Sergio Bondanza

P63 IDENTIFIES KERATINOCYTE STEM CELLS

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 14-3-3σ 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.

Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state

This report describes the clinical results obtained from a multicentre experience of the use of autologous and allogenic cultured human epidermal cells in the treatment of partial and full skin thickness burns. A laboratory has been organized to supply cultured epithelium to Burns Units in different cities. From May 1986 to December 1988, 58 patients with an age range of 1 to 59 years, and with burns covering between 7 and 95 per cent of the body surface area, have been treated. Graftable cultured epithelium can be frozen and remain viable if stored in a skin bank. Such grafts were used successfully to treat patients with partial and full skin thickness wounds.

Cultivation of human keratinocyte stem cells: current and future clinical applications.

Cultured human keratinocytes have a wide spectrum of clinical applications. Clinical results reported by several investigators are, however, contradictory. In this review, the authors discuss the biological and surgical issues which play a key role in the clinical outcome of cultured epidermal autografts used for the treatment of massive full-thicknes burns. The importance of cultivation of epidermal stem cells and of their transplantation onto a wound bed prepared with donor dermis is emphasised. The paper also reviews recent data showing that: (i) cultured epidermal autografts bearing melanocytes can be used for the treatment of stable vitiligo; (ii) keratinocytes isolated from other lining epithelia, such as oral, urethral and corneal epithelia, can be cultivated and grafted onto patients suffering from disabling epithelial defects; (iii) keratinocyte stem cells can be stably transduced with retroviral vectors and are therefore attractive targets for the gene therapy of genodermatoses.

Regeneration of the entire human epidermis using transgenic stem cells

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Bmi-1 Reduction Plays a Key Role in Physiological and Premature Aging of Primary Human Keratinocytes

Accumulation of senescent cells contributes to the reduced regenerative capacity in aged tissues. By evaluating the molecular pathways of senescence in relation to proliferative potential of primary keratinocyte cultures from young and old healthy donors, and from young patients with inherited defects leading to premature aging, we demonstrated that p16(INK4a) is a reliable marker of both physiological and premature epidermal aging. Analysis of the expression and activity of p16(INK4a) regulators showed that stem cell depletion, reduced proliferation, and p16(INK4a) upregulation in keratinocytes derived from the chronologically and prematurely aged epidermis strongly correlate with Bmi-1 downregulation. In highly proliferative tissues, replicative and premature senescence participate in determining senescent cell accumulation. Our findings demonstrated that Bmi-1 is downregulated in human keratinocytes during both in vitro processes, in parallel with p16(INK4a) upregulation and accomplishment of clonal conversion. When premature senescence was induced by specific exogenous stimuli, concomitant Ets-1 upregulation was also observed. Moreover, Bmi-1 inhibited Ets-1-mediated p16(INK4a) upregulation. Finally, Bmi-1 overexpression reduced p16(INK4a) promoter activity and decreased protein expression in aged and diseased keratinocytes, inducing a delay of clonal conversion and an increase of cell clonogenic ability. Altogether these findings underline a key role of Bmi-1 downregulation in enforcing aging in primary human keratinocytes.

Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) Immortalizes Primary Human Keratinocytes By Maintaining Cells in the Stem Cell Compartment

Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin‐dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B‐Cell Specific Moloney murine leukemia virus site 1 (Bmi‐1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi‐1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down‐regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining sternness.—Maurelli, R., Zambruno, G., Guerra, L., Abbruzzese, C., Dimri, G., Gellini, M., Bondanza, S., Dellambra, E. Inactivation of p16INK4a (inhibitor of cyclin‐dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment. FASEB J. 20, E742–E756 (2006)

Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis

Background  Several surgical techniques have been proposed for the treatment of piebaldism. These procedures, however, are poorly suited for the treatment of large leucodermal lesions, can cause scars and require multiple donor sites. Recently, it has been reported that autologous cultured epidermis induces scarless repigmentation of large vitiligo lesions, using a single small donor site.

The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.

Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells

Figure 1. Regeneration of a transgenic functional epidermis on the skin wound of the JEB patient. (a) The long-standing ulceration on the lower right leg of the patient 2 days before transplantation. (b) Western blot analysis of cell lysates (20 mg protein, 30 seconds exposure time) from (lane 1) normal control and patient keratinocyte cultures (lane 2) before and (lane 3) after gene correction, probed with a monoclonal antibody against laminin 332-b3. (lane 4) Western blot analysis of a higher amount of loaded protein (65 mg, 5 seconds exposure time) of uncorrected patient keratinocytes, and (lane 5) normal keratinocyte cultures using the same laminin-332-b3 antibody. The 75-kD band in lane 4 is consistent with the truncated laminin-332-b3 generated by the c.1903C>T; p.R635X mutation. (c) Transplantation of cultured transgenic epidermal sheets (asterisks) on the prepared wound bed. Grafts are overlaid with petrolatum gauze. (d) Initial epidermal regeneration at 14 days. (e) Complete epidermal regeneration at 3.5 months. (f) Stable epidermal regeneration at 16 months. Note crusting and erosions outside of the grafted area. JEB, junctional epidermolysis bullosa. TO THE EDITOR Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3, LAMB3, or LAMC2, which together encode laminin-332, a heterotrimeric protein consisting of a3, b3, and g2 chains (Fine et al., 2014). In nonlethal generalized intermediate JEB, laminin332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basementmembrane uponminor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life (Fine et al., 2014; Laimer et al., 2010; Nakano et al., 2002). There is no cure for JEB; treatments are symptomatic and aimed at relieving the devastating clinical manifestations (Carulli et al., 2013). The only published evidence for the possibility of a permanent local treatment of JEB was provided by a phase I/II trial showing that autologous epidermal cultures containing geneticallymodified epidermal stem cells were able to restore a normal epidermis on a JEB patient (De Rosa et al., 2014; Mavilio et al., 2006). However, the transgenic epidermis was applied in areas still covered by a diseased but apparently functional epidermis, which was surgically removed before grafting (Mavilio et al., 2006). Although it is clear that the ideal clinical application of transgenic epidermis would aim at preventing the development of devastating chronic lesions,manypatients suffer from therapyresistant chronic ulcerations that are highly predisposed to cancer development and need timely closure (Goldberg

Nonirradiated human fibroblasts and irradiated 3T3-J2 murine fibroblasts as a feeder layer for keratinocyte growth and differentiation in vitro on a fibrin substrate.

The standard method for producing graftable epithelia relies on the presence of a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (Rheinwald and Green technique). Here, we studied a new keratinocyte culture system, which envisages the utilization of nonirradiated human fibroblasts embedded into a fibrin substrate, in cultures destined for a future clinical application. We tested this culture system using keratinocytes grown on a fibrin gel precoated with 3T3-J2 murine fibroblasts as a control. In order to evaluate the new technology, we compared the clonogenic potential and the proliferative, differentiative and metabolic characteristics of keratinocytes cultured on the fibrin gel under the two culture conditions. The results demonstrated that the proposed technology did not impair the behavior of cultured keratinocytes and revealed that cells maintained their proliferative potential and phenotype under the experimental conditions. In particular, the demonstration of stem cell maintenance under the adopted culture conditions is very important for acute burn treatment with skin substitutes. This work is a first step in the evaluation of a new keratinocyte culture system, which has been studied in order to take advantage of an additional human cell population (i.e. nonirradiated, growing fibroblasts) for future transplantation purposes in acute and chronic wounds. Additional research will allow us to attain (1) the removal of murine cells in the initial phase of keratinocyte cultures, and (2) the removal of other potentially dangerous animal-derived materials from the entire culture system.