which are antimicrobial peptides produced by the skin, play a role in the pathogenesis of acne vulgaris (AV). Objective The aim of this study was to determine the role of antimicrobial peptides in the pathogenesis of AV and enlighten the effects of doxycycline and isotretinoin in the expression of these defensins in AV. Materials and Methods A total of 44 patients (22 patients in each group) with Grade 6 and 8 AV who were indicated doxycycline or isotretinoin for their treatment, and 20 healthy volunteers were included in this study. Pretreatment cutaneous samples were obtained from pustular lesions and uninvolved skin of AV patients and were repeated after the treatment. Only one biopsy was obtained from controls. Results Cutaneous levels of hBD-1 and hBD-2 were significantly increased in AV patients when compared with healthy controls (P<0.05). Doxycycline therapy achieved a decrease in hBD-1 levels (P<0.05), whereas isotretinoin therapy achieved a reduction in hBD-2 levels when compared with pretreatment levels (P<0.05). Posttreatment hBD-1 and hBD-2 levels were not different between doxycycline and isotretinoin groups (P>0.05). Conclusion In the light of these results, it was reasonable to assume the role of hBD-1 and hBD-2 in the pathogenesis of AV. Our results showing a significant reduction in hBD-1 staining with doxycycline treatment and in hBD-2 with isotretinoin suggested that some part of their anti-acne effect worked through these mechanisms.
- Background Miliacin, the main triterpenoid from millet, is known to stimulate keratinocyte metabolism and proliferation. Polar lipids are able to form vesicles with active compounds and to improve their bioavailability. Objectives We aimed to demonstrate potential benefits of a solution of miliacin encapsulated within polar lipids (MePL) on telogen effluvium prevention and hair condition in women. METHODS After preliminary cell proliferation studies, a placebo‐controlled, multicentric, randomized, double‐blind trial was performed on sixty‐five nonmenopausal women affected by telogen effluvium, to assess the efficacy of a 12‐week oral supplementation with MePL. Telogen and anagen densities were determined by phototrichogram analysis. Scalp dryness and hair brightness were clinically evaluated using a Likert scale. Results MePL further enhanced cell proliferation in hair bulb from human scalp than miliacin alone. Compared to the placebo treatment, MePL supplementation significantly reduced telogen density after 12 weeks of treatment. An increase of anagen density was observed in both groups, although there was no significant difference between the two treatments. Scalp dryness was more decreased in the MePL group than in the placebo group. A better improvement of hair brightness was also observed after 12 weeks of supplementation with MePL. Conclusion Twelve weeks of MePL supplementation significantly reduced the hair density in the telogen phase and, in parallel, improved scalp dryness and hair condition. These effects could be linked to MePL activity on cell proliferation in hair bulb. MePL is an original association of plant extract that could help to prevent and/or limit hair loss in women.
- Single-cell cloning is essential in stem cell biology, cancer research, and biotechnology. Regulatory agencies now require an indisputable proof of clonality that current technologies do not readily provide. Here, we report a one-step cloning method using an engineered pipet combined with an impedance-based sensing tip. This technology permits the efficient and traceable isolation of living cells, stem cells, and cancer stem cells that can be individually expanded in culture and transplanted.
- Although the loss of scalp hair is distressing and many medical treatments focus on its restoration, the removal of body hair has been adopted since ancient times. Beauty standards, which r eflect the culture of each society, have been presenting the depilated body as absolutely desirable. Through the ages various methods of hair removal have been used depending on the requirements of the individuals. In recent years, Laser and Intense Pulse Light devices have been considered as the most promising solution for excess hair growth, without excluding the efficacy of other methods to induce satisfactory epilatory results. The enzyme-based hair removal method has received little recognition even though experimental and clinical data support its efficacy to provide long term or even permanent epilation. The present review presents these data and examines the likelihood of considering the aforementioned method as ideal.
