P63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2001; 98(6):3156-61. DOI: 10.1073/pnas.061032098
Source: PubMed


The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

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Available from: Graziella Pellegrini
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    • "The present study was aimed to compare the effects human versus mouse EGF activity on cell confluency and culture duration, since it has previously shown that the affinities of mEGF for human low affinity receptors and high affinity receptors are much lower than those of hEGF (Connolly and Rose, 1987). Also we have quantified the effect of human EGF on the expression of corneal stem cell marker (P63) (Pellegrini et al., 2001) versus corneal epithelial differentiation marker (K3) (Rodriguez et al., 1987) in order to define optimal culture conditions to obtain high quantities of undifferentiated cells for autologous transplantation. "
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    ABSTRACT: We evaluate the efficacy and safety of Keraring 355° intrastromal corneal ring segment (ICRS) implantation aided by PocketMaker microkeratome for the correction of keratoconus. Patients underwent ICRS insertion using mechanical dissection with PocketMaker microkeratome and completed 6 months of follow-up. Uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), refraction, topographic findings, safety, efficacy index, and adverse events were reported for six months postoperatively. We evaluated 15 eyes of 15 patients (12 men) with a mean age of 28.87 ± 6.94 years (range 21–49 years). At final postoperative examination, there was a statistically significant reduction in the spherical equivalent refractive error compared to preoperative measurements (−5.46 ± 1.52 to −2.01 ± 1.63 D, ). Mean preoperative UCVA (logMAR) before implantation was 0.79 ± 0.48, and postoperative UCVA was 0.28 ± 0.15, . Mean preoperative BSCVA (logMAR) before implantation was 0.36 ± 0.21; at final follow-up examination BSCVA was 0.18 ± 0.9, . Mean decreased from 48.33 to 43.31 D, . All patients were satisfied with ICRS implantation; 86.7% were moderately to very happy with the results. No intraoperative or postoperative complications were demonstrated. This preliminary study shows that ICRS (Keraring 355°) implantation is an efficient, cost-effective, and minimally invasive procedure for improving visual acuity in nipple type keratoconic corneas.1. IntroductionKeratoconus is a bilateral, progressive, noninflammatory disease of the cornea which often leads to high myopia and astigmatism with an estimated prevalence of approximately 1 in 2000 [1]. In the general population, the incidence of keratoconus is estimated to be between 50 and 230 per 100,000 [2–4]. It seems to be a multifactorial disease with an unknown exact etiology which impairs the quantity and quality of vision secondary to thinning in and protrusion of the cornea. This results in an irregular astigmatism with or without myopia [5–7]. Despite the fact that only one eye may be affected initially, keratoconus ultimately affects both eyes [8]. The conservative management of keratoconus in early stages consists of spectacle correction or rigid contact lenses. In more advanced stages with severe corneal irregular astigmatism and stromal opacities, surgical treatment with deep lamellar keratoplasty and penetrating keratoplasty (PK) should be considered [9–13].Intrastromal corneal ring segments (ICRSs) represent a substantial evolution in the management of keratoconus. Moreover, long-term data on ICRS procedures demonstrated promising results in topographic regularity and uncorrected visual acuity (UCVA), indicating the “possibility of putting back or even replacing keratoplasty in keratoconus patients” [14, 15].Different brands of ICRSs are currently on the market, including Intacs (Addition Technology, Inc.), Ferrara (Ferrara Ophthalmics Ltd.), and Keraring (Mediphacos Ltd.). Kerarings are made of medical grade polymethyl methacrylate (PMMA) with a UV blocker. They are characterized by a triangular cross section with variable thickness and an arc length that induces a flattening effect on the cornea. Keraring 355° intrastromal corneal ring (ICR; Mediphacos, Minas Gerais, Brazil) is a new unique intracorneal ring design especially developed for a nipple type keratoconus. It is available in a diameter of 5.7 mm and a thickness range of 200 and 300 μm. To our knowledge, there are no reports on the effect of insertion or implantation of Keraring 355° on the postoperative outcome. To investigate the short-term visual and refractive outcomes after implantation of Keraring 355°, we conducted the current study in which all eyes had a 6-month follow-up.2. Materials and Method This prospective, consecutive, interventional study included 15 eyes from 15 patients (12 men, 3 women) with a mean age of years (range 21 to 49 years) with keratoconus. It was approved by The