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ARTICLE doi:10.1038/nature24487

Regeneration of the entire human epidermis using transgenic stem cells Tobias Hirsch1*, Tobias Rothoeft2*, Norbert Teig2*, Johann W. Bauer3*, Graziella Pellegrini4,5*, Laura De Rosa5*, Davide Scaglione6, Julia Reichelt3, Alfred Klausegger3, Daniela Kneisz3, Oriana Romano7, Alessia Secone Seconetti5,

Roberta Contin5, Elena Enzo5, Irena Jurman8, Sonia Carulli9, Frank Jacobsen1, Thomas Luecke10, Marcus Lehnhardt1,

Meike Fischer2, Maximilian Kueckelhaus1, Daniela Quaglino7, Michele Morgante8, Silvio Bicciato7, Sergio Bondanza9 &

Michele De Luca5

Generalized JEB is a severe and often lethal genetic disease that is char- acterized by structural and mechanical fragility of the integuments. Blisters and erosions of the skin and mucosa occur within the lamina lucida of the basement membrane in response to minor trauma. Massive chronic skin wounds greatly impair patients’ quality of life, lead to recurrent infections and scars, and predispose patients to skin cancer. JEB is caused by mutations in three genes—LAMA3, LAMB3 or LAMC2—that jointly encode laminin-332 (a heterotrimeric protein, also known as laminin 5, consisting of α3, β 3, and γ 2 chains) and in genes that encode collagen XVII and α 6β 4 integrins1. Deleterious mutations that cause an absence of laminin-332 are usually lethal early in life. In nonlethal cases of JEB, laminin-332 is strongly reduced and hemidesmosomes are rudimentary or absent. There is no cure for JEB and more than 40% of patients die before adolescence1,2. The available symptomatic treatments can only relieve the devastating clinical manifestations.

Monthly renewal and timely repair of the human epidermis is sus- tained by epidermal stem cells, which generate colonies known as holoclones3,4. Holoclones produce meroclone- and paraclone-forming cells, which behave as transient amplifying progenitors3,4. Epithelial cultures harbouring holoclone-forming cells can permanently restore massive skin and ocular defects5–9. A phase I/II clinical trial (one patient) and a single-case study provided compelling evidence that local transplantation of transgenic epidermal cultures can generate a func- tional epidermis, leading to permanent (the longest follow-up being of 12 years) correction of skin lesions in patients with JEB10–12. However, owing to the paucity of treated areas (a total of around 0.06 m2), the treatment did not substantially improve the patients’ quality of life10–12.

A major criticism of this therapeutic approach has been its supposed unsuitability for the massive skin lesions marking generalized JEB. Here we demonstrate life-saving regeneration of virtually the entire epidermis (approximately 0.85 m2) of a seven-year-old child suffering from a devastating form of JEB, by means of autologous transgenic keratinocyte cultures. The regenerated epidermis remained robust and resistant to mechanical stress and did not develop blisters or erosions during the 21-month follow-up. This fully functional epidermis is entirely sustained by a limited number of transgenic epidermal stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo.

The patient In June 2015, a seven-year-old child was admitted to the Burn Unit of the Children’s Hospital, Ruhr-University, Bochum, Germany. He carried a homozygous acceptor splice site mutation (C1977-1G> A, IVS 14-1G> A) within intron 14 of LAMB3. Since birth, the patient had developed blisters all over his body, particularly on his limbs, back and flanks. His condition deteriorated severely six weeks before admission, owing to infection with Staphylococcus aureus and Pseudomonas aeruginosa. Shortly after admission, he suffered complete epidermal loss on about 60% of his total body surface area (TBSA). During the following weeks, all therapeutic approaches failed and the patient’s short-term prognosis was unfavourable (Methods). After the patient’s parents had provided informed consent, the regional regulatory authorities and the ethical review board of the Ruhr-University authorized the compassionate use of combined ex vivo cell and gene therapy. The parents of the patient also consented

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.