- Stem cells are by definition present in all self-renewing tissues and are believed to play a central role in cell growth and differentiation. Existing evidence suggests that a subpopulation of epidermal basal keratinocytes represents stem cells; however, these cells have never been positively identified. In this paper we review evidence that in monkey palm epidermis there exist two morphologically distinct subpopulations of basal keratinocytes that are spatially segregated. One population, located in the shallow rete ridges, is characterized by a cytoplasm filled with tonofilaments and a highly convoluted ("serrated") dermal-epidermal junction; these cells may play a role in anchoring the epidermis to the dermis. In contrast, the other population, located at the tips of deep rete ridges, is characterized by a "primitive" cytoplasm containing abundant melanosomes and a relatively flattened ("nonserrated") dermal-epidermal junction. Tritiated thymidine labeling experiments suggest that the nonserrated basal keratinocytes are slow-cycling; however, a highly proliferative population of keratinocytes can be identified immediately above these basal cells. These findings are consistent with the concept that the nonserrated basal keratinocytes may represent stem cells that give rise to suprabasally located, transient amplifying cells before undergoing terminal differentiation. Monkey palm epidermis provides a model system for further studies of primate epidermal stem cells.
- We have used tritiated thymidine (3HT) labelling and immunoperoxidase staining of 67 K polypeptide in human epidermis to study normal skin before and after irritation induced by application of croton oil (20% and 50% in olive oil). By this double labelling method, it was possible to identify, on the same skin section, DNA-synthesizing nuclei and epidermal cells which contained 67 K polypeptide. Our results clearly indicate that the germinative compartment of normal epidermis is a mixed population comprising basal and adjacent suprabasal cells; as a rule, absence of expression of 67 K polypeptide, which characterizes all basal cells, could not be regarded as a good marker to distinguish the germinative from the differentiating compartments. In mild primary irritation, the ratio of 3HT-labelled undifferentiated cells to keratinizing ones was similar to that observed in normal epidermis. In severe irritation, this homeostatic process was altered, as 3HT-labelled cells were mainly 67 K negative.
- The events in epidermal development are distinctive for each trimester. During the first trimester, the epidermis changes from a single-layered covering to a stratified epithelium. Many of the structural changes that occur establish cellular integration and support to stabilize this barrier between the embryo and its environment. The morphologic and physiologic evidence, indicates, however, that the epidermis is not a functional barrier to diffusible compounds during this period, a time when the environment is comparable and perhaps important in providing substances needed for development. This is also a period in which all of the primordia of the epidermal appendages are initiated and the immigrant cells begin to migrate in among the keratinocytes. In the second trimester, the system is established and the pattern for the adult epidermis is laid out. Further development and differentiation are required to refine the pattern into a functional system which will acquire the properties of the adult tissue. The work of the second trimester then is to expand and elaborate certain structures and to change and modify others. The functional state is altered as glands become secretory and the epidermis differentiates into a functional barrier, first by regression of the periderm and secondly with keratinization of the epidermis proper. Exclusion of the amniotic fluid occurs at a time when this formerly compatible environment becomes hostile. The epidermis, thus formed in nearly its final form, ceases, new developmental events and subsists throughout the third trimester until near birth in basically the same condition. Most of the data to support these generalizations have come from an impressive body of morphologic information. From the present review, it should be quite apparent that the quantitative and functional aspects of epidermal development are areas where inquiry is needed so that speculation may be substantiated by data and replaced with fact.
