Title: Generation of Human Induced Pluripotent Stem Cells from Unaffected Neonatal Skin Cells
Legend: We have generated and characterized iPSC clones from three unaffected, neonatal individuals using non-integrating episomal reprogramming plasmids expressing OCT4, SOX2, LIN28, KLF4, and c-Myc. Figure A shows representative immunohistochemistry detecting the common pluripotent stem cell markers OCT4 (Green), SOX2 (Red), and NANOG (Magenta) with a Hoechst DNA counterstain (Blue). Figure B shows quantification of the stem cell markers from Figure A using a Perkin-Elmer Operetta high-content imaging platform. A minimum of 10,000 cells were counted for each data set. Figure C shows PCR results depicting the depletion of the episomal reprogramming plasmids by passage 11 in all eight clones. In an episomal-specific PCR, strong PCR product bands are seen for the episomal reprogramming plasmids E1, E2, and E3 but not in the iPSC lanes. PCR for genomic DNA shows products in the iPSC lanes as a positive control. Figure D shows representative, normal karyotyping results for these lines. Figure E shows representative quantitative PCR (qPCR) results that demonstrate the ability of these iPSCs to differentiate to the three germlayers (ectoderm, endoderm, and mesoderm). Following directed differentiation with a tri-lineage differentiation kit (StemCell Technologies), these cells expressed common lineage markers (compared to iPSC controls).
Citation: Yin Y, Petersen AJ, Soref C, Richards W, Berndt E, Zhang S-C, and Bhattacharyya A. Generation of human induced pluripotent stem cells from unaffected neonatal skin cells. (2018) Stem Cell Res – Pending Submission.
Abstract: Human induced pluripotent stem cells (iPSCs) hold great promise for basic research and regenerative medicine. Many iPSCs have been generated from healthy donors for basic research and for use as controls for disease modeling using disease specific pluripotent stem cells. Most iPSCs – both control and disease – that are publicly available are derived from adult donors. Here, we have generated and characterized multiple iPSC clones from three unaffected individuals from skin fibroblasts collected following birth. These iPSC lines offer age-matched control cell lines for developmental disease research and comparison cells for age-related research. Furthermore, the neonatal donor cells have not been exposed to postnatal environmental conditions that may give rise to epigenetic modifications or de novo genetic mutations. iPSCs generated from neonates provide a valuable resource for studies of human developmental biology, developmental disorders, and aging.
About the investigator: Anita Bhattacharyya’s lab examines how brain development is altered in developmental disorders characterized by intellectual impairment. The cerebral cortex is the most complex area of the brain and is responsible for functions unique to humans, such as language and abstract thought. Problems in any of the crucial cerebral cortex formation steps can lead to intellectual impairment.