Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions

Abstract

The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and aintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency- associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.