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.
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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 the average number of integrations in pre- and post-graft cultures; M. Forcato for feedback on the bioinformatics analyses; and G. De Santis for control skin specimens.
G.P. and M.D.L. are co-founders and members of the Board of Directors of Holostem Terapie Avanzate (HTA), s.r.l, Modena, Italy; Chiesi Farmaceutici S.p.A. (a co-founder of HTA), holds an Orphan Drug Medicinal Product designation (EU/3/15/1465) for the transgenic cultures used in this paper.
Reviewer Information Nature thanks A. Aiuti, C. Blanpain, D. Strech and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
The scheme shows the entire procedure, from skin biopsy to transplantation and follow-up. The total number of keratinocytes, corresponding clonogenic fraction and days of cultivation are shown for each passage. All analyses performed at each follow-up are indicated. Immunofluorescence (IF), in situ hybridization (ISH) and transmission electron microscopy (TEM) were performed on randomly taken 0.2–0.4-mm2 punch biopsies. Genome-wide analysis (NGS) was performed on pre-graft cultures (PGc) and on primary cultures initiated from approximately 0.5-cm2 biopsies taken from the left leg (4Mc and 8Mc2) and the left arm (8Mc1). Clonal analysis and tracing were performed on PGc, 4Mc and 8Mc1.
a, Preparation of a dermal wound bed at the time of transplantation. b, Transplantation onto the left arm of plastic-cultured epidermal grafts, mounted on a non-adhering gauze (asterisks). c, The engrafted epidermis (asterisks) is evident upon removal of the gauze (arrows) ten days after grafting. d, Regenerated epidermis on the left arm after 1 month. e, f, Transplantation (e) and engraftment (f) of both plastic-cultured (asterisk) and fibrin-cultured (arrow and inset in e) grafts on the left leg. f, Inset, complete epidermal regeneration is evident after 1 month. g, The back of the patient was covered with fibrin-cultured grafts (inset). h, Complete epidermal regeneration was observed after 1 month, with the exception of areas marked with asterisks. Islands of epidermis were observed inside those denuded areas (arrows). i, Within 4 months, the regenerated epidermis surrounding the open lesions and the epidermal islands detected within those open lesions had spread and covered the denuded areas.
a, Haematoxylin and eosin staining of skin sections (7-μm thick) prepared from normal skin and from the patient at admission and at 4, 8 and 21 month follow-ups. Black arrows show ruptures at the epidermis–dermis junction. Scale bar, 20 μm. b, Sections (7-μm thick) from normal skin, the patient’s skin at admission and the patient’s skin 21 months after transplantation were immunostained using antibodies against laminin 332-α3, laminin 332-γ2, α6 integrin and β4 integrin. c, Adhesion of cohesive cultured epidermal sheets. Top, spontaneous detachment (arrow) of confluent culture of laminin 332-β3 null keratinocytes from the patient. Bottom, culture of patient’s genetically corrected keratinocytes remained firmly attached to the substrate. As with normal control cells, detachment would have required prolonged enzymatic treatment. d, The absence of a humoral immune response to the transgene product was verified by indirect immunofluorescence performed on monkey oesophagus and normal human split skin (NH-SS) sections, using the patient’s plasma taken 21 months after transplantation. An anti-human laminin-332 antibody (anti-GB3) was used as a positive control (C+). A healthy donor’s plasma was used as negative control (C−). Arrows denote the expected localization of the laminin-332 labelling. Scale bar, 100 μm.
Clonogenic progenitors (blue cells) contained in the original skin biopsy and in 8,472 cm2 of transgenic epidermis are indicated. Stem cells, detected as holoclones (pink cells), were identified by clonal analysis (Methods and Extended Data Fig. 5). The number of holoclones contained in the primary culture has been estimated. The schematic model posits the existence of specific long-lived stem cells generating pools of short-lived progenitors (Hypothesis 1) or a population of equipotent epidermal progenitors (Hypothesis 2). The number of integrations predicted by the Chapman–Wilson capture–re-capture model and formally detected by NGS analysis in 4Mc, 8Mc1 and 8Mc2 (right) is consistent with the number of transplanted holoclones and therefore supports Hypothesis 1.
Sub-confluent cultures were trypsinized, serially diluted and inoculated (0.5 cells per well) onto 96-multiwell plates containing irradiated 3T3-J2 cells. After 7 days of cultivation, single clones were identified under an inverted microscope (scale bar, 100 μm), trypsinized, transferred to two dishes and cultivated. One dish (one-quarter of the clone) was fixed 12 days later and stained with rhodamine B for the classification of clonal type. The clonal type was determined by the percentage of aborted colonies formed by the progeny of the founding cell. The clone was scored as a holoclone when 0–5% of colonies were terminal. When 95–100% of colonies were terminal (or when no colonies formed), the clone was classified as a paraclone. When the amount of terminal colonies was between 5% and 95%, the clone was classified as a meroclone. The second dish (three-quarters of the clone) was used for integration analysis after 7 days of cultivation.
Quantitative PCR was performed on genomic DNA from pre-graft cultures (PGc), primary cultures generated at 4 months (4Mc) and 8 months (8Mc1, 8Mc2) follow-up and selected holoclones (PRE.G_H1, PRE.G_H10, FU4m_H1-11, PRE.G_H7). The experiment was performed in duplicate and the plot is presented as two individual data points for each sample. Source data
Transgenic epidermal cultures (PGc) contain a mixed population of clonogenic basal stem cells (blue) and transient amplifying progenitors (grey). Upon engraftment and initial epidermal regeneration, both stem and transient amplifying cells can proliferate and eventually generate suprabasal terminally differentiated cells. Upon epidermal renewal (after 4 and 8 months), the short-lived transient amplifying progenitors (grey) are progressively lost. The long-lived stem cells then generate new pools of transient amplifying progenitors (now blue basal cells), which will produce terminally differentiated cells (suprabasal blue cells).
During his time in hospital, the patient’s inflammatory and nutritional status was documented by blood concentration of C-reactive protein (CRP; upper panel) and albumin (lower panel). The times of biopsy sampling (B) and epidermal culture transplantation are shown with arrows. The linear regressions visualize the trend of pre-graft (dotted) and post-graft (black line) progressions. The red line within the CRP time course demonstrates the CRP limit, which is considered a criterion for severe inflammation. These data demonstrate the critical situation of the patient at admission and before transplantation and the improvement of his general status upon epidermal regeneration. Source data
This file contains Supplementary Tables 1-3 and Supplementary Data. (PDF 70 kb)
A list of independent integrations identified by NGS analysis. The libraries of integrations were obtained using two independent LTR-primers (3pIN, 3pOUT). The file contains the list of independent integrations found in PGc, 4Mc, 8Mc1 and 8Mc2 and merged data (all_integrations) showing integrations retrieved across samples. (XLSX 1599 kb)
Removal of adherent dressing from sites of follow-up biopsies shows stability of the regenerated epidermis. (MOV 9088 kb)
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Hirsch, T., Rothoeft, T., Teig, N. et al. Regeneration of the entire human epidermis using transgenic stem cells. Nature 551, 327–332 (2017). https://doi.org/10.1038/nature24487
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