Year 1

Generation of pluripotent human stem cell lines (iPS) requires the expression of 3-5 transcription factors. However, pre-existing somatic mutations and mutations acquired from integrated viral or naked DNA vectors used to introduce these transcription factors pose a considerable risk for cancer and represent the major obstacle in translating stem cell therapy to the clinic. To overcome this obstacle, we proposed an alternative approach to generating patient-derived stem cell lines using cell-permeable, pluripotent-inducing transcription factors. Introducing active proteins into cells avoids the long-term risk of genetic mutations caused by DNA based expression vectors of these transcription factors. Our labs pioneered the cell-permeable delivery of proteins, including transcription factors into cells. After mastering the delivery approach in tester human fibroblasts, we will focus our efforts on various easily accessible cell types from skin biopsies.

To use our protein delivery technology for the generation of iPS cells from skin cells, several milestones need to be achieved. First, proteins of five pluripotent transcription factors (Oct4, Nanog, Sox2, Klf4, Myc, and Lin28) were engineered to contain the cell-permeable delivery domain or Protein Transduction Domain (PTD) and produced in large quantities in the lab. Second, to test the activity of each cell-permeable pluripotent transcription factor, we have assessed their ability to enter the nucleus and to activate specific gene targets. We have found several problems, relating primarily to expression levels and retention of transcriptional activity. Our current work suggests that the placement or physical location of the cell-permeable delivery domain may interfere with optimal activity of the transcription factor. To optimize activity, additional fusion proteins have been generated and the intracellular levels and transcriptional activity will be determined. Another possibility for poor activity is the trapping of cell-permeable proteins in endosomal compartments, preventing efficient nuclear trafficking. We have previously utilized additional proteins to facilitate escape of cell-permeable proteins from these compartments and, thus, several peptides and drugs are being screened for their ability to improve targeting by recombinant cell-permeable proteins.

Another area of progress has been the isolation of cells from different compartments of the skin. Fibroblasts from the skin are extremely heterogeneous, thus fibroblasts from different layers of the skin, e.g. papillary vs. reticular dermis, are being isolated and assessed for differences in their efficiency in cell-permeable protein targeting. Current cultures are being assayed to confirm the origin of these cells and will be tested for activity against cell-permeable transcription factors described above. In addition, over the past year, several new adult-stem cell populations have been identified in the hair follicle. Efforts in the upcoming year will be to isolate these populations for similar tests of cell-permeable protein targeting and efficiency in becoming iPS cells.

Another important aspect of iPS generation is the ratio of each pluripotent transcription factor to the other. Using a surrogate approach, we have performed extensive matrices of varying each transcription factor relative to the other and have now identified the optimal ratio. This is a key step in optimize the generation of iPS cell induction. Overall, we have made significant, albeit slower than expected, progress in the design and purification of the PTD-transcription factor fusion proteins. We anticipate that as we perform more experiments with these PTD fusion proteins that work will rapidly accelerate during the second year.