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Schematic representation of human skin and epidermolysis bullosa (EB). (A) Epidermal layers (left) and the epidermal-dermal junction (middle) are designated. The right panel denotes the hemidesmosome and its components, in relation to EB simplex (EBS), junctional EB (JEB), and dystrophic EB (DEB). (B) Proteins involved in the pathogenesis of EBS, JEB, and DEB and site of blister formation.
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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 st...
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... are >200 monogenic skin diseases, featuring either dominant or recessive transmission patterns ( Tadini et al. 2015). Most genodermatoses are caused by mutations affecting genes mainly expressed by epidermal keratinocytes, although some of them can also be expressed by dermal fibroblasts (Fig. 2). Some genetic skin disorders are marked by a mosaic pattern, often with alternating stripes of affected and unaffected skin that follow the lines of Blaschko, which can be caused by a postzygotic mutation during embryogenesis (Paller et al. ...
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... EB encompasses a heterogeneous group of rare, genetic disorders characterized by recurrent blistering of the skin and other stratified epithelia (Fig. 2). Blisters arise spontaneously or on minimal mechanical stress or trauma as a result of increased epithelial fragility, caused by molecular defects within genes encoding various structural proteins of the epidermal-dermal junction ( Fig. 2; Fine et al. 2014). The incidence and prevalence of EB in the United States are ∼1/53,000 and ...
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... of rare, genetic disorders characterized by recurrent blistering of the skin and other stratified epithelia (Fig. 2). Blisters arise spontaneously or on minimal mechanical stress or trauma as a result of increased epithelial fragility, caused by molecular defects within genes encoding various structural proteins of the epidermal-dermal junction ( Fig. 2; Fine et al. 2014). The incidence and prevalence of EB in the United States are ∼1/53,000 and 1/125,000, respectively, and similar estimates have been obtained in some European countries (Fine 2010(Fine , 2016. EB affects individuals from all ethnic origins without gender predilection and displays either dominant or recessive patterns ...
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... United States are ∼1/53,000 and 1/125,000, respectively, and similar estimates have been obtained in some European countries (Fine 2010(Fine , 2016. EB affects individuals from all ethnic origins without gender predilection and displays either dominant or recessive patterns of inheritance. More than 1000 mutations in at least 14 structural genes (Fig. 2) have been documented causing distinct clinical manifestations, ranging from mild to severe, with local or generalized involvement and significant morbidity and mortality. This variety depends on several factors, both phenotypic (distribution and severity of the lesions, involvement of mucosae, age of onset) and molecular (targeted ...
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... EB encompasses three major forms, primarily based on the level of skin cleavage (and subsequent blistering formation), that is intraepidermal in EB simplex (EBS), within the lamina lucida in JEB and beneath the lamina densa in DEB ( Fig. 2; Intong ...
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... JEB is a chronic, devastating, and life-threatening condition caused by mutations in LAMA3, LAMB3, or LAMC2-encoding, respectively, α3, β3, and γ2 chains of laminin-332 (also known as laminin 5)-and in genes encoding collagen XVII and α6β4 integrins ( Fig. 2; Fine et al. 2008Fine et al. , 2014). Hemidesmosomes are rudimentary or absent, hence blisters occur within the lamina lucida of the basement membrane ( Fig. 2) and predominate at sites exposed to friction, trauma, or heat. In addition, hair, nail, and enamel defects are common, along with the involvement of the ocular surface and ...
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... by mutations in LAMA3, LAMB3, or LAMC2-encoding, respectively, α3, β3, and γ2 chains of laminin-332 (also known as laminin 5)-and in genes encoding collagen XVII and α6β4 integrins ( Fig. 2; Fine et al. 2008Fine et al. , 2014). Hemidesmosomes are rudimentary or absent, hence blisters occur within the lamina lucida of the basement membrane ( Fig. 2) and predominate at sites exposed to friction, trauma, or heat. In addition, hair, nail, and enamel defects are common, along with the involvement of the ocular surface and gastrointestinal and renal systems ( Laimer et al. 2010;Yancey and Hintner 2010). Deleterious mutations in genes encoding laminin 332 are usually lethal early in ...
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... is caused by mutations in COL7A1, the gene encoding collagen VII, the major component of dermal anchoring fibrils, whose absence (or alteration) induces blister formation just beneath the lamina densa of the basement membrane (Fig. 2). Dominant DEB (DDEB) generally displays a mild phenotype, with blisters occurring on areas of trauma (with a predilection for the extremities), often resulting in scars, milia (keratin-filled cysts of sweat glands), and loss of nails ( Das and Sahoo 2004). Instead, generalized RDEB is more severe. Blisters develop at birth and ...