- A study was made on the stem cell concepts. When we are talking about stem cells, we do not necessarily, and certainly not always talk about hemopoietic stem cells. There are many kinds of stem cells in the adult organism and there is one definition only which can apply to all these stem cells, and that is: a self-maintaining capacity. This means that stem cells can maintain their numbers for at least one life span of the organism. This means that 'stemness' implies an extensive self-maintaining capacity, and we mean self-maintaining capacity for many cell cycles, which in the mouse in most cases of 200 cell cycles, and in man - especially with a hemopoietic or intestinal stem cell system - well in excess of a thousand cell cycles. Every cell in the body, after the zygote and the first few cleavage divisions, is already a differentiated cell. That is: stem cells in the late embryonic crypt stem cells, skin epithelial stem cells or liver stem cell, are already differentiated cells. It is true that there are some stem cells which can give rise to further differentiated products, but not all stem cells do so. The author has given in his paper some of his ideas, about G(o) and genetic housekeeping, proliferation control and cell interaction, which like all concepts are pure fiction until such time that appropriate experimental evidence corroborates them. But at least, with the hemopoietic stem cells assay methods are available and with a little bit of clear thinking, clear definition - according to the author - and proper use of the available assays, the ideas can be tested. (E. Szirmai - Stuttgart) - Stuttgart, GFR)
- During keratinocyte maturation, individual cells undergo an orderly succession of biochemical and structural changes. In certain skin disorders alterations in keratinocyte numbers, volumes, and epidermal skin thickness occur. To assess such alterations and to provide base line values for normal human epidermis, a computer assisted histologic technique was developed. Skin biopsies were taken from normal skin on the forearm, back and thigh of 6 adult men. Whole specimens of epidermis were separated from the dermis with collagenase, fixed, stained, and mounted for microscopic examination. From the three dimensional coordinates of epidermal nuclei, epidermal cell volumes, surface density of epidermal cells, and epidermal thickness were determined. Measurement of cell volumes in this way compared favorably with electronic cell sizing of disaggregated epidermal cells in matched samples. The mean densities of nucleated cells per 10(4) mu2 surface area were 452, 483, and 487 for the forearm, back and thigh respectively. This technique will be used to make similar assessments in disorders of abnormal keratinocyte maturation.
- Human diploid epidermis epidermal cells have been successfully grown in serial culture. To initiate colony formation, they require the presence of fibroblasts, but proliferation of fibroblasts must be controlled so that the epidermal cell population is not overgrown. Both conditions can be achieved by the use of lethally irradiated 3T3 cells at the correct density. When trypsinized human skin cells are plated together with the 3T3 cells, the growth of the human fibroblasts is largely suppressed, but epidermal cells grow from single cells into colonies. Each colony consists of keratinocytes ultimately forming a stratified squamous epithelium in which the dividing cells are confined to the lowest layer(s). Hydrocortisone is added to the medium, since in secondary and subsequent subcultures it makes the colony morphology more oderly and distinctive, and maintains proliferation at a slightly greater rate. Under these culture conditions, it is possible to isolate keratinocyte clones free of viable fibroblasts. Like human diploid fibroblasts, human diploid keratinocytes appear to have a finite culture lifetime. For 7 strains studied, the culture lifetime ranged from 20-50 cell generations. The plating efficiency of the epidermal cells taken directly from skin was usually 0.1-1.0%. On subsequent transfer of the cultures initiated from newborns, the plating efficiency rose to 10% or higher, but was most often in the range of 1-5% and dropped sharply toward the end of their culture life. The plating efficiency and culture lifetime were lower for keratinocytes of older persons.
- A microcolony technique is described for measuring epidermal cell survival 3 days after whole-body X-irradiation. This assay provides a cell D0 value of 233 +/- 11 rad and a zero-dose extrapolate of 1-23 x 10(4) cells/cm2 for mice irradiated in oxygen 20 hours after hair plucking. The microcolony cellularity had an apparent doubling-time of 25 hours which may be an upper limit at least for some clones. The clones appeared to fragment continually and form new daughter clones, suggesting that few would form macroscopic nodules. Many of the clones were also apparently associated with hair follicles.
- Adult rat pelage follicle dermal papilla cells induced follicle neogenesis and external hair growth when associated with adult footpad skin epidermis. They thus demonstrated a capacity to completely change the structural arrangement and gene expression of adult epidermis--an ability previously undocumented for cultured adult cells. Isolation chambers ensured that de novo follicle formation must have occurred by eliminating the possibility of cellular contributions, and/or inductive influences, from local skin follicles. These findings argue against previous suggestions of vibrissa follicle specificity, and imply that the potential for hair follicle induction may be common to all adult papilla cells.
