ABSTRACT
We present Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a scalable platform producing autologous organotypic iPS cell-derived induced skin composite (iSC) grafts for definitive treatment. Clinical-grade manufacturing integrates CRISPR-mediated genetic correction with reprogramming into one step, accelerating derivation of COL7A1-edited iPS cells from patients. Differentiation into epidermal, dermal and melanocyte progenitors is followed by CD49f-enrichment, minimizing maturation heterogeneity. Mouse xenografting of iSCs from four patients with different mutations demonstrates disease modifying activity at 1 month. Next-generation sequencing, biodistribution and tumorigenicity assays establish a favorable safety profile at 1-9 months. Single cell transcriptomics reveals that iSCs are composed of the major skin cell lineages and include prominent holoclone stem cell-like signatures of keratinocytes, and the recently described Gibbin-dependent signature of fibroblasts. The latter correlates with enhanced graftability of iSCs. In conclusion, DEBCT overcomes manufacturing and safety roadblocks and establishes a reproducible, safe, and cGMP-compatible therapeutic approach to heal lesions of DEB patients.
Subject(s)
Cell- and Tissue-Based Therapy , Collagen Type VII , Epidermolysis Bullosa Dystrophica , Induced Pluripotent Stem Cells , Humans , Epidermolysis Bullosa Dystrophica/therapy , Epidermolysis Bullosa Dystrophica/genetics , Animals , Induced Pluripotent Stem Cells/transplantation , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Mice , Collagen Type VII/genetics , Collagen Type VII/metabolism , Cell- and Tissue-Based Therapy/methods , Fibroblasts/metabolism , Cell Differentiation , Keratinocytes/metabolism , Keratinocytes/transplantation , Skin/metabolism , Transplantation, Autologous , Male , Mutation , Female , Skin Transplantation/methods , Gene Editing/methods , CRISPR-Cas SystemsABSTRACT
Type 1 regulatory T (Tr1) cells are inducible, interleukin (IL)-10+FOXP3− regulatory T cells that can suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have optimized an in vitro protocol to generate a Tr1-enriched cell product called T-allo10, which is undergoing clinical evaluation in patients with hematological malignancies receiving a human leukocyte antigen (HLA)mismatched allo-HSCT. Donor-derived T-allo10 cells are specific for host alloantigens, are anergic, and mediate alloantigen-specific suppression. In this study, we determined the mechanism of action of T-allo10 cells and evaluated survival of adoptively transferred Tr1 cells in patients. We showed that Tr1 cells, in contrast to the non-Tr1 population, displayed a restricted T cell receptor (TCR) repertoire, indicating alloantigen-induced clonal expansion. Tr1 cells also had a distinct transcriptome, including high expression of cytotoxic T lymphocyteassociated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). Blockade of CTLA-4 or PD-1/PD-L1 abrogated T-allo10mediated suppression, confirming that these proteins, in addition to IL-10, play key roles in Tr1-suppressive function and that Tr1 cells represent the active component of the T-allo10 product. Furthermore, T-allo10derived Tr1 cells were detectable in the peripheral blood of HSCT patients up to 1 year after T-allo10 transfer. Collectively, we revealed a distinct molecular phenotype, mechanisms of action, and in vivo persistence of alloantigen-specific Tr1 cells. These results further characterize Tr1 cell biology and provide essential knowledge for the design and tracking of Tr1-based cell therapies.
Subject(s)
Isoantigens , Programmed Cell Death 1 Receptor , CD4-Positive T-Lymphocytes , CTLA-4 Antigen , Humans , T-Lymphocytes, RegulatoryABSTRACT
Human T lymphocytes were stimulated using phorbol myristate acetate and ionomycin. Twenty-four hours post-activation the cells were harvested for DNA content and for measurements using a newly developed cell profiling system employing dielectrophoresis. This system provides individual cell size and dielectrophoresis data for statistically relevant numbers of control and activated cells. From this it was determined that the mean membrane specific capacitance decreased from 13.49 (+/- 4.72) mF/m(2) to 10.62 (+/- 5.13) mF/m(2). This can be related to a 21.3% reduction in the effective membrane surface area associated with membrane topography (e.g. reduction of membrane associated microvilli, blebs and folding), or to other changes of membrane architecture, following cell activation. From cytometric determinations of DNA content, it was concluded that these effects were related to a 3.0-fold decrease of cells in S-phase, and a 1.5-fold increase in G1 cells. This work demonstrates the powerful potential of using dielectrophoresis as a noninvasive tool to follow physiological changes that accompany transmembrane signaling events.