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... but not least, hurdles also relate to intrinsic challenges in understanding the mechanisms underlying many rare diseases, which complicates the design of efficacious therapies. Particularly in RDEB, where skin cleavage is deeper than in JEB (Fig. 2), the clinical success of epidermal cultures strictly relies on proper selection of the patients including their immunological picture ( Siprashvili et al. 2016). It also depends on control of inflammation and microenvironment of the receiving wound bed (which varies between JEB and RDEB and can hamper the engraftment of the culture), ...
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... This approach has recently been combined with gene therapies for rare genetic skin diseases, in which ex vivo gene-corrected epidermal sheets generated from patients' keratinocytes, including keratinocyte stem cells (KSCs), are cultured and grafted onto patients with severe skin damage [2][3][4]. Ex vivo gene-corrected epidermal sheet therapy has been applied for junctional epidermolysis bullosa (JEB), recessive dystrophic epidermolysis bullosa (RDEB), and Netherton syndrome (NS) [2][3][4][5][6][7]. However, the success We investigated these questions by culturing human primary keratinocytes including KSCs obtained from human interfollicular skin with Y-27632 (hereafter referred as 'ROCKi'). ...
Keratinocyte stem cells (KSCs) can be cultured as ex vivo epidermal sheets and are attractive for cell and gene therapy. However, the growth and expansion of primary keratinocytes (including KSCs) in in vitro culture can be inefficient as KSCs undergo apoptotic cell death, known as anoikis when isolated from skin biopsies and dissociated from their extracellular environment. This further affects the survival and stemness of KSCs in in vitro culture and hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle. However, it risks changing the characteristics of stem cells. In this study, we studied the effect of the Y-27632 ROCKi on cultured primary human keratinocytes. Following a short-term (6 days) ROCKi treatment, we assessed keratinocyte proliferation and differentiation potential and KSC stemness at the molecular-, cellular- and single-cell levels. A significant increase in colony-forming efficiency associated with increased expression of proliferation markers and mitochondrial mass was observed in treated cells vs. untreated cells. Similarly, treated cells showed a distinct transcriptional profile at the single-cell level, but these changes became indistinguishable after discontinuation of ROCKi treatment. Single-cell transcriptome analysis further confirmed that short-term ROCKi treatment did not affect stem cell characteristics. Finally, we showed that ROCKi rapidly activated Akt and upstream of ERK signalling pathways in primary human keratinocytes. In conclusion, rapid proliferation due to short-term ROCKi treatment was transient and reversible, and did not affect the KSC population or their characteristics. We thus present a safer approach for the use of ROCKi in ex vivo clinical applications.
... This approach has recently been combined with gene therapies for rare genetic skin diseases, in which ex vivo gene-corrected epidermal sheets generated from patients' keratinocytes, including keratinocyte stem cells (KSCs), are cultured and grafted onto patients with severe skin damage [2][3][4]. Ex vivo gene-corrected epidermal sheet therapy has been applied for junctional epidermolysis bullosa (JEB), recessive dystrophic epidermolysis bullosa (RDEB), and Netherton syndrome (NS) [2][3][4][5][6][7]. However, the success We investigated these questions by culturing human primary keratinocytes including KSCs obtained from human interfollicular skin with Y-27632 (hereafter referred as 'ROCKi'). ...
Primary keratinocytes including keratinocyte stem cells (KSCs) can be cultured as epidermal sheets in vitro and are attractive for cell and gene therapies for genetic skin disorders. However, the initial slow growth of freshly isolated keratinocytes hinders clinical applications. Rho-associated kinase inhibitor (ROCKi) has been used to overcome this obstacle, but its influence on the characteristics of KSC and its safety for clinical application remains unknown. In this study, primary keratinocytes were treated with ROCKi Y-27632 for six days (short-term). Significant increases in colony formation and cell proliferation during the six-day ROCKi treatment were observed and confirmed by related protein markers and single-cell transcriptomic analysis. In addition, short-term ROCKi-treated cells maintained their differentiation ability as examined by 3D-organotypic culture. However, these changes could be reversed and became indistinguishable between treated and untreated cells once ROCKi treatment was withdrawn. Further, the short-term ROCKi treatment did not reduce the number of KSCs. In addition, AKT and ERK pathways were rapidly activated upon ROCKi treatment. In conclusion, short-term ROCKi treatment can transiently and reversibly accelerate initial primary keratinocyte expansion while preserving the holoclone-forming cell population (KSCs), providing a safe avenue for clinical applications.