1Department of Plastic Surgery, Burn Centre, BG University Hospital Bergmannsheil, Ruhr University Bochum, 44789 Bochum, Germany. 2Department of Neonatology and Pediatric Intensive Care, University Children’s Hospital, Ruhr University Bochum, 44791 Bochum, Germany. 3EB House Austria and Department of Dermatology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria. 4Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, 41124 Modena, Italy. 5Center for Regenerative Medicine “Stefano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy. 6IGA Technology Services s.r.l., 33100 Udine, Italy. 7Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy. 8Istituto di Genomica Applicata and Dipartimento di Scienze Agroalimentari, Ambientali e Animali, University of Udine, 33100 Udine, Italy. 9Holostem Terapie Avanzate s.r.l., 41125 Modena, Italy. 10Department of Neuropaediatrics, University Children’s Hospital, Ruhr University Bochum, 44791 Bochum, Germany. *These authors contributed equally to this work.

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to the publication of the photographs and medical information included in this paper.

At the time of the first surgery, the patient had complete epidermal loss on approximately 80% TBSA (Fig. 1a, b).

Regeneration of epidermis by transgenic cultures In September 2015, a 4-cm2 biopsy, taken from a currently non- blistering area of the patient’s left inguinal region, was used to estab- lish primary keratinocyte cultures, which were then transduced with a retroviral vector expressing the full-length LAMB3 cDNA under the control of the Moloney leukaemia virus long terminal repeat13 (MLV-RV; Methods, Extended Data Fig. 1). Sufficient 0.85-m2 trans- genic epidermal grafts, enough to cover all of the patient’s denuded body surface, were applied sequentially on a properly prepared dermal wound bed (Extended Data Fig. 2a). All limbs, flanks and the entire back were treated with grafts during October and November 2015. Some of the remaining denuded areas received grafts during January 2016.

Previously, transgenic epidermal sheets have been cultivated on plastic, enzymatically detached from the vessel and mounted on a non-adhering gauze10–12. Keratinocyte cultivation on a fibrin substrate—currently used to treat massive skin and ocular burns6,8,9— eliminates cumbersome procedures for graft preparation and trans- plantation and avoids epidermal shrinkage, allowing the production of larger grafts from the same number of clonogenic cells as are needed to produce plastic-cultured grafts. Because degradation of fibrin after transplantation, which is critical to allow cell engraftment, had never been assessed in a wound bed of a patient with JEB, at the first surgery we compared plastic- and fibrin-cultured grafts (Methods, Extended Data Fig. 1).

The left arm received plastic-cultured grafts (Extended Data Fig. 2b, asterisks). Upon removal of the non-adhering gauze (ten days after grafting, Extended Data Fig. 2c, arrows), epidermal engraftment was evident (asterisks). One month after grafting, epidermal regeneration was stable and complete (Extended Data Fig. 2d). The left leg received both plastic- and fibrin-cultured grafts (Extended Data Fig. 2e, asterisk and arrow, respectively), both of which showed full engraftment after ten days (Extended Data Fig. 2f, asterisk and arrow, respectively) and complete epidermal regeneration after one month (Extended Data Fig. 2f, inset). Similar data were obtained on the other limbs. Thus,

the patient’s denuded back (Extended Data Fig. 2g) was treated with only fibrin-cultured grafts (inset). As shown in Extended Data Fig. 2h, virtually complete epidermal regeneration was observed after 1 month, with the exception of some areas (asterisks), some of which contained islands of newly formed epidermis (arrows). Over the following weeks, the regenerated epidermis surrounding the open lesions and the epidermal islands spread and covered most of the denuded areas (Extended Data Fig. 2i). We then transplanted grafts onto the remaining defects on the patient’s flanks, thorax, right thigh, right hand and shoulders. Epidermal regeneration was attained in most of those areas.

