There is a general movement towards generating induced pluripotent stem (iPS) cells in more defined, feeder-free, and clinically relevant systems, as we move closer to realizing the use of iPS-derived cells in cellular therapies. Moreover, there is growing need to reprogram cells from easily accessible tissues such as skin (fibroblasts) and blood, to develop disease models and patient-specific therapeutics. TeSR™-E7™medium, based on a formulation by Chen et al. (2011), is a low-protein, xeno-free, and defined medium for feeder-free reprogramming. Here, we demonstrate the use of TeSR™-E7™ in reprogramming of human fibroblast cells into fully characterized iPS cells, and furthermore extend its utility to include reprogramming of other somatic cell types such as bone marrow-derived mesenchymal stromal cells (MSCs), CD34+ hematopoietic cells, and culture expanded erythroid cells. Fibroblast suspensions from different sources were transfected with episomal vectors containing Oct4, Sox2, Klf4, L-Myc, and then plated onto Matrigel®-coated plates containing either TeSR™-E7™, TeSR™-E8™, or KnockOut Serum Replacement (KOSR)-containing human embryonic stem (hES) cell maintenance medium (KOSR-hES). Cells were cultured in each medium with daily changes and iPS cell colonies exhibiting human pluripotent stem cell (hPSC)-like colony morphology were enumerated after 21 - 28 days. A range of reprogramming efficiencies were observed in TeSR™-E7™ from human fibroblasts of adult (0.125 ± 0.05%, n=3), neonatal (0.016 ± 0.003%, n=2) and fetal (0.015%, n=1) origin. Notably, reprogramming efficiencies in TeSR™-E7™ were 2-fold higher compared to KOSR-hES for neonatal fibroblasts (n=6, p<0.05) but equivalent for adult fibroblasts (n=6). Reprogramming efficiencies were on average 9.4- and 6.1-fold higher in TeSR™ E7™ compared to TeSR™-E8™ for both neonatal and adult fibroblasts (n=6, p<0.05). TeSR™-E7™-derived iPS cell colonies were then picked and sub-cultured in mTeSR™1 or TeSR™-E8™ maintenance media on Matrigel or Vitronectin-XF™ matrices for further characterization. iPS cell lines generated in TeSR™-E7™ exhibited characteristic hPSC-like morphology, >95% positivity for Oct4, SSEA-3, Tra-1-81 and were karyotypically normal. In addition, iPS cells derived in TeSR™-E7™ exhibited the capacity to differentiate efficiently to cells of the three germ-layers using STEMdiff™ Neural Induction Medium, STEMdiff™ Definitive Endoderm kit, or STEMdiff™ APEL™ Medium supplemented with cytokines. Finally, we show reprogramming efficiencies for multiple somatic cell types, including MSCs (0.001%, n=2), CD34+ hematopoietic cells (0.015%, n=1), and culture expanded erythroid cells (0.007%, n=1) with successful establishment of iPS cell lines from these cell sources. All iPS cell lines derived from these cell sources exhibited hPSC morphology and are now being further characterized for pluripotency. Overall, our studies demonstrate the robustness and versatility of TeSR™-E7™ for reprogramming multiple human somatic cell types.

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4th International Conference on Stem Cell Engineering

Induced Pluripotent Stem Cells Generated from Multiple Somatic Cell Types Via Feeder-Free Reprogramming in TeSR™-E7™ Medium

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