- We report for the first time the successful maintenance and growth of human hair follicles in vitro. Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers' forceps. Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. The increase in length was seen to be attributed to the production of a keratinised hair shaft, and was not associated with the loss of hair follicle morphology. [methyl-3H]thymidine autoradiography confirmed that in vitro the in vivo pattern of DNA synthesis was maintained; furthermore, [35S]methionine labelling of keratins showed that their patterns of synthesis did not change with maintenance. The importance of this model to hair follicle biology is further demonstrated by the observations that TGF-beta 1 has a negative growth-regulatory effect on hair follicles in vitro and that EGF mimics the in vivo depilatory effects that have been reported in sheep and mice.
- In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.
- Germinative epidermal cells in the lower end bulb region of anagen hair follicles are highly active, and give rise to hair fibres through rapid proliferation and complex differentiation. They have often been termed hair follicle stem cells, but owing to difficulties in isolation and identification their properties have previously only been clearly documented in vivo. We aimed to isolate and culture germinative cells in vitro, and used microdissection methods to dissect a small but identifiable group of cells from complete follicles. Transmission electron microscopy confirmed that the isolated cells were identical to germinative epidermal cells in situ. SDS-PAGE was used to show that they did not have the same protein composition as epidermis from their immediate proximity (overlying hair matrix), or from other follicular (outer root sheath) and interfollicular (skin basal) regions. Moreover, the germinative cells were found to display morphology and in vitro behaviour that distinguished them from comparative epidermal cells. When cultured in media and on substrata normally conducive to epidermal cell growth they remained in a quiescent state, and did not divide or differentiate. In contrast to other epidermal cells that formed typical pavement-like arrangements, germinative cells remained uniformly small, round and closely packed. However, when cultured in association with hair follicle dermal papilla cells they were radically stimulated into proliferative and aggregative behaviour. Furthermore, they were able to form organotypic-like structures, and exceptionally for skin-derived cell recombinations, a distinct basal lamina at the papilla-germinative cell junction. These results provide evidence that hair follicle germinative cells have intriguing properties that distinguish them from other follicular epidermis. The finding that they can be activated by dermal papilla cells reflects the intimate nature of the papilla-germinative cell relationship in situ, and should facilitate research into hair growth control mechanisms. The nature of germinative cells is discussed in the wider context of hair follicle stem-cell terminology.
- Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
- Heterokaryons provide a model system in which to examine how tissue-specific phenotypes arise and are maintained. When muscle cells are fused with nonmuscle cells, muscle gene expression is activated in the nonmuscle cell type. Gene expression was studied either at a single cell level with monoclonal antibodies or in mass cultures at a biochemical and molecular level. In all of the nonmuscle cell types tested, including representatives of different embryonic lineages, phenotypes, and developmental stages, muscle gene expression was induced. Differences among cell types in the kinetics, frequency, and gene dosage requirements for gene expression provide clues to the underlying regulatory mechanisms. These results show that the expression of genes in the nuclei of differentiated cells is remarkably plastic and susceptible to modulation by the cytoplasm. The isolation of the genes encoding the tissue-specific trans-acting regulators responsible for muscle gene activation should now be possible.
- Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co-expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.
- Living hair-forming cells (trichocytes) were obtained from basal portions of human, bovine and ovine hair-follicles, free from contaminations of root-sheath epithelia. Their intermediate filament (IF) cytoskeleton was studied by gel electrophoresis of the native, i.e. non-S-carboxymethylated polypeptides, by peptide-map analysis of the individual components, by reconstitution experiments and by immunological methods. The IF protein complement of trichocytes from all three species is characterized by a very similar set of eight highly conserved alpha-keratin polypeptides, comprising four members of the basic (type II; Mr 56,500-60,000) and four members of the acidic (type I; Mr 41,000-44,000) cytokeratin subfamily. None of these eight trichocyte alpha-keratin polypeptides, which form heterotypic complexes and IF in vivo and in vitro, is identical to any of the epithelial cytokeratins of the same species. All the trichocyte-specific cytokeratins are native polypeptides encoded by different mRNAs, as demonstrated by in vitro translation of hair follicle mRNA. The same polypeptides are also found in mature hairs, although with different patterns of modification. Our study provides the first analysis of the native unmodified alpha-keratin polypeptides of trichocytes and hairs and therefore allows a direct comparison of these with the epithelial cytokeratins and other IF proteins from the same species. These findings indicate that, during fetal hair-follicle formation, the differentiation of trichocytes from epithelial cells involves a complete cessation of the synthesis of epithelial cytokeratins and a marked induction of the synthesis of a complex set of trichocyte-specific cytokeratins.