Thus, approximately 80% of the patient’s TBSA was restored by the transgenic epidermis (Fig. 1c). During the 21-month follow-up (more than 20 epidermal renewing cycles), the regenerated epidermis adhered firmly to the underlying dermis, even after induced mechanical stress (Fig. 1d and Supplementary Video), healed normally and did not form blisters, including in areas where follow-up biopsies were taken (Fig. 1e, arrow).

The patient was discharged in February 2016. His epidermis is currently stable and robust, and does not blister, itch, or require ointment or medications.

Ten punch biopsies were taken randomly, 4, 8 and 21 months after grafting. The epidermis had normal morphology and we could not detect blisters, erosions or epidermal detachment from the underlying dermis (Extended Data Fig. 3a). In situ hybridization using a vector-specific t-LAMB3 probe showed that the regenerated epider- mis consisted only of transgenic keratinocytes (Fig. 2a). At admission, laminin 332-β 3 was barely detectable in the patient’s skin (Fig. 2b). By contrast, after grafting, control epidermis (obtained from surgical waste, typically from abdominoplasties or mammoplasty reduction) and transgenic epidermis expressed virtually identical amounts of laminin 332-β 3, which was properly located at the epidermis– dermis junction (Fig. 2b). In addition, the basal lamina contained normal amounts of laminin 332-α 3 and γ 2 chains and α 6β 4 integrins, all of which had been strongly decreased at admission (Extended Data Fig. 3b). Thus, transduced keratinocytes restored a proper adhesion machinery (Extended Data Fig. 3c). Indeed, the transgenic epidermis showed normal thickness and continuity of the basement membrane (Fig. 2c, arrowheads) and normal morphology of hemidesmosomes (Fig. 2c, arrows). Twenty-one months after surgery, the patient’s serum did not contain autoantibodies directed against the basement membrane zone (Extended Data Fig. 3d).

In summary, transgenic epidermal cultures generated an entire functional epidermis in a patient with JEB. This is consistent with the use of keratinocyte cultures for decades to successfully treat victims of life-threatening burns on up to 98% of TBSA5,6,9,14. It can be argued that the patient’s clinical picture (massive epidermal loss, critical conditions, poor short-term prognosis) was unusual and our aggressive surgery (mandatory for this patient) unthinkable for the clinical course of most patients with epidermolysis bullosa. However, progressive replacement of diseased epidermis can be attained in multiple, less- invasive surgical interventions on more limited body areas. Epidermolysis bullosa has the advantage of a preserved dermis (not available in deep burns), which allows good functional and cosmetic outcomes. This approach would be optimal for newly diagnosed patients early in their childhood. A bank of transduced epidermal stem cells taken at birth could be used to treat skin lesions while they develop, thus preventing, rather than restoring, the devastating clinical manifestations that arise in these patients through adulthood. Currently, combined ex vivo cell and gene therapy cannot be applied to lesions of the internal mucosae; however, such lesions are usually more manageable than those on the skin, perhaps with the exception of oesophageal strictures.

Integration profile of transgenic epidermis Pre-graft transgenic cultures (PGc) were generated from about 8.7 × 106 primary clonogenic cells and consisted of 2.2 × 108 keratinocytes

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Figure 1 | Regeneration of the transgenic epidermis. a, Clinical picture of the patient showing massive epidermal loss. b, Schematic representation of the clinical picture. The denuded skin is indicated in red; blistering areas are indicated in green. Flesh-coloured areas indicate currently non- blistering skin. Transgenic grafts were applied on both red and green areas. c, Restoration of patient’s entire epidermis, with the exception of very few areas on the right thigh, buttocks, upper shoulders/neck and left axilla (white circles, altogether ≤ 2% of TBSA). d, Normal skin functionality and elasticity. e, Absence of blister formation at sites where post-graft biopsies were taken (arrow).

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(divided into 36 vials), around 45% of which were seeded to prepare 0.85-m2 transgenic epidermal grafts (Extended Data Fig. 1).