Institutional Review Board of the Eye Research Center, Bina Eye Hospital, and followed the tenets of the Declaration of Helsinki. After fully explaining the purpose and procedures of the study, all patients were asked to sign an informed consent form before treatment. Inclusion criteria were nipple type keratoconic eyes with clear central cornea, age between 21 and 49 years, minimum corneal thickness of 360 microns, mean keratometry between 45 and 52 D, contact lens intolerance, an uncorrected visual acuity (UCVA) not better than 20/50, and no visual dysfunctions other than keratoconus. Contact lens wear was discontinued three weeks prior to the exams. Exclusion criteria were positive pregnancy test, breast-feeding, history of vernal and atopic keratoconjunctivitis, history of keratorefractive surgery on the operative eye, patients with dry eye, history of corneal stromal disorders, nystagmus, immunosuppressive drugs users, hyperopia, advanced keratoconus with inferior corneal thinning less than 360 μm, and patients with severe ocular and systemic pathologies (e.g., history of herpes keratitis, diagnosed autoimmune disease, systemic connective tissue disease, glaucoma, cataract, diabetic retinopathy, and age-related macular degeneration). A complete ophthalmic examination was performed preoperatively and postoperatively, including uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), manifest refraction, spherical equivalent (SE), keratometry () readings, and ultrasound pachymetry. Corneal topography was measured using the Orbscan II Slit Scanning Corneal Topography/Pachymetry System (Orbscan II, Bausch & Lomb). Visual acuity was measured using Snellen notation and then converted to logMAR for statistical analysis. Diagnosis of keratoconus was established by the combination of computerized video keratography of the anterior and posterior corneal surfaces (Orbscan IIz), readings, and corneal pachymetry [16, 17]. The safety of implantation of Keraring 355° in patients with keratoconus was assessed using a refractive surgery safety index (safety index = postoperative best-corrected visual acuity ÷ preoperative best-corrected visual acuity). Efficacy was assessed using a refractive surgery efficacy index (efficacy index = postoperative uncorrected visual acuity ÷ preoperative best-corrected visual acuity) [18, 19].Furthermore, we assessed patient satisfaction with three different questions. We asked every patient about their overall satisfaction with ICRS implantation after three years based on a six-point Likert scale ((0) no satisfaction; (1) very little satisfaction; (2) little satisfaction; (3) moderate satisfaction; (4) high satisfaction; (5) very high satisfaction). We also asked patients the questions “Would you recommend this procedure to other patients?” And “Would you have ICRS implantation for the other eye?”2.1. Surgical ProcedureAll surgical procedures were performed by the same experienced surgeon (Khosrow Jadidi) in an operating room under topical anesthesia with proparacaine hydrochloride 0.5% (Alcaine, Alcon) drops. In order to mark the central point of intrastromal corneal ring implantation, the operation microscope (OMS-800 Standard TOPCON Corporation, Japan) was used. In addition to the above, the pupil center was marked for proper centralization. The surgical procedure included creation of a pocket within the corneal stroma of 8.5 mm in diameter at 300-micron depth using a PocketMaker microkeratome (Dioptex GmbH) as described elsewhere [28, 38, 39] with a minor modification: when correct position of the blade was determined, the microvibrating diamond blade was set at 300 μm of the measured corneal thickness and a single 2 mm radial incision was made at the steepest meridian. Then, the applicator was fixated to the eye by the suction ring. The suction ring was removed from the eye after creating a closed intrastromal pocket of 8.5 mm diameter and 300 μm depth through the small incision tunnel. The Keraring 355° segment was inserted at the steepest meridian into the circular channel via the notch, using implantation forceps. The appropriate Keraring 355° segment thickness was selected and then implanted in the eye according to the new nomogram designed based on the author’s experiences (Table 1 and Figure 1). The centration of the implant was adjusted using keratoscope. Subsequently, a silicone-hydrogel bandage contact lens (Bausch & Lomb) was placed on the cornea. Postoperatively, patients were prescribed betamethasone drops (Sina Darou) four times a day, chloramphenicol drops (Sina Darou) four times a day, and nonpreserved artificial tears (Artelac, Bausch & Lomb, France) six times per day. Chloramphenicol drops were discontinued one week postoperatively, but betamethasone drops were tapered after four to six weeks. The bandage contact lens was removed one day postoperatively. Patients were then scheduled for postoperative clinical examinations at one month and three and six months.