- During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.
- A transglutaminase-catalyzed cross-linking process characteristic of keratinocytes leads to the formation of the insoluble corneocyte envelope. The essentials of this process take place in vitro in a reconstituted system derived from subcellular fractions. A particulate fraction containing membrane-bound envelope precursor proteins and the enzyme transglutaminase is combined with cytosolic proteins; when the enzyme is activated by Ca++, cytosolic proteins are removed from solution and cross-linked to particulate proteins. This interaction is cell-type-specific, since particulates derived from fibroblasts and also containing transglutaminase activity cannot substitute for those of keratinocytes. Involucrin, a cytosolic protein known to be a precursor of the envelope, is more efficiently cross-linked than other cytosolic proteins. The cross-linking of proteins of the particulate fraction (membrane proteins) is promoted by the presence of involucrin.
- Cultures of human epidermal cells may be used to generate epidermis on athymic recipients. We describe two novel techniques for grafting such cultures. Both techniques permit the generation of typical human epidermis within 7 d. Both techniques result in less graft contraction than conventional grafting, and there is no difficulty in distinguishing the human epidermis generated by the graft from the epidermis of the recipient animal. Starting with a single human biopsy, epidermis may be generated on a great many experimental animals; such grafts should therefore provide uniform material for investigation of the properties of human epidermis.
- Hemopoiesis is regulated by a complex series of interactions, including interactions among hemopoietic cells themselves, hemopoietic cells and the extracellular matrix, hemopoietic cells and marrow stromal cells, and hemopoietic cells and growth factors. In vitro culture systems have allowed a reductionist approach to the solution of these various problems and have facilitated experiments at the mechanistic level. The hemopoietic system is organized hierarchically with multipotential self-renewing stem cells, committed progenitor cells, and mature cells. The various stimuli necessary for growth and development of these cells are rapidly being elucidated. The nature of commitment (or differentiation) remains an enigma, but model systems have been developed in which various aspects of this problem can be investigated. In this respect, growth and differentiation factors obviously have a major role to play. Now that many of these factors have been molecularly cloned (and pure target cell populations are available) their role in vivo and their mode of action can be examined.
- A method for the isolation of outer root sheath keratinocytes from plucked human hair follicles and for their subsequent cultivation has been developed. The selective trypsinization of outer root sheath keratinocytes provided a single cell suspension of defined origin within the hair follicle. The 3T3 feeder layer technique supports sustained growth of these cells in that as little as one single plucked hair follicle (yielding approximately 1.5 X 10(4) cells) consistently gave rise to a confluent 35-mm culture dish (with approximately 1.5 X 10(6) cells) within about 2 weeks. The outer root sheath keratinocytes can be serially passaged for up to 3 times and also cryopreserved.
- The purpose of this investigation was to characterize the [3H]thymidine label-retaining and the "maturing" classes of basal cells from the dorsal epidermis of adult SENCAR mice and to compare their early cellular kinetic responses to topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Autoradiography of epidermal whole mounts and cross sections demonstrated that injection of [3H]thymidine every 6 h for 1 week labeled 95% of the basal nuclei, including those in the central region of the epidermal proliferative units. One month later, the labeling index was reduced to 2%; 90% of the label-retaining cells were within a nuclear diameter of the central suprabasal column of the proliferative units. When mice were treated with 2 micrograms of TPA 1 month after labeling, mitotic label-retaining cells were found within 22 h after treatment. Seventy-five percent of the label-retaining cells remained on the basal layer through the 28-h experimental period. In contrast, the basal labeling index following a 1-h pulse of [3H]thymidine was 5%. Eighty-five percent of the labeled cells were found in the periphery of the proliferative units. By 4 days after pulse labeling, most of the originally labeled cells had divided, although vertical cross sections indicated that 92% remained on the basal layer. When mice were treated with TPA on day 4, labeled cells were rarely found in mitosis. Instead, about 60% of the labeled cells were displaced to the suprabasal layers. These observations suggest that 2 classes of epidermal basal cells have different early responses to TPA treatment: the label-retaining cells proliferate, and most of the "maturing" cells continue to differentiate.