To investigate the genome-wide integration profile, we sequenced three PGc samples using two independent long terminal repeat (LTR) primers (3pIN and 3pOUT, Supplementary Table 1) for library enrich- ment (n = 12; see Methods). High-throughput sequencing recovered a total of 174.9 million read pairs and the libraries obtained using the two LTR primers showed a similar number of reads and comparable insertion counts (Pearson R > 0.92, P < 0.005). After merging all inte- gration sites from the two independent priming systems, we identified 27,303 integrations in PGc (Fig. 3a, bars) with an average coverage of 2.5 reads per insertion (Fig. 3a, lines and Supplementary Table 4). The same analysis was performed on primary cultures initiated from three biopsies (approximately 0.5 cm2 each) taken four (left leg) and eight (left arm and left leg) months after grafting (4Mc, 8Mc1, and 8Mc2, respectively; see Methods).

Notably, we detected only 400, 206, and 413 integrations in 4Mc, 8Mc1, and 8Mc2, respectively (Fig. 3a, bars), with average coverage of 27.3, 19.5, and 20.4 reads per insertion (Fig. 3a, lines).

To exclude the possibility that the large difference in the number of integrations between pre- and post-graft samples could be attributable to PCR reactions causing unbalanced representation of event-specific

amplicons, or to a spatiality effect of punch biopsies, we estimated the expected number of PGc, 4Mc, 8Mc1, and 8Mc2 integrations using the Chapman–Wilson capture–recapture model on the data obtained from the independent libraries (Methods)15. In PGc, the model estimated 65,030 ± 2,120 integrations (approximately twice the actual number of detected insertions). The same model estimated 457 ± 31, 323 ± 50, and 457 ± 24 independent integrations in 4Mc, 8Mc1, and 8Mc2, respectively (confidence level of 99%, α = 0.01), which is consistent with the number of events detected. Of note, 58%, 43%, and 37% of 4Mc, 8Mc1, and 8Mc2 integrations, respectively, were identified in PGc (Fig. 3b), which is consistent with the percentage (approximately 50%) of insertions detected in PGc by next-generation sequencing (NGS) analysis.

Integrations were mapped to promoters (defined as 5-kb regions upstream of the transcription start site of RefSeq genes), exons, introns, and intergenic regions. In all pre- and post-graft samples, about 10% of events were located within promoters. The majority of integrations were either intronic (approximately 47%) or intergenic (approximately 38%) and less than 5% were found in exons (Fig. 3c, left). We also anno- tated integrations in epigenetically defined transcriptional regulatory elements (Methods). As shown in Fig. 3c (right), about 27% of inte- grations were associated with active promoters or enhancers and there was no significant difference in the distribution of insertions between pre- and post-graft samples (P > 0.05; Pearson’s χ 2 test). Thus, the inte- gration pattern was maintained in vivo and epidermal renewal did not determine any clonal selection.

Genes containing an integration were not functionally enriched in Gene Ontology categories related to cancer-associated biological processes16, with the exception of cell migration and small GTPase- mediated signal transduction (Fig. 3d and Extended Data Table 1a).

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Figure 2 | Restoration of a normal epidermis–dermis junction. Skin sections were prepared from skin from a healthy control subject (surgical waste from abdominoplasties or mammoplasty reduction), the patient’s affected skin (admission) and transgenic skin at 4, 8 and 21 months (M) follow-up. a, In situ hybridization was performed using a transgene-specific probe (t-LAMB3) on 10-μ m-thick skin sections. An E-cadherin-specific probe (Cdh1) was used as a control. Scale bars, 40 μ m. b, Immunofluorescence for laminin 332-β 3 was performed with 6F12 monoclonal antibodies on 7-μ m-thick skin sections. DAPI (blue) stains nuclei. Dotted line marks the epidermis–dermis junction. Scale bars, 20 μ m. c, Electron microscopy was performed on 70-nm-thick skin sections. A regular basement membrane (arrows) and normal hemidesmosomes (arrowheads, higher magnification in the inset) can be seen in sections of the patient’s transgenic skin. Scale bars, 1 μ m.

