Derivation of human iPS supported by isogenic fibroblasts

Abstract

OBJECTIVE: Pluripotent stem cells have the potential to differentiate in vitro in many, if not all, functional cell types. Therefore, these cells may be ideally suited for cellular therapy of diseases for which the loss of functional cells is at the center of the physiopathology. Induced pluripotent stem cells (iPS) have recently emerged as a reproducible model of pluripotent stem cells that can be generated from post-natal tissues. This new technology opens the way to the production of pluripotent cells autologous to each patient. DESIGN: We designed a protocol to generate human iPS (hiPS) were the feeder layer is the same as the cells being reprogrammed, i.e. a human newborn foreskin fibroblast cell line (hFF1). MATERIALS AND METHODS: This cell line at passage 8 was subjected to one round of transduction during 24 hours with four lentiviruses coding for the transcription factors OCT4/POU5F1, NANOG, SOX2 and LIN28. The transduced cells were switched to human embryonic stem cells culture condition 1 or 7 days after the transduction: KO-DMEM + KO-SR + 10 ng bFGF medium on a feeder layer composed of irradiated hFF1. RESULTS: Starting from day 40 posttransduction, we obtained 11 colonies with a typical phenotype of pluripotent cells including a high nucleus-to-cytoplasm ratio and a well-defined colony border, with occasional central patches of differentiating cells. These colonies grow rapidly, are currently at p25 to p28, are alcaline phosphatase positive and express Tra-1-81, marker of pluripotency. At p4 and p15, transgenic expression was not detected by RT PCR, but we observed endogenous expression of Oct4, Sox2, Lin28 and Nanog. Furthermore, expression microarray analysis showed clustering of our reprogramming cells with human ES cells and other hiPS, but not with the original hFF cells among 252 human tissues samples. CONCLUSIONS: In conclusion, we reproducibly generated cells with many characteristics of pluripotent hiPS cells, using an isogenic human fibroblastic feeder layer.