    Full-text · Article · Jan 2015 · Journal of Ophthalmology
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    • "The interaction between the APM and the follicle mesenchyme might be an essential part of the hair follicle cycle. The DP and dermal sheath include a population of mesenchymal stem cells that contribute to follicle the homeostasis.[646566] Follicle cycling is associated with the movement of cells between the DP and dermal sheath.[6768] "
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    ABSTRACT: The arrector pili muscle (APM) consists of a small band of smooth muscle that connects the hair follicle to the connective tissue of the basement membrane. The APM mediates thermoregulation by contracting to increase air-trapping, but was thought to be vestigial in humans. The APM attaches proximally to the hair follicle at the bulge, a known stem cell niche. Recent studies have been directed toward this muscle's possible role in maintaining the follicular integrity and stability. This review summarizes APM anatomy and physiology and then discusses the relationship between the follicular unit and the APM. The potential role of the APM in hair loss disorders is also described, and a model explaining APM changes in hair loss is proposed.
    Full-text · Article · Jul 2014 · International Journal of Trichology
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    • "p63, which is routinely used as a marker of myoepithelial cells, is strongly expressed by the K-HME cells. However, it is also a stem cell marker in the epidermis and limbal epithelium [45]. In p63-null mice, the epithelium fails to stratify, and mammary buds or other epidermal appendages do not form [46]. Pellegrini and colleagues have argued that the phenotype of p63-null mice should be ascribed to a failure to maintain the stem cell compartment. "
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    ABSTRACT: Background Normal, healthy human breast tissue from a variety of volunteer donors has become available for research thanks to the establishment of the Susan G. Komen for the Cure® Tissue Bank at the IU Simon Cancer Center (KTB). Multiple epithelial (K-HME) and stromal cells (K-HMS) were established from the donated tissue. Explant culture was utilized to isolate the cells from pieces of breast tissue. Selective media and trypsinization were employed to select either epithelial cells or stromal cells. The primary, non-transformed epithelial cells, the focus of this study, were characterized by immunohistochemistry, flow cytometry, and in vitro cell culture. Results All of the primary, non-transformed epithelial cells tested have the ability to differentiate in vitro into a variety of cell types when plated in or on biologic matrices. Cells identified include stratified squamous epithelial, osteoclasts, chondrocytes, adipocytes, neural progenitors/neurons, immature muscle and melanocytes. The cells also express markers of embryonic stem cells. Conclusions The cell culture conditions employed select an epithelial cell that is pluri/multipotent. The plasticity of the epithelial cells developed mimics that seen in metaplastic carcinoma of the breast (MCB), a subtype of triple negative breast cancer; and may provide clues to the origin of this particularly aggressive type of breast cancer. The KTB is a unique biorepository, and the normal breast epithelial cells isolated from donated tissue have significant potential as new research tools.
    Full-text · Article · Jun 2014 · BMC Cell Biology
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