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Figure 3 | Integration profile of transgenic epidermis. a, Integrations were identified in libraries obtained using two LTR primers (3pIN, light grey bars; 3pOUT, dark grey bars; Supplementary Table 1) and in the merged set (black bars). Lines (right axis) depict the average integration coverage, calculated after removal of PCR duplicates. b, Venn diagram of the number of shared integrations across samples. c, Percentage of integrations mapped to: promoters, exons, introns, and intergenic regions (left); epigenetically defined active and weak promoters and enhancers, or genomic regions with no histone marks (right). P > 0.05; Pearson’s χ 2 test. d, Dot plot of the top five enriched Gene Ontology biological process terms for each sample. Dot colour indicates statistical significance of the enrichment (q value); dot size represents the fraction of genes annotated to each term.

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These findings were expected, as our culture conditions were optimized to foster keratinocyte proliferation and migration, to sustain clonogenic cells and to avoid premature clonal conversion and terminal differenti- ation, all of which are instrumental for the proper clinical performance of cultured epidermal grafts14. Thus, similar to what has been reported in transgenic haematopoietic stem cells17,18, our high-throughput analyses revealed a cell-specific vector preference that is related to the host cell status in terms of chromatin state and transcriptional activity at the time of transduction19.

Concerns have been raised about insertional genotoxicity arising from MLV-RV vector use; this has been reported with haematopoietic stem cells in specific disease contexts17,20–22. A γ RV vector, similar to ours, obtained marketing authorization for ex vivo gene therapy of adenosine deaminase severe combined immunodeficiency and has been approved for phase I/II clinical trials on recessive dystrophic epidermolysis bullosa (RDEB)23 (https://clinicaltrials.gov/ct2/show/ NCT02984085).

The patient’s integration profile confirmed the absence of clonal selection both in vitro and in vivo. Likewise, we never observed immor- talization events related to specific proviral integrations in many serially cultivated MLV-RV-transduced keratinocytes. Two patients with JEB who received a total of around 1 × 107 clonogenic transgenic keratino- cytes at selected body sites (3.5 and 12 years follow-up)10–12, and our patient, who received around 3.9 × 108 transgenic clonogenic cells all over his body (Extended Data Fig. 1), did not manifest tumour development or other related adverse events. Therefore, on the basis of in vivo data, the frequency of a detectable transformation event (if any) in MLV-RV-transduced keratinocytes would be less than 1 in 1 × 107 during the first 12 years of follow-up. Although the follow-up of this patient was shorter and does not allow us to draw definitive conclu- sions, the frequency of detectable insertional mutagenesis events to date is less than 1 in 3.9 × 108. In evaluating the risk/benefit ratio, it should also be considered that patients with severe JEB are likely to develop aggressive squamous cell carcinoma as a consequence of the progression of the disease.

The transgenic epidermis is sustained by holoclones The percentage of clonogenic cells, including holoclones, remained rela- tively constant during the massive cell expansion needed to produce the grafts (Extended Data Fig. 1 and Extended Data Table 2). The patient received approximately 3.9 × 108 clonogenic cells, about 1.6 × 107 of which were holoclone-forming cells, to cover around 0.85 m2 of his body (Extended Data Figs 1, 4 and Extended Data Table 2). Thus, approximately 4.6 × 104 cm−2 clonogenic cells or approximately 1.8 × 103 cm−2 stem cells were transplanted onto the patient’s body surface (Extended Data Fig. 4).