- A model for epidermal proliferation of a subpopulation of clonogenic cells is briefly discussed and tested in relation to published results of fractionated irradiation survival for mouse epidermis. It is suggested that since the computed average values for the initial number of clonogenic cells differ by an order of magnitude for the measured values for basal cells in mouse skin, one in ten of the basal cells might be clonogenic. It is pointed out that this explanation is compatible with the observation of hexagonal epidermal units, the central stem cell cycling at a slower rate than the rest. (UK)
- A detailed examination of cell proliferation kinetics in normal human epidermis is presented. Using tritiated thymidine with autoradiographic techniques, proliferative and differentiated cell kinetics are defined and interrelated. The proliferative compartment of normal epidermis has a cell cycle duration (Tc) of 311 h derived from 3 components: the germinative labeling index (LI), the duration of DNA synthesis (ts), and the growth fraction (GF). The germinative LI is 2.7% +/- 1.2 and ts is 14 h, the latter obtained from a composite fraction of labeled mitoses curve obtained from 11 normal subjects. The GF obtained from the literature and from human skin xenografts to nude mice is estimated to be 60%. Normal-appearing epidermis from patients with psoriasis appears to have a higher proliferation rate. The mean LI is 4.2% +/- 0.9, approximately 50% greater than in normal epidermis. Absolute cell kinetic values for this tissue, however, cannot yet be calculated for lack of other information on ts and GF. A kinetic model for epidermal cell renewal in normal epidermis is described that interrelates the rate of birth/entry, transit, and/or loss of keratinocytes in the 3 epidermal compartments: proliferative, viable differentiated (stratum malpighii ), and stratum corneum. Expected kinetic homeostasis in the epidermis is confirmed by the very similar "turnover" rates in each of the compartments that are, respectively, 1246, 1417, and 1490 cells/day/mm2 surface area. The mean epidermal turnover time of the entire tissue is 39 days. The Tc of 311 h in normal cells in 8-fold longer than the psoriatic Tc of 36 h and is necessary for understanding the hyperproliferative pathophysiologic process in psoriasis.
- Two structurally distinct populations of basal keratinocytes, nonserrated and serrated, were observed in cynomolgus monkey and human palm epidermis. Anatomical location, fine structural features, and kinetic properties suggest that nonserrated cells represent a stem cell population and that serrated cells help anchor the epidermis to the dermis.
- We and others have shown previously that corneal keratinocyte stem cells can proliferate in vitro better than their progeny cells. In this paper, we applied this approach to the identification of hair follicular stem cells. When human scalp hair follicles were placed in explant culture, the bulge area yielded best outgrowths. In another experiment, we isolated different subpopulations of human follicular keratinocytes by micro-dissection, dispersed them by trypsin/EDTA into single cells, and grew them in the presence of 3T3 feeder cells. The keratinocytes were then subcultured under identical conditions to compare their in vitro life span. Our results indicate that the life span of keratinocytes of the upper follicle (containing mainly the isthmus area) > sebaceous gland > lower follicle (between the bulge and bulb) > bulb (containing the matrix cells). The cultured upper follicular keratinocytes tend to be small and relatively uniform in size. The poor in vitro growth of matrix cells may reflect their non-stem cell nature and/or special growth requirement(s) satisfied in vivo by the neighboring dermal papilla cells. Unexpectedly, we found that the upper follicular keratinocytes grow even better than epidermal keratinocytes. The existence of a subpopulation of keratinocytes with an in vitro growth potential superior than other known keratinocytes of the skin supports the hypothesis that follicular stem cells reside in the upper follicle. Our data also raise the possibility that putative follicular stem cells are involved not only in forming the follicle, but also in the long-term maintenance of the epidermis. Finally, we discuss the possibility that keratinocyte stem cells, as defined by their in vivo slow-cycling nature, are absent in culture.
