(F1244) INTER- AND INTRA-DONOR VARIABILITY OF IPSC GENERATED FOM HUMAN SKIN PUNCH BIOPSIES VIA AN ACCELERATED AND VALIDATED PIPELINE USING NON-INTEGRATIVE RNA-BASED REPROGRAMMING
Miltenyi Biotec B.V. & Co. KG Bergisch Gladbach, Nordrhein-Westfalen, Germany
Abstract: Human induced pluripotent stem cells with their potential to differentiate into countless different cell types hold great potential for allogenic as well as autologous cellular therapy. However, many traditional techniques used for generation of iPSCs entail the risk for genomic integration and therefore limit their use in a clinical setting. In addition, the established protocols are often lengthy and require the subsequent screening for unwanted vector integration. Transient mRNA based reprogramming eliminates this risk and is one of the fastest and most efficient reprogramming technologies available today.
We hereby present an accelerated and robust protocol to generate cryopreserved and quality controlled stocks of monoclonal iPSC lines from human skin biopsies within 6-7 weeks. Fibroblasts were isolated from abdominal skin via combined mechanical and enzymatic dissociation. The cells were expanded for only one passage before starting the reprogramming routine, eliminating the need for extensive upstream expansion of fibroblast and thus minimizing the risk for cultivation induced aberrations. For reprogramming, cells were transfected for five consecutive days with an mRNA cocktail containing the transcription factors OCT3/4, SOX2, KLF4, MYC, NANOG and LIN28A. During a short growth phase iPSC colonies formed with high efficiency and could be further expanded into stable monoclonal cell lines.
We derived and characterized 36 iPSC lines from five healthy donors (age 50-62). All tested lines showed characteristic stem cell morphology, typical pluripotency-associated marker expression and successfully differentiated into the three germ layers endoderm, ectoderm and mesoderm. While phenotypically all iPSC lines revealed low intra- and inter-donor variability, the verification of genetic integrity (digital PCR / SNP-based CNV analysis) and the assessment of their oncogenic risk profile (whole exome sequencing) was more selective, highlighting the need for comprehensive quality control of newly generated iPSC lines.
In summary, the described protocol allows for efficient and reliable integration-free generation of high quality monoclonal iPSC lines from human skin punch biopsies within only 6-7 weeks and will therefore facilitate the generation of iPSCs from individual donors.
