Generation of germline-competent induced pluripotent stem cells

Abstract

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.

How iPS cells changed the world

“…Over the next year, three laboratories, including Yamanaka's, confirmed the results and improved the reprogramming method [2][3][4] . Within another six months, Yamanaka and James Thomson at the University of Wisconsin-Madison managed to reprogram adult cells from humans 5,6 .…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…Unfortunately, genetic manipulation used to generate iPS cells can cause unexpected effects in activation of oncogens, such as c-MYC and lead to cancer formation. In addition, the viral vectors after integration with the host may lead to unexpected genetic disorder (Okita et al 2007;Takahashi et al 2007;Tateishi et al 2008;Yu et al 2009). Therefore, new and improved methods of generating iPS cells, including minimal or no genetic modification are needed (Huangfu et al 2008;Kaji et al 2009;Okita et al 2008;Stadtfeld et al 2008;Woltjen et al 2009;Yu et al 2009;Zhou et al 2009).…”

Can we grow sperm? A translational perspective on the current animal and human spermatogenesis models

“…63 To circumvent this contentious issue, induced pluripotent stem cells (iPSC) have been developed by activating the pluripotency transgenes such as Yamanaka factors (Oct3/4, Sox2, Klf4 and c-Myc) in adult somatic cells to express embryonic stem cell-like properties. 64 Many strategies including both viral transfection and non-viral delivery of the reprogramming factor transgenes have been established to provide a source of pluripotent cells from adult tissues. 64,65 Detailed historical and current methods of nuclear reprogramming of adult cells to pluripotent state can be found in an excellent review by Yamanaka and Blau.…”

“…64 Many strategies including both viral transfection and non-viral delivery of the reprogramming factor transgenes have been established to provide a source of pluripotent cells from adult tissues. 64,65 Detailed historical and current methods of nuclear reprogramming of adult cells to pluripotent state can be found in an excellent review by Yamanaka and Blau. 66 Human iPSC can be obtained using the same approaches, 67,68 and reprogrammed human iPSCs apparently display similar potential to hESCs in their developmental capacity to generate PGCs.…”