- In the present paper, we show that a multilayered and well-differentiated epidermis can easily and rapidly be generated in vitro from the outer root sheath of human hair follicles deposited on de-epidermized demis. Histologically, this epidermis presented characteristic features of normal human epidermis in vivo. Moreover, markers specific for interfollicular keratinocyte terminal differentiation, such as the K10 keratin, involucrin, membrane-bound transglutaminase, filaggrin and loricrin, were expressed in the reconstructed tissue. By in situ hybridization, keratin K5 and K10 mRNAs were detected in the basal and suprabasal cells, respectively, as in normal human epidermis. The differentiation pattern achieved in this reconstructed epidermis confirms the already reported phenotypical shift from outer root sheath cells to interfollicular keratinocytes and shows that this transition takes place in the absence of living fibroblasts. The differentiation of the reconstructed epidermis thus obtained was modulated by retinoic acid in a dose-dependent manner. This culture system on dead dermis is easier to handle than similar cultures on collagen-fibroblast lattices because of the resistance of dermis to mechanical forces and to collagenolysis. It could represent a valuable wound-healing model and a promising tool for pharmacological studies on in vitro reconstructed skin.
- The epidermis and its related appendages such as the hair follicle constitute the epithelial compartment of the skin. The exact location and distribution of the keratinocyte colony-forming cells within the epidermis or its appendages are unknown. We report that in the rat vibrissa, keratinocyte colony-forming cells are highly clustered in the bulge-containing region. Approximately 95% of the total colonies formed in culture from fractionated vibrissae were in this location and fewer than 4% were located in the matrix area of the follicle. Finer dissection of the bulge-containing region located the colony-forming cells in the small part containing the bulge itself. The segregation of keratinocyte colony-forming cells in the bulge confirms the hypothesis that the bulge is the reservoir of the stem cells responsible for the long-term growth of the hair follicle and perhaps of the epidermis as well.
- Isolated human hair follicle germinative epidermal cells were observed in vitro for the first time. When cultured alone, this small, round, novel cell type did not grow, divide, take on an outer root sheath-type appearance, or display any obvious signs of epidermal differentiation. We have previously described comparable cells from rat vibrissa follicles. However, in combination with human hair follicle dermal papilla populations, the germinative epidermal cells were stimulated into proliferative and complex interactive behaviors. This included the formation of composite organotypic structures containing not only impressively intact basement membrane, but also the hair-specific form, glassy membrane.
- The epidermis is believed to contain two types of proliferating cells: stem cells and cells with a lower capacity for self-renewal and higher probability of undergoing terminal differentiation (transit amplifying cells). We report that keratinocytes with characteristics of stem cells can be isolated from cultured human epidermis on the basis of high surface expression of beta 1 integrins and rapid adhesion to extracellular matrix (ECM) proteins. Among keratinocytes there was a log linear relationship between the relative level of beta 1 integrins on the cell surface and proliferative capacity; furthermore, the cells with the highest colony-forming efficiency adhered most rapidly to type IV collagen, fibronectin, or keratinocyte ECM. Proliferating keratinocytes that adhered more slowly had characteristics of transit amplifying cells: after one to five rounds of division, all of their daughters underwent terminal differentiation. Since stem cells can be isolated to greater than 90% purity on the basis of their adhesive properties, it will now be possible to investigate the mechanisms that regulate the fate of their progeny.
- This essay is a critique of a revolutionary hypothesis, and of the language used to frame it, that offers a novel interpretation for the dynamics of the follicular cycle by distinguishing, distinctly, germinative cells in the bulge from matrical cells in the bulb. Curiously, this intriguing "bulge-activation hypothesis" elicited practically no response in the scientific literature, and our critique is designed to rectify that.
- 1. A direct technique for the measurement of the number of cells in a bone marrow suspension capable of continued proliferation is described. 2. The technique was used to measure the radiation sensitivity of normal mouse bone marrow cells and yielded a D0 of 115 ± 8 rads. © 2012 by Radiation Research Society. All rights of reproduction in any form reserved.
- The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
