Article

Regeneration of the entire human epidermis using transgenic stem cells

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Abstract

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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Sequence Read Archive

Referenced accessions

GenBank/EMBL/DDBJ

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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 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.

Author information

Author notes

    • Tobias Hirsch
    • , Tobias Rothoeft
    • , Norbert Teig
    • , Johann W. Bauer
    • , Graziella Pellegrini
    •  & Laura De Rosa

    These authors contributed equally to this work.

Affiliations

  1. Department of Plastic Surgery, Burn Centre, BG University Hospital Bergmannsheil, Ruhr University Bochum, 44789 Bochum, Germany

    • Tobias Hirsch
    • , Frank Jacobsen
    • , Marcus Lehnhardt
    •  & Maximilian Kueckelhaus
  2. Department of Neonatology and Pediatric Intensive Care, University Children’s Hospital, Ruhr University Bochum, 44791 Bochum, Germany

    • Tobias Rothoeft
    • , Norbert Teig
    •  & Meike Fischer
  3. EB House Austria and Department of Dermatology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria

    • Johann W. Bauer
    • , Julia Reichelt
    • , Alfred Klausegger
    •  & Daniela Kneisz
  4. Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, 41124 Modena, Italy

    • Graziella Pellegrini
  5. Center for Regenerative Medicine “Stefano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy

    • Graziella Pellegrini
    • , Laura De Rosa
    • , Alessia Secone Seconetti
    • , Roberta Contin
    • , Elena Enzo
    •  & Michele De Luca
  6. IGA Technology Services s.r.l., 33100 Udine, Italy

    • Davide Scaglione
  7. Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy

    • Oriana Romano
    • , Daniela Quaglino
    •  & Silvio Bicciato
  8. Istituto di Genomica Applicata and Dipartimento di Scienze Agroalimentari, Ambientali e Animali, University of Udine, 33100 Udine, Italy

    • Irena Jurman
    •  & Michele Morgante
  9. Holostem Terapie Avanzate s.r.l., 41125 Modena, Italy

    • Sonia Carulli
    •  & Sergio Bondanza
  10. Department of Neuropaediatrics, University Children’s Hospital, Ruhr University Bochum, 44791 Bochum, Germany

    • Thomas Luecke

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Contributions

T.H., T.R., N.T., J.W.B. and G.P. defined strategic procedures and performed transplantation of the transgenic grafts, surgical and medical procedures and clinical follow-up; L.D.R. performed immunofluorescence data and imaging analysis, analysed the data and assembled all input data, prepared the figures and edited the manuscript; D.S., I.J. and M.M. performed integration profiles of transgenic epidermis; R.C., J.R., A.K. and D.K. performed clonal tracing in epidermal cells; O.R. and S.Bi. conducted all bioinformatics analyses; A.S.S. and E.E. performed in situ hybridization; S.C. and S.Bo. performed all culture procedures and preparation of genetically modified epidermal graft; F.J., T.L., M.L., M.F. and M.K. carried out the follow-up on the patient; D.Q. performed electron microscopy analysis; M.D.L. coordinated the study, defined strategic procedures, administered the experiments and wrote the manuscript.

Competing interests

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.

Corresponding author

Correspondence to Michele De Luca.

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

Supplementary information

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    Supplementary Information

    This file contains Supplementary Tables 1-3 and Supplementary Data.

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    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 4

    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.

Videos

  1. 1.

    Mechanical stress

    Removal of adherent dressing from sites of follow-up biopsies shows stability of the regenerated epidermis.