... 18 In contrast, the human IFE appears to be maintained by a hierarchy of SCs that generates actively dividing progenitor cells which ultimately commit to terminal differentiation. [18][19][20] In wounded mouse skin, several epidermal cell populations, including those in HFs distal to the wound site, contribute to the skin wound repair process. 15,16 Interestingly, lineage restriction and spatial confinement of HF-resident SC pools are transiently lost during wound repair, allowing contribution of multiple SC populations. ...
Skin cancers are by far the most frequently diagnosed human cancers. The closely related transcriptional co‐regulator proteins YAP and TAZ (WWTR1) have emerged as important drivers of tumor initiation, progression and metastasis in melanoma and non‐melanoma skin cancers. YAP/TAZ serve as an essential signaling hub by integrating signals from multiple upstream pathways. In this review, we summarize the roles of YAP/TAZ in skin physiology and tumorigenesis and discuss recent efforts of therapeutic interventions that target YAP/TAZ in in both preclinical and clinical settings, as well as their prospects for use as skin cancer treatments.
... They suggested that substitution of the missing or mutated gene with the normal allele via gene addition could provide a therapeutic effect, at least for recessively inherited, monogenic genetic disorders. Indeed, remarkable success has been obtained for severe immunodeficiencies and is about to be achieved for other genetic diseases, such as EB [1,2]. ...
... Cultures containing transgenic epidermal stem cells can be used to prepare grafts able to permanently restore a functional epidermis in patients with EB [5]. Indeed, combined ex vivo cell and gene therapy has been proposed to tackle both RDEB and the junctional form of EB (JEB) ( Figure 1B) [2]. ...
... Since this strategy envisages the transplantation of transgenic, long-lived, self-renewing epidermal stem cells, a single transplantation is sufficient to permanently (to date, up to 16 years' follow-up) restore a fully functional epidermis. Importantly, despite the very high number (up to 4 × 10 8 ) of transgenic clonogenic keratinocytes transplanted per patient, no adverse events, including insertional mutagenesis, have been observed in any of the patients treated so far [2]. However, combined ex vivo cell and gene therapy has its own pitfalls. ...
Efforts are dedicated to definitively tackle skin lesions plaguing patients with epidermolysis bullosa (EB), a devastating genetic disorder affecting the integumentary system. Both in vivo gene therapy, as recently reported by Gurevich et al., and combined ex vivo cell and gene therapy strategies are under investigation. Here, we address the advantages and disadvantages of these different approaches.
... Recently, several informative review articles in dermatology journals have focused on ex vivo DNA/gene-editing therapies for rare genodermatoses (De Rosa et al., 2020;Jayarajan et al., 2021;March et al., 2018). In this study, we include both ex vivo and in vivo gene-editing applications in dermatology and further focus our attention on incorporating all classes of CRISPR nucleases-DNA and RNA targeting-into the repertoire of clinical tools available to the dermatologist. ...
Over the past decade, CRISPR has rapidly made its way from the bench to the bedside – providing a newfound therapeutic avenue to not only treat genetic diseases, but to permanently cure them. While there are several clinical trials in early stages, there are so far no CRISPR-based clinical trials for cutaneous disease. In this review, we describe multiple cutaneous diseases that represent ideal targets for CRISPR based therapeutics due to known single-gene-causing-mutations. We also explore the potential of CRISPR nucleases to treat inflammatory disorders such as eczema and psoriasis, which are not classically categorized as genodermatoses. We describe therapeutic solutions for these diseases that are guided by various CRISPR-associated (Cas) effector proteins – for example, using Cas9 to permanently edit the DNA of somatic cells, Cas3 to target foreign DNA to combat viral/bacterial skin infections, and Cas13 to edit mutated RNA transcripts in diseases where permanent DNA editing is untenable. Furthermore, we discuss various drug delivery modalities for CRISPR therapeutics – including transdermal patches and microneedles – that are uniquely suited for dermatological disease. In sum, we highlight the potential of CRISPR-based therapeutics to revolutionize the treatment of cutaneous disease with a goal of being accessible to the practicing dermatologist.
