Reconstitution of T-lineage differentiation potential of induced pluripotent stem cells derived from severe combined immunodeficiency patients by retroviral gene transfer.
Zusammenfassung der Projektergebnisse
The aim of this project was to complement iPSCs generated from fibroblasts obtained from patients with severe combined immunodeficiency with lenti-viral vectors and test their T-cell differentiation potential in vitro. Finally, generation, complementation and characterization of iPSC with NHEJ defects became the focus of this work. Fibroblast cell lines obtained from patients with radiosensitive SCID caused by NHEJ defects, including Artemis, Ligase 4 and DNA-PKcs deficiency, were reprogrammed into induced pluripotent stem cells using lenti-viral vector transfer of the four transcription factors Oct4, Sox2, Klf4, and cMyc. While reprogramming efficiency of Artemis-deficient fibroblasts was similar to control cells, it was severely impaired in LIG4- and also significantly reduced in DNA-PKcs-deficient cells. Furthermore, iPSC derived from LIG4-deficient fibroblasts acquired karyotypic abnormalities during reprogramming. All NHEJ-deficient cell lines showed impaired DNA repair kinetics studied by yH2AX foci formation at various time points after irradiation. NHEJ activity was analyzed in fibroblasts and iPSC using a plasmid based assay and revealed impaired direct joining, but increased microhomoly joining in NHEJ-deficient cells using alternative pathways. Gene complementation with codon-optimized Ligase 4 completely restored DNA repair and NHEJ activity and partially restored reprogramming efficiency in LIG4-deficient fibroblasts. But, since the transgene was silenced in most of the iPSC clones obtained from complemented fibroblasts, only one LIG4-deficient iPSC clone could be expanded under antibiotic selection. NHEJ activity was impaired in iPSC in the same way as in parent fibroblasts, however, was completely restored in the LIG4 complemented iPSC clone. Radiosensitivity in iPSC was studied on synchronized cells in G0/G1 and G2/M distinguished by CENP-F expression and revealed impaired DNA repair in NHEJ-deficient lines. Besides this, irradiation resulted in a cell cycle block in G0/G1 and impaired proliferation in NHEJ-deficient iPSC compared to control iPSC. Although the differentiation towards the myeloid lineage does not seem to be affected in Artemis- and DNA-PKcs-deficient iPSC, LIG4-deficient cells show impaired survival in developing embryoid bodies and less efficient maturation into granulocytes. However, DNA repair in irradiated myeloid cells was impaired with similar kinetics as observed in parent fibroblasts. Overall, these findings suggest a predominant role of Ligase 4 in reprogramming and differentiation, which is not or only partially affected in Artemis- and DNA-PKcs-deficient iPSC, respectively.
Projektbezogene Publikationen (Auswahl)
- The role of induced pluripotent stem cells in research and therapy of primary immunodeficiencies; Curr Opin Immunol, 2012 (5): 617-24
Weinacht KG, Brauer PM, Felgentreff K, Devine A, Gennery AR, Giliani S, Al-Herz W, Schambach A, Zúñiga-Pflücker JC, Notarangelo LD
(Siehe online unter https://doi.org/10.1016/j.coi.2012.07.001)