If the originally transduced clonogenic cells were all long-lived equi- potent progenitors, we would have recovered thousands of integrations per cm2 of regenerated epidermis, and all clonogenic cells contained in 4Mc, 8Mc1 and 8Mc2 cultures would have had independent integra- tions, irrespective of clonal type. Instead, if the transgenic epidermis were sustained by only a restricted number of long-lived stem cells (continuously generating pools of transient amplifying progenitors), we would have recovered, at most, only a few hundred integrations per cm2, and meroclones and paraclones contained in 4Mc, 8Mc1 and 8Mc2 cultures would have had the same integrations as were found in the corresponding holoclones.

The number of integrations detected in post-graft cultures (Fig. 3a) is consistent with the number of stem cells that have been trans- planted (Extended Data Fig. 4), and therefore strongly supports the latter hypothesis, which was verified by proviral analyses at clonal level (Extended Data Fig. 5) on PGc, 4Mc and 8Mc1. A total of 687 clones (41 holoclones and 646 meroclones or paraclones) were analysed. PGc, 4Mc and 8Mc1 generated 20, 14 and 7 holoclones and 259, 264 and 123 meroclones or paraclones, respectively. Thus, PGc, 4Mc and 8Mc1 contained 7.2%, 5.0% and 5.4% holoclone-forming cells, respectively

(Extended Data Table 2). Each clone was cultivated for further analysis. Libraries of vector–genome junctions, generated by linear- amplification-mediated (LAM) PCR followed by pyrosequencing, retrieved 31 independent integrations unambiguously mapped on the genomes of holoclones (Extended Data Table 1b). One holoclone (4Mc) was untransduced, and 28, 11 and 1 holoclones contained 1, 2 and 3 integrations, respectively. Eleven holoclones in 4Mc shared the same integration pattern. The same happened for two pairs of holoclones in 8Mc1. The copy numbers of holoclones were confirmed by quantitative PCR with reverse transcription (RT–qPCR) (Extended Data Fig. 6). Notably, 75% and 80% of integrations found in 4Mc and 8Mc1 holoclones, respectively, were retrieved in PGc (Fig. 4a), supporting the NGS-based survey as well as a representative sampling. The integration pattern observed in holoclones confirms the absence of selection of specific integrations during epidermal renewal in vivo (Fig. 4b) and mirrors the pattern found in their parental cultures (Fig. 3c), including the absence of genes associated with cell cycle control, cell death, or oncogenesis (Fig. 3d and Extended Data Table 1a).

We then performed clonal tracing by PCR, using the genomic coor- dinates of holoclone insertions. As expected, the majority (91%) of PGc meroclones and paraclones did not contain the same integrations as were detected in the corresponding holoclones (Fig. 4c, PGc). This per- centage had decreased to 37% by 4 months after grafting (Fig. 4c, 4Mc). Notably, virtually the entire clonogenic population of primary keratino- cyte cultures established at 8 months contained the same integrations as were detected in the corresponding holoclones (Fig. 4c, 8Mc1). Thus, the in vivo half-life of transient amplifying progenitors is approximately 3–4 months. These data formally show that the regenerated epidermis is sustained only by long-lived stem cells (holoclones) and underpins the notion that meroclones and paraclones are short-lived progenitors that are continuously generated by the holoclones, both in vitro and in vivo. The high percentage of holoclone integrations retrieved in PGc, together with the number of shared events across cultures (Fig. 3b), suggests that the average coverage of the NGS analysis in PGc allowed

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Figure 4 | Integration profile of stem and transient amplifying cells. a, Percentage of holoclone integrations recovered in the PGc bulk population. b, Holoclone integrations mapped to: promoters, exons, and introns, and intergenic regions (left); epigenetically defined active and weak promoters and enhancers, or genomic regions with no histone marks (right). c, The PGc pie chart shows that 91% of meroclones and paraclones (grey segment) did not contain the same integrations as detected in the corresponding holoclones (each indicated by different blue segments). The 4Mc and 8Mc1 pie charts how that this percentage decreased to 37% and 13%, respectively. TA, transient amplifying.

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us to preferentially identify integrations in holoclones and in transient amplifying cells deriving from such holoclones during the cultivation process.

