We are still waiting for formal ethical clearance to undertake work using my fibroid (fibroblast) cells. In the interim, Brad sent through a Nature protocol detailing options for producing iPSCs from human keratinocytes derived from plucked hair follicles or skin biopsies. I have isolated keratinocytes from hair follicles before at QUT when I was part of the Tissue Repair and Regeneration Group for the HSE (Human Skin Equivalent/Experience Project). It was quite a mission as keratinocytes require a feeder layer of fibroblast cells. These needed to be irradiated to ensure that they did not outgrow the keratinocytes 🙁
Since we are only doing a skin biopsy if the fibroid cells are not viable, I am parking this option and scouting for protocols that are fibroblast specific. With that in mind, the company Sigma-Aldrich has specified an iPSC reprogramming protocol – Reprogramming of Human Fibroblasts using Non-Integrating Self-Replicating RNA Vectors – designed for fibroblasts and with a 30-day creation estimate. Of course, you always have to double or triple timeframes when you are undertaking a protocol for the first time. Of course, I will need to discuss this option with Brad and seek a pricing and availability estimate.
Fisher Scientific also has a reprogramming kit – the CytoTune-iPS Sendai Reprogramming kit. However, the latest version and full kit carries a hefty price tag at over $18, 500. They also offer a potentially more affordable option via ‘Episomal Vectors’ or Epi5™ Episomal iPSC Reprogramming Kit. These could be an option but Brad is of course the best advisor.
There are also a number of journal articles detailing iPSC reprogramming including: Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1 [Nature], Human Pluripotent Stem Cells (iPSC) Generation, Culture, and Differentiation to Lung Progenitor Cells [Methods Mol Biol.], Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells [Science Direct], Generation of human iPSCs from cells of fibroblastic and epithelial origin by means of the oriP/EBNA-1 episomal reprogramming system [Stem Cell Research & Therapy].
Looks like I’ve got some reading to do…