Takahashi and Yamanaka first reported

Takahashi and Yamanaka (2006) first reported the establishment of murine iPSCs in 2006, by transducing mouse embryonic fibroblasts with retroviruses encoding c-Myc, Oct3/4, Sox2, and Klf4. Subsequently, the first hiPSC lines were derived in 2007 (Yu et al., 2007; Takahashi et al., 2007), again by employing retroviral transduction of fibroblasts. Since this pioneering research, the field of iPSC research continues to expand rapidly and several attempts have been made to develop methods that minimise the random integration of transgenes into the genome, which is considered necessary for clinical applications. These include excisable viral cassettes (Kaji et al., 2009; Woltjen et al., 2009), and nonintegrating adenoviral vectors (Zhou and Freed, 2009; Stadtfeld et al., 2008), together with nonviral methods such as repeated plasmid transfection (Okita et al., 2008) and protein transductions (Kim et al., 2009; Zhou et al., 2009). However, to date, these methods are associated with reprogramming efficiencies of as low as 0.0001–0.001%, which is between 1000-fold and 100-fold lower than retroviral transduction. Moreover, they are often restricted to embryonic or fetal tissue, or to mouse cells, whereas the desired target for clinical relevance would be p450 inhibitors isolated from adult humans. Therefore, for in vitro biomedical applications, such as disease modeling or drug screening where random viral integrations are of lesser significance, retroviral transduction is still the method of choice. Although the use of retroviruses in hiPSC production is now well established, the method is constrained by the ability to cost-effectively produce lentivirus on a scale large enough to meet the requirements of the experiment. Furthermore, the common method for concentrating the virus by ultracentrifugation requires specialist equipment, and can be prohibitive due to this cost. Here we demonstrate that viral yields can be increased 100-fold by optimising the production conditions, and in addition, the transduction efficiency can be improved a further 6-fold by conjugating the virus produced to streptavidin superparamagnetic particles. This means that a single well within a 6-well plate can produce between 0.5×107 and 1×107 infectious units, enough for 10–20hiPSC experiments in which 50 000 somatic cells are transduced with a multiplicity of infection of 10. As a result, the labor and cost burden of this element of hiPSC line production are greatly reduced. An additional obstacle we have addressed in the production of sustainable hiPSC lines is the identification and subsequent isolation of truly reprogrammed hiPSCs from a heterogeneous derivation culture that also contains mouse embryonic fibroblast feeders, somatic cells, and partially reprogrammed cells of similar morphology to hiPSCs. This process remains highly subjective, but is critical for the production of high-quality hiPSC lines. Judgments are made on the basis of appearance with the “best looking,” and often faster growing colonies being selected for propagation. In addition, this process of mechanical dissection and passage to culture the new hiPSC line is highly labor-intensive. Therefore, we have developed an isolation method involving the positive selection of “true” hiPSCs based on the cell surface expression of the pluripotency marker TRA-1-81, previously shown to be the most appropriate marker for identification of fully reprogrammed hiPSCs in culture (Chan et al., 2009). Not only does this eliminate the selection bias, but because manual identification and mechanical dissection are replaced with bulk trypsin passaging, the process is simplified and allows many lines to be derived at once.
Results and discussion
Materials and methods
Acknowledgments
Introduction Embryonic stem cells (ESCs) are cells derived from the inner cell mass (ICM) of preimplantation blastocysts with the ability to produce any cell type in the adult animal. While ESCs can differentiate to form various cell types, they can also divide to form two daughter ESCs via self-renewal. Although self-renewal encompasses both growth and maintenance of developmental potential, most research has focused on understanding the signaling and transcriptional networks involved in the latter, i.e., maintenance of the ESC pluripotent state (Liu et al., 2007). In contrast, comparatively few studies have investigated pathways involved in ESC growth and death under conditions that have been shown to maintain pluripotency. Studies have shown that the addition of EGF, ANG-2, Activin, Nodal, LIF, or BMP-4 proteins to mouse ESC (mESC) cultures results in improved growth (Heo et al., 2006; Han et al., 2007; Ogawa et al., 2007; Viswanathan et al., 2002; Qi et al., 2004). However, these studies did not include testing for secretion of these proteins by ESCs; i.e., they did not attempt to fully demonstrate that these pathways are constitutively active in mouse ESC cultures and that the factors are acting in an autocrine fashion. SDF-1 has been shown to act as an autocrine survival factor for mESCs, but only in the absence of serum (i.e., during serum withdrawal) (Guo et al., 2005).