In summary, as depicted in Extended Data Fig. 7, these findings demonstrate that (i) PGc consisted of a mixture of independent trans- genic holoclones, meroclones and paraclones; (ii) meroclones and paraclones (which can be isolated directly from a skin biopsy) are tran- sient amplifying progenitors, do not self-renew and are progressively lost during cultivation and in vivo epidermal renewal, and therefore do not contribute to the long-term maintenance of the epidermis; (iii) the transgenic epidermis is sustained only by long-lived stem cells detected as holoclones; and (iv) founder stem cells contained in the original primary culture must have undergone extensive self-renewal (in vitro and in vivo) to ultimately sustain the regenerated epidermis, as confirmed by the number of shared events across samples and across holoclones.

Discussion The entire epidermis of a patient with JEB can be replaced by autolo- gous transgenic epidermal cultures harbouring an appropriate number of stem cells. Both stem cells and transient amplifying progenitors are instrumental for proper tissue regeneration in mammals24. However, the nature and the properties of mammalian epidermal stem cells and transient amplifying progenitors are a matter of debate25,26. Although epidermal cultures have been used for 30 years in the clinic14, formal proof of the engraftment of cultured stem cells has been difficult to obtain. Similarly, the identification of holoclones as human epithelial stem cells and of meroclones and paraclones as transient amplifying progenitors, and their role in long-term human epithelial regeneration, have been inferred from compelling but indirect evidence6,8,9,27. Using integrations as clonal genetic marks, we show that the vast majority of transient amplifying progenitors are progressively lost within a few months after grafting and that the regenerated epidermis is sustained only by a limited number of long-lived, self-renewing stem cells. Similar data have been produced with transgenic haematopoietic stem cells28. This notion argues against a model in which a population of equipotent epidermal progenitors directly generate differentiated cells during the lifetime of the animal25, and supports a model in which specific stem cells persist during the lifetime of the human and contribute to both renewal and repair by giving rise to pools of progenitors that persist for various periods of time, replenish differentiated cells and make short- term contributions to wound healing26. Hence, the essential feature of any cultured epithelial graft is the presence (and preservation) of an adequate number of holoclone-forming cells. The notion that the transgenic epidermis is sustained only by engrafted stem cells further decreases the potential risk of insertional oncogenesis.

In conclusion, transgenic epidermal stem cells can regenerate a fully functional epidermis virtually indistinguishable from a normal epidermis, in the absence of related adverse events so far. The different forms of epidermolysis bullosa affect approximately 500,000 people worldwide (http://www.debra.org). The successful outcome of this study paves the way for gene therapy to treat other types of epidermo- lysis bullosa and provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

Received 19 June; accepted 10 October 2017.

Published online 8 November 2017.

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Supplementary Information is available in the online version of the paper.

Acknowledgements Holostem Terapie Avanzate s.r.l. met all costs of GMP production and procedures of transgenic epidermal grafts. This work was partially supported by the Italian Ministry of Education, University and Research (MIUR), no. CTN01_00177_888744; Regione Emilia-Romagna, Asse 1 POR-FESR 2007-13; Fondazione Cassa di Risparmio di Modena; DEBRA Südtirol - Alto Adige; DEBRA Austria; European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 670126-DENOVOSTEM); ERC under the European Union’s Seventh Framework Programme (grant agreement no. 294780-NOVABREED); and Epigenetics Flagship project CNR-MIUR grants. We thank H. Green for continuous support; O. Goertz for his contribution to the surgical procedures; the Department of Anaesthesiology, in particular P. Zahn and T. Maecken, and the entire OR staff, in particular S. Taszarski and V. Stroh, for their dedicated perioperative care; the nurses of ward PÄD1 for continuous and devoted assistance; A. Neumayer and J. Frank for technical assistance in defining clone integrations; B. Mussnig for performing indirect immunofluorescence; M. C. Latella for determining

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