... The remarkable clinical outcomes of regenerative medicine in renewing tissues, such as blood and squamous epithelia, accrue from a thorough characterization of their specific stem cells (De Luca et al., 2019;De Rosa et al., 2020). Thus, targeting stem cells is the cornerstone for successful ex vivo gene therapy of genetic diseases affecting the epidermis. ...
Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients’ skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm²). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses.
Clinical Trial Registration: EudraCT Number: 2018-000261-36.
... Technologische Herausforderungen der Ex-vivo-Strategien limitieren derzeit ihre klinische Anwendung. Insbesondere Vektorensicherheit (Insertionsmutagenese), Schwierigkeiten des zielgerichteten Transfers großer Gene wie COL7A1, die Identifizierung und effiziente Transduktion von Holoclones, potenzielle Autoimmunphänomene gegen therapeutisch induzierte Neoantigene oder auch die Invasivität und damit Belastung des operativen Eingriffs bei hochvulnerablen PatientInnen bedürfen der Beachtung und methodischen Weiterentwicklung [9,23]. ...
Zusammenfassung
Unter Epidermolysis bullosa (EB) subsumiert man genetische Erkrankungen, die durch gesteigerte Hautfragilität mit Blasenbildung nach bereits geringen mechanischen Belastungen gekennzeichnet sind. Hochvariable kutane, extrakutane und Organmanifestationen verursachen eine signifikante Morbidität und Mortalität sowie eine hohe Krankheitslast für Betroffene und Angehörige. Obwohl derzeit nicht heilbar, eröffnen Fortschritte in der molekularen Charakterisierung pathogenetischer Prozesse, in den diagnostischen Techniken und molekularen Therapieansätzen neue Perspektiven. Neben korrektiven, potenziell kurativen Behandlungszugängen mit dem Ziel der Wiederherstellung der Funktion von Gen bzw. Protein stellen krankheitsmodifizierende Strategien eine wertvolle Ergänzung dar. Unter Letztere fallen symptomatische Therapien, die sekundär dysregulierte, den Phänotyp modulierende Entzündungskaskaden adressieren oder zielgerichtete Interventionen hinsichtlich bestimmter Symptome wie Fibrosierung, Juckreiz oder kanzerogener Zelltransformation. Molekulare Verfahren erlauben heute zudem, eine Diagnose und damit Prognose früher und präziser zu stellen, was die genetische Beratung erleichtert.
Das Management von EB-Patienten ist komplex und bedarf einer Spezialexpertise und multidisziplinär akkordierten Versorgung. Entsprechende Ressourcen halten designierte Expertisezentren wie das EB-Haus Austria vor, das als Mitglied des Europäischen Referenznetzwerks für Seltene Hauterkrankungen (ERN Skin) sowohl klinische Versorgung, Grundlagen- und klinische Forschungsaktivitäten sowie Zugang zu Aus- und Weiterbildungsprogrammen für Betroffene, Betreuende und medizinisches Fachpersonal gewährleistet.
... Locally damaged adult skin can heal with scars but it lacks the capacity to fully regenerate the complex composition of larger skin areas [14]. Severe scarring and chronic wounds after major skin burns [15], surgeries or devastating skin diseases [16] limit skin mobility, respiration and light protection, resulting in body fluid loss, life-threatening infections and skin cancer [17,18]. The current gold standard for extended area epidermal replacement is transplantation of ex vivo engineered epidermal sheets [19,20]. ...
Self-assembly of solid organs from single cells would greatly expand applicability of regenerative medicine. Stem/progenitor cells can self-organize into micro-sized organ units, termed organoids, partially modelling tissue function and regeneration. Here we demonstrated 3D self-assembly of adult and induced pluripotent stem cell (iPSC)-derived fibroblasts, keratinocytes and endothelial progenitors into both, planar human skin in vivo and a novel type of spheroid-shaped skin organoids in vitro, under the aegis of human platelet lysate.
Methods: Primary endothelial colony forming cells (ECFCs), skin fibroblasts (FBs) and keratinocytes (KCs) were isolated from human tissues and polyclonally propagated under 2D xeno-free conditions. Human tissue-derived iPSCs were differentiated into endothelial cells (hiPSC-ECs), fibroblasts (hiPSC-FBs) and keratinocytes (hiPSC-KCs) according to efficiency-optimized protocols. Cell identity and purity were confirmed by flow cytometry and clonogenicity indicated their stem/progenitor potential. Triple cell type floating spheroids formation was promoted by human platelet-derived growth factors containing culture conditions, using nanoparticle cell labelling for monitoring the organization process. Planar human skin regeneration was assessed in full-thickness wounds of immune-deficient mice upon transplantation of hiPSC-derived single cell suspensions.
Results: Organoids displayed a distinct architecture with surface-anchored keratinocytes surrounding a stromal core, and specific signaling patterns in response to inflammatory stimuli. FGF-7 mRNA transfection was required to accelerate keratinocyte long-term fitness. Stratified human skin also self-assembled within two weeks after either adult- or iPSC-derived skin cell-suspension liquid-transplantation, healing deep wounds of mice. Transplant vascularization significantly accelerated in the presence of co-transplanted endothelial progenitors. Mechanistically, extracellular vesicles mediated the multifactorial platelet-derived trophic effects. No tumorigenesis occurred upon xenografting.
Conclusion: This illustrates the superordinate progenitor self-organization principle and permits novel rapid 3D skin-related pharmaceutical high-content testing opportunities with floating spheroid skin organoids. Multi-cell transplant self-organization facilitates development of iPSC-based organ regeneration strategies using cell suspension transplantation supported by human platelet factors.
... The goal of these strategies is the sustained or permanent restoration of functional protein expression in EB, through addition, replacement, modification or correction at the DNA, RNA, protein, or cellular level [22]. While these molecular approaches are already under evaluation in clinical trials, the methodological challenges involved, as well as the genotypic heterogeneity of EB, pose major hurdles that make case-specific, tailored approaches mandatory [21,23]. ...
... Current gene delivery techniques are incapable of specifically targeting holoclones in vivo. As such, ensuring efficient targeting of these epidermal stem cells first requires their isolation prior to introduction of the replacement gene ex vivo [23]. This strategy was first implemented in 2006, in a JEB patient harboring mutations in the LAMB3 gene. ...
... Ex vivo gene replacement strategies have advanced the furthest in clinical trials and have enabled insight into patient perspective and experience. The therapy comes with certain drawbacks, including the invasiveness of the approach, which can require multiple biopsies to ensure successful (stem) cell isolation, and the harsh debridement needed for wound bed preparation, which has been shown to increase engraftment success [22,23,36]. This places significant burden on this vulnerable patient population. ...
New insights into molecular genetics and pathomechanisms in epidermolysis bullosa (EB), methodological and technological advances in molecular biology as well as designated funding initiatives and facilitated approval procedures for orphan drugs have boosted translational research perspectives for this devastating disease. This is echoed by the increasing number of clinical trials assessing innovative molecular therapies in the field of EB. Despite remarkable progress, gene-corrective modalities, aimed at sustained or permanent restoration of functional protein expression, still await broad clinical availability. This also reflects the methodological and technological shortcomings of current strategies, including the translatability of certain methodologies beyond preclinical models as well as the safe, specific, efficient, feasible, sustained and cost-effective delivery of therapeutic/corrective information to target cells. This review gives an updated overview on status, prospects, challenges and limitations of current gene-targeted therapies.
... Despite this fundamental difference, similar clusters of basal keratinocytes have been identified in both conditions: the BAS-II cluster is similar to our H cluster and the BAS-I cluster is similar to our M cluster 45 . The notion that the BAS-II cluster contains more cells than BAS-I is consistent with the notion that, as opposed to primary cultures, single clonogenic keratinocytes directly analyzed from a skin biopsy generate more holoclones than meroclones 46 . Similarly, and despite the notion that murine keratinocytes do not initiate the same clonal types identified in human cultures, both human holoclone-forming cells and the population of K14+ cells containing murine epidermal stem cells upregulate genes regulating DNA repair, cell cycle, chromosome segregation 3 . ...
... Computational analysis of scRNA-seq data identified a continuous trajectory showing that holoclone-forming cells generate meroclone-and paraclone-forming cells (eventually producing terminally differentiated cells), strengthening the notion that long-lived self-renewing stem cells generate pools of TA progenitors 5 , which are known to play a role in epidermal regeneration during wound-healing processes. Indeed, meroclones and paraclones were enriched in sets of genes related to wound healing, corroborating the notion that clonogenic keratinocytes derived directly from a biopsy taken from normal, unwounded skin generate mostly holoclones, whereas keratinocytes cloned from wounded skin generate predominantly meroclones and paraclones 46 . ...
Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.
