Platform for Induction and Maintenance of Transgene-free hiPSCs Resembling Ground State Pluripotent Stem Cells

• Published in: Stem cell reports 2014-03, Vol.2 (3), p.366-381
• Main Authors: Valamehr, Bahram, Robinson, Megan, Abujarour, Ramzey, Rezner, Betsy, Vranceanu, Florin, Le, Thuy, Medcalf, Amanda, Lee, Tom Tong, Fitch, Michael, Robbins, David, Flynn, Peter
• Format: Article
• Language: English
• Subjects: Abnormal Karyotype; Animals; Cell Culture Techniques; Cell Differentiation; Cell Transdifferentiation; Cells, Cultured; Cellular Reprogramming; Chromosome Aberrations; Cluster Analysis; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation, Developmental; Genomic Instability; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Mice; Pluripotent Stem Cells - cytology; Pluripotent Stem Cells - metabolism; Transcription Factors - genetics; Transcription Factors - metabolism; Transgenes
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2014.01.014

Cell banking, disease modeling, and cell therapy applications have placed increasing demands on hiPSC technology. Specifically, the high-throughput derivation of footprint-free hiPSCs and their expansion in systems that allow scaled production remains technically challenging. Here, we describe a platform for the rapid, parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and expedited episomal reprogramming using just OCT4/SOX2/SV40LT combination (0.5%–4.0%, between days 12 and 16) in a completely feeder-free environment. The resulting hiPSCs are transgene-free, readily cultured, and expanded as single cells while maintaining a homogeneous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions described exhibit properties associated with the ground state of pluripotency. The simplicity and robustness of the system allow for the high-throughput generation and rapid expansion of a uniform hiPSC product that is applicable to industrial and clinical-grade use. [Display omitted] •FRM supports highly efficient minimal factor episomal reprogramming•Clonal derivation of hiPSCs through high-resolution single-cell sorting•FMM supports long-term maintenance of genomically stable, transgene-free hiPSCs•Maintenance of homogeneous pluripotent culture resembling the ground state Cell therapy and banking applications have placed increasing demands on hiPSC technology. Here, Flynn and colleagues demonstrate a simple multiplex selection and maintenance platform utilizing stage-specific small molecule media additives and flow cytometry sorting to derive minimal factor episomal hiPSCs that are genomically stable and resemble ground state pluripotent stem cells. This platform may serve as a path to generate clinically relevant hiPSCs.