Small Molecules Improve Episomal Reprogramming Figure 4. Optimizing episomal vector and transgene combinations for small molecule-aided reprogramming of different human somatic cells. (A) Effects of different episomal vector combinations and transformation-deficient LMYC on the reprogramming of human fibroblasts. Transfected human foreskin fibroblasts (HFFs) were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 13 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ,0.336106 input cells) was counted on day 21 post-transfection. Data shown are mean 6 SEM (n = 3). (B) Effects of different transgene combinations on the reprogramming of human adult adipose tissue-derived stem cells (AdSCs). Transfected adipose tissue-derived stem cells were plated to matrigel-coated 10-cm dishes. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post- transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ,0.356106 input cells) was counted on day 21 post-transfection. (C) Effects of transformation-deficient LMYC on the reprogramming of human cord blood (CB) cells. Purified human cord blood CD34+ cells were expanded in culture for 7 days (,75 fold expansion). Following expansion, the cord blood cells were nucleofected with episomal vectors and plated to fibronectin/matrigel-coated 6-well plates. N2B27-100 medium supplemented with PCALH was used to support reprogramming between day 2 and 11 post-transfection, followed by mTeSR1 for expansion. The number of iPSC colonies (from ,0.176106 post-expansion input cells) was counted on day 17 post-transfection. Data shown are mean 6 SEM (n = 6). doi:10.1371/journal.pone.0017557.g004 removing the bacterial vector component using site-specific recombination to produce minicircle oriP/EBNA-1 episomal vectors. Nevertheless, the current method is robust enough for the routine derivation of footprint-free iPSCs from different somatic cell types and from many human donor samples. Selection of the right episomal vector combination and transgene combination appeared to be important for achieving optimal reprogramming efficiency for different somatic cell types (Fig. 4A and 4B), likely reflecting their different requirement of balanced transgene expression. Since only a single transfection is needed for episomal reprogramming, this method could be readily applied to many cell types including blood cells such as cord blood cells, although peripheral blood cells might represent an ideal cell source for large-scale studies as established procedures are readily available for blood handling (e.g. collection, cryopreservation, shipping and cell isolation). It is interesting to see how well this method can be applied to peripheral blood cells, particularly cells without genomic rearrangements. The current episomal repro- gramming method used matrigel as the matrix to support reprogramming and iPSC expansion due to its affordability. This, however, introduced undefined components into the method. For the production of clinical-grade human iPSCs, the matrigel can most likely be replaced with defined matrix that supports both donor cell and iPSC growth, such as vitronectin [32]. The disadvantage with the episomal method, compared to mRNA- mediated reprogramming [17], is that the iPSCs will need to undergo multiple passages to completely lose episomal vectors (e.g. ,14 passages for fibroblast-derived iPSCs). This, however, will unlikely present a problem in applications, since iPSCs will need to undergo multiple passages to be rid of epigenetic memory of the donor cells [18], and to be fully characterized for downstream applications. Episomal reprogramming in the presence of small molecules appeared to progress through a distinct intermediate stage - piPSCs (Fig. 2B), as the same cells were obtained when the N2B27 medium was supplemented with bFGF in test 2 (Fig. 2A). These piPSCs expressed alkaline phosphatase, could be continuously passaged in the presence of small molecules, and retained episomal reprogramming vectors, transgene expression and the ability to give rise to human ESC-like iPSCs upon withdrawal of small molecules. The generation of these piPSCs depended on both the SV40LT expression and the small molecule treatment, since none of these piPSCs could be observed in the absence of the SV40 LT expression or the small molecules. In our previous report, we included SV40LT to counteract the toxic effects caused by c-MYC overexpression [19]. The role of SV40LT in reprogramming, however, is rather controversial. It was initially used in combination with OCT4, SOX2, c-MYC and KLF4 to reprogram human fibroblasts, but was found to be absent from the iPSCs generated [33]. This suggested that the SV40LT expression did not contribute to iPSC derivation. A second report showed that the SV40LT expression enhanced both the reprogramming efficiency and the reprogramming pace [34]. Results from our own work showed that although the SV40LT expression greatly facilitated the appearance of reprogramming intermediates, its continuous expression blocked the iPSC generation (unpublished data). With the small molecule-aided episomal reprogramming in defined media, the SV40LT expression seemed critical, since no PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e17557