title: T cells fail to develop in the human skin-cell explants system; an inconvenient truth.
abstract: BACKGROUND
  Haplo-identical hematopoietic stem cell (HSC) transplantation is very successful in eradicating haematological tumours, but the long post-transplant T-lymphopenic phase is responsible for high morbidity and mortality rates. Clark et al. have described a skin-explant system capable of producing host-tolerant donor-HSC derived T-cells. Because this T-cell production platform has the potential to replenish the T-cell levels following transplantation, we set out to validate the skin-explant system.
  
  
  RESULTS
  Following the published procedures, while using the same commercial components, it was impossible to reproduce the skin-explant conditions required for HSC differentiation towards mature T-cells. The keratinocyte maturation procedure resulted in fragile cells with minimum expression of delta-like ligand (DLL). In most experiments the generated cells failed to adhere to carriers or were quickly outcompeted by fibroblasts. Consequently it was not possible to reproduce cell-culture conditions required for HSC differentiation into functional T-cells. Using cell-lines over-expressing DLL, we showed that the antibodies used by Clark et al. were unable to detect native DLL, but instead stained 7AAD+ cells. Therefore, it is unlikely that the observed T-lineage commitment from HSC is mediated by DLL expressed on keratinocytes. In addition, we did confirm expression of the Notch-ligand Jagged-1 by keratinocytes.
  
  
  CONCLUSIONS
  Currently, and unfortunately, it remains difficult to explain the development or growth of T-cells described by Clark et al., but for the fate of patients suffering from lymphopenia it is essential to both reproduce and understand how these co-cultures really "work". Fortunately, alternative procedures to speed-up T-cell reconstitution are being established and validated and may become available for patients in the near future.
authors.affiliation: Department of Internal Medicine, Division of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands.
authors.forename: Bob
authors.initials: B
authors.lastname: Meek
authors.forename: Catharina H M J
authors.initials: CH
authors.lastname: Van Elssen
authors.forename: Mirelle J A J
authors.initials: MJ
authors.lastname: Huijskens
authors.forename: Sjoukje J C
authors.initials: SJ
authors.lastname: van der Stegen
authors.forename: Siebe
authors.initials: S
authors.lastname: Tonnaer
authors.forename: Stijn B J
authors.initials: SB
authors.lastname: Lumeij
authors.forename: Joris
authors.initials: J
authors.lastname: Vanderlocht
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kirkland
authors.forename: Reinout
authors.initials: R
authors.lastname: Hesselink
authors.forename: Wilfred T V
authors.initials: WT
authors.lastname: Germeraad
authors.forename: Gerard M J
authors.initials: GM
authors.lastname: Bos
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000166:cytology*; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C